Note: Descriptions are shown in the official language in which they were submitted.
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NASAL-ADMINISTERED VACCINES USING
MULTI-SCREENED NALT-TARGETING AND PHAGOCYTIC
POLYPEPTIDE TRANSPORT SEQUENCES
FIELD
[0001 ] This invention is in the fields of medicine, biochemistry, and
vaccines. In
particular, it relates to vaccines that can be manufactured very rapidly and
in huge
quantities by using specialized viruses that grow in bacteria (rather than
requiring bird eggs
or other eukaryotic cells for viral incubation), and that can be administered
by spraying a
mist into the nasal cavities, without requiring needles or syringes.
BACKGROUND
[0002 ] Reference to any prior art in this specification is not, and should
not be taken as, an
acknowledgment or any form of suggestion that this prior art forms part of the
common
general knowledge in any country.
[0003 ] At the most basic level, vaccines function by presenting foreign
antigens, to cells
that function as part of a mammalian immune system. This process allows an
immune
system to lay the groundwork for accelerated formation of antibodies that will
help block
and kill invading pathogens, if a need ever arises in the future. Beyond that
basic level, the
immune system is quite complex, and uses multi-step sequences involving
numerous
different types of cells. Chapter-length descriptions that provide good
overviews are
available in books such as Alberts et al, Molecular Biology of the Cell, or
Guyton and Hall,
Textbook of Medical Physiology. More detailed analyses are available in books
such as
Kuby Immunology, 6th edition (Kindt et al, W.H. Freeman, 2006), Immunology,
5th
Edition (Goldsby et al, W.H. Freeman, 2002), and Immunobiology, 6th edition
(Janeway,
Garland Science, 2004). In addition, numerous review articles (which can be
located
through the U.S. National Library of Medicine database, at
www.ncbi.nlm.nih.gov/entrez/query.fcgi) provide more information on specific
aspects of
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vaccines or immunology. As examples, Thomas et al 2005 reviews genetically-
engineered
vaccines, while articles such as Gaubin et al 2003, Clark et al 2004, Wang et
al 2004, and
Miedzybrodzki et al 2005 focus on bacteriophages as vaccine carriers or
delivery vehicles.
Various materials also are available via the Internet. For example, the
materials for
numerous college or medical school courses on immunology are available (one
example is
at http://uhaweb.hartford.edu/BUGL/immune.htm). Glossaries also are available,
such as at
http://users.path.ox.ac.uk/-scobbold/tig/gloss.html. Accordingly, background
information
herein on the immune system is intended only as an introduction and overview,
and more
detailed information is available from other sources.
[0004 ] Assuming that a reader understands the basic cell types and processes
that are
involved in immune responses, attention must be focused on vaccines that can
be presented
via nasal sprays, or other methods that involve contact with "mucosal"
surfaces, discussed
below. This route of administration has several potential advantages, compared
to vaccines
that require injection using needles (injection using needles is often
referred to as
"parenteral" administration, and often involves intramuscular injection).
However, under
the prior art, nasal and other mucosal vaccines have not reached a point where
they are as
effective as injected vaccines, and they have not been highly successful. The
obstacles that
must be overcome by nasal vaccines, and the progress they have made in recent
years, are
discussed in articles such as Eriksson et al 2002, Kiyono et al 2004, and
Mestecky et al
2005. A mucosal vaccine that has been approved for use protects against
influenza, and is
sold under the trademark FLUMIST(TM) by Medlmmune Vaccines, Inc., as described
in
articles such as McCarthy et al 2004, and on websites such as www.flumist.com,
www.fda.gov/cber/flu/flumistqa.htm, and
www.rxlist.com/cgi/generic3/flumist.htm. Failed
efforts to develop mucosal vaccines are also known, and include, for example,
Oravax Inc.,
which effectively collapsed in 1998 and was acquired by Peptide Therapeutics
Group PLC,
which later was taken over by Acambis PLC (www.acambis.com), which apparently
is no
longer attempting to develop any mucosal vaccines.
[0005 ] In the field of nasal vaccines, mucoadherents and adjuvants require
attention.
Mucoadherents (also called "absorption enhancers" or similar terms) will cause
a nasal
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spray or similar formulation to either: (i) cling to the nasal sinuses (or
other mucous
membranes) for a longer period of time, or (ii) penetrate through a mucous
membrane
more rapidly, and in higher quantities. Either of those effects can increase
the likelihood
that an antigen in a vaccine will be noticed and recognized as foreign, by the
immune
system, in ways that will provoke a desired immune response. Accordingly,
mucoadherents can be used with nasal vaccines as disclosed herein. Examples
include
chitosan and certain types of cyclodextrins, phospholipids, and other
"bioadhesive"
compounds, described in articles such as Davis et al 2003 and Zuercher 2003,
and powders
that convert into gels when they become wet, such as a compound from aloe vera
plants
sold by DelSite Biotechnologies under the trademarks GELVAC and GELSITE
(www.delsite.com). More information on vaccines formulated as aerosols,
powders, or
similar forms is in articles such as LiCalsi et al 1999 and Chan et al 2006.
It should also be
noted that the nasal membranes are negatively charged; therefore, use of
"cationisation"
(described below) to impart a positive charge to vaccine particles may also be
able to
increase the contact time between the vaccine particles and their targeted
membrane
surfaces.
[0006 ]The subject of adjuvants is more complex, and requires careful
attention.
ADJUVANT DEFINITIONS, TYPES, AND EFFECTS
[0007 ] In the field of immunology, definitions of "adjuvant" have changed and
evolved
over time, and early definitions (which began to appear in the 1920's) may not
encompass
recent developments or definitions. Two functional definitions need to be
considered; they
say essentially the same thing, but with subtle differences.
[0008 ] In a first widely-used definition, an adjuvant is an agent or
substance which, while
not having any specific antigenic effect in itself, can stimulate an immune
system in a
manner that increases a response to a vaccine. In a second definition, an
adjuvant is an
agent or substance which, when combined with an antigen, reduces the amount of
antigen
that is needed in order to stimulate an effective immune response. A subtle
distinction
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between those definitions is described below, since it is relevant to this
invention.
[0009 ] In prior decades, and in most relevant articles and books, most
adjuvants were
separate compounds that were mixed with an antigen-carrying vaccine, to create
an
enhanced formulation. If an antigen in a vaccine formulation is accompanied by
an
adjuvant that functions, in effect, as an irritant that will provoke
inflammation, swelling, or
similar responses, the immune system will be alerted more rapidly to the type
of challenge
that arises when something foreign enters the body at a certain location. When
an immune
system has been "alerted" by an adjuvant that provokes or increases localized
inflammation, it will use signaling and response mechanisms to send large
numbers of
immune cells to that site, to help fight the invasion. If numerous immune
cells are sent to
the site where a vaccine was administered, those immune cells will encounter
and detect
the antigen more rapidly, and in larger numbers. This will lead to a larger,
faster, more
effective response to the antigenic molecule, which is also present in the
vaccine mixture,
and which is injected into the same site as the adjuvant (this also explains
why nearly all
injectable vaccines are injected into muscle or skin tissue, rather than
intravenously).
Accordingly, an adjuvant can increase both the speed and the magnitude of an
immune
response to a vaccine.
[00010 ] Similar effects explain how adjuvants can help immune systems mount a
more effective and consistent responses to a "weak" antigen. In this context,
"weak"
antigens are classified based on their effects in lab animals; weak antigens
are those that
are not strong enough to trigger full-scale antibody-producing immune
responses, in the
majority (or in a sizable fraction) of inoculated animals, unless an adjuvant
is also
administered. In this field, terms such as "weak" versus "strong" will depend
on the
animals used, the dosages used, and similar factors.
[00011 ]For decades, immunologists used a mixture called Freund's complete
adjuvant
(FCA), when they injected animals with a foreign antigen to provoke an immune
response.
The "complete" mixture contained pathogenic microbes called mycobacteria,
which had
been killed by heat or chemicals. However, FCA tends to provoke painful
inflammatory
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responses, as part of its mode of action. Therefore, it is not used in human
medicine, and
when laws were passed in most countries (mostly in the 1980's) requiring that
any pain
and suffering imposed on lab animals must be minimized, other adjuvants began
to be
actively developed, as described in Eriksson et al 2002 (which focuses on
mucosal
adjuvants) and Guy et al 2005. As examples, Yuki et al 2003 and Lycke 2004
describe
adjuvants in which a cholera toxin protein fragment is combined with an E.
coli toxin
fragment or a Staphylococcus aureus toxin fragment.
[00012 ] Cholera toxin (CT) and heat-labile E. coli toxin (HT) have been
studied
extensively, as potential mucosal adjuvants, because of several natural
activities and
effects they have. Since both toxins are polypeptides, the hope is that
limited amino acid
sequences from either or both of the CT and HT toxins can be isolated, and
then
incorporated into mucosal vaccines, to give such vaccines an enhanced ability
to reach
submucosal tissues containing large numbers of macrophages or other immune
cells.
Accordingly, animal tests and human clinical trials were commenced during the
1990's,
using CT and HT as nasal adjuvants. However, the human trials had to be
terminated,
when animal tests indicated that vaccines carrying CT or HT sequences could
travel
through certain types of nerve fibers and enter the brain, causing brain
inflammation.
Those problems are described in a report from a July 2001 conference,
entitled, "Safety
Evaluation of Toxin Adjuvants Delivered Intranasally", issued by the U.S.
National
Institute of Allergy and Infectious Diseases (NIAID), available at
http://www.niaid.nih.gov/dmid/enteric/intranasal.htm. That report contains an
extensive
description of concerns and requirements that need to be addressed before any
human
clinical trials can begin again, using either CT or HT-derived toxins.
[00013 ] Rather than overcoming those problems, subsequent research identified
even more
problems that occurred when CT and HT toxin fragments when used as adjuvants
for nasal
vaccines. For example, Yoshino et al 2004 and van Ginkel et al 2005 reported
that in
animal tests, CT and HT toxin adjuvants created the following problems and
warning
signals: (i) they altered the normal patterns of antigen travel and immune
cell responses
(collectively referred to as "antigen trafficking"), which raises questions
about whether an
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immune response to a vaccine would properly prepare a host to withstand an
actual
pathogenic infection; and, (ii) they provoked inflammation in the nasal tract.
[00014 ] Accordingly, the Inventor herein took a completely different route,
and avoided
any work with known toxins or toxin fragments as candidate adjuvants.
[00015 ] Genetically-engineered adjuvants offer the promise of incorporating a
polypeptide
sequence having adjuvant activity, into a "disarmed" (nonpathogenic) viral
particle or
fragment (or into an engineered chimeric protein) that also carries an
antigenic polypeptide
sequence. Therefore, in modern vaccines, adjuvants can be agents (such as
known
polypeptide sequences that have adjuvant-like activity) that are incorporated
into the same
particle or protein that also carries an antigen (such as a polypeptide
sequence derived
from a surface protein of a pathogen).
[00016 ] Returning to the two compatible definitions of "adjuvant" listed
above, it recently
has become clear that the formulation of vaccines as "microparticles", having
sizes that
emulate various types of pathogenic microbes, can help trigger an effective
immune
response to a vaccine. That is not surprising, since macrophages and other
immune cells
evolved in ways that are ideally suited to combat such microbes. Therefore, by
giving
vaccine particles a certain physical size (measured in micrometers, or in some
cases
nanometers), the efficacy and speed of an immune response to an antigen can be
increased., when the antigen is carried by a vaccine having an optimized
particulate size.
Accordingly, if the sizes of vaccine particles have been optimized, then the
particles
themselves (with optimal dimensions) can be regarded as having adjuvant
activity and
efficacy.
[00017 ] Finally, it also should be noted that adjuvants can be divided into
two classes,
based on their mechanism of action. "Delivery" adjuvants increase. immune
responses by
means that increase the amount of contact between antigens, and "antigen-
presenting cells"
(APC's), using mechanisms such as described above. By contrast,
"immunostimulatory"
adjuvants activate the immune system by stimulating certain types of cells to
release
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cytokines (i.e., hormone-type messenger molecules that trigger various types
of cellular
responses), or by other similar actions as described in articles such as
Hunter (2002).
NASAL AND OTHER MUCOSAL VACCINES
[00018 ] One field that requires particular attention involves mucosal
vaccines. This
includes vaccines that are administered topically to a "mucous membrane", also
called an
"epithelial" membrane. Such membranes include the mouth, nasal sinuses,
vagina, rectum,
etc.
[00019 ] The type of skin that covers most of the body and the limbs is made
of epidermal
cells. These cells are created by a "budding" process, in which an underlying
layer of cells
continuously creates a set of "partial" offspring cells. These cells become
dehydrated and
flat ("squamous") as they move closer to the outer skin surface, and by the
time they reach
a subsurface layer called the "stratum corneum", they are "enucleated" (i.e,
they no longer
contain a nucleus or chromosomes). By the time they reach the outer epidermal
surface,
they are effectively dehydrated and nonviable, and cannot support viral
replication. This
makes them ideal as an outer protective layer. They typically last for only a
few days on
the surface, then they peel and flake off, in the form of dried single cells
or tiny clusters
that usually are too small to be seen by the naked eye.
[00020 ] In contrast to dry epidermal skin, mucous membranes are covered by
epithelial
cells. Instead of providing a protective coating of flattened and dehydrated
dead or dying
cells, epithelial cells remain hydrated, to sustain their viability. After
they reach an outer
surface of a mucous membrane, they have a relatively short lifespan (typically
about 4 to
about 8 days), and during that period, they are much more active than
epidermal cells, and
generally are capable of supporting viral infections, especially if any
lesions, abrasions, or
other breaches in a mucous membrane enable viruses to penetrate through the
outermost
layers.
[00021 ] Accordingly, there is a high level of interest in mucosal vaccines.
Among other
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advantages, mucosal vaccines can avoid any need for needles or syringes, which
pose
various problems (including hazardous waste disposal, theft by drug users,
accidents that
can infect healthcare workers, etc.), and which offend or intrude on the
cultural norms of
some societies. In addition, mucosal vaccines that are incorporated into
genetically
engineered plants offer the potential for helping reduce animal-related and
food-related
diseases.
[00022 ] Because of their potential, much effort and attention has been
devoted to mucosal
vaccines, as described in, e.g., Vajdy et al 2004, O'Hagan et al 2004, and
Holmgren et al
2005. Reviews that focus on animal usage include Meeusen et al 2004; reviews
that focus
on efforts to develop mucosal HIV/AIDS vaccines include Belyakov et al 2004
and
Stevceva et al 2004.
PHAGES AND PHAGE VACCINES
[00023 ] Since this invention involves a class of microbes called "phages", as
both active
agents and adjuvants, background information needs to be provided on phages,
and on
prior efforts to use phages as vaccines.
[00024 ] The term phage is a shortened form of bacteriophage, derived from the
Greek
words for "eats bacteria". Most phages will kill and destroy their bacterial
hosts. However,
a special class of viruses was discovered that can infect bacteria and then
emerge, in large
numbers, without killing the host cells. These specialized viruses are long
and thin
"filaments" which, after reproducing in a cell, are thin enough to emerge
through pores or
other outlets in a cell's membranes, without damaging the cell. Even though
these types of
filamentous viruses do not "eat" or kill their hosts, they were nevertheless
called
bacteriophages, or simply phages. Since they do not kill their hosts, they can
be grown in
bacterial cell culture at enormously high rates, so long as the bacteria are
cultured with
shaking or stirring, to prevent the phages from smothering the bacteria by
sheer volume
and bulk.
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[00025 ] Several types of phages have been manipulated in ways that render
them highly
useful for genetic engineering. The most popular phages, used in laboratories
around the
world, usually combine all of the following traits:
(i) the DNA they carry is small enough to be easily manipulated;
(ii) they allow small or moderate-sized foreign polypeptide sequences to be
inserted
into segments of a first "coat" protein that is present in more than a
thousand copies in
each phage particle, without disrupting the ability of the modified phages to
replicate in
bacteria;
(iii) they can allow even larger foreign polypeptide sequences to be inserted
into a
second type of protein that is positioned near one end of the phage filament;
(iv) their DNA has been modified in ways that allow the phages to function
both:
(1) as bacterial plasmids, in double-stranded DNA form, or (2) as phage DNA,
in single-
stranded DNA form. This provides various advantages; for example, it allows
antibiotic-resistance genes to be incorporated into the phages, in ways that
allow host
bacteria which have been infected by phages to be easily selected, from among
large
populations of bacteria, by culturing the bacteria in media containing the
corresponding
antibiotics;
(v) finally, these types of phages cannot infect mammals. They are totally
nonpathogenic in animals and humans, and they can be used without the
expensive and
cumbersome precautions that are required when pathogenic microbes are
involved.
[00026 ] Based on these and other factors, phage preparations have been
developed in two
different forms. Both forms must be understood, and kept distinct.
[00027 ] One form is a preparation containing numerous copies of single
specific phage
carrying a single foreign gene sequence (or "insert"). This type of
preparation, often called
a clonal or monoclonal phage, can be used for various purposes, such as for
making
vaccines. As an example, clonal phages containing a protein sequence from the
human
beta-amyloid protein (which forms beta-amyloid plaques, in the brains of
people suffering
from Alzheimer's disease) have been created by Beka Solomon, Dan Frenkel, and
their
coworkers at Tel Aviv University. These are described in a number of issued
patents
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(including US 6,919,075, by Solomon et al 2005, and US 6,703,015, by Solomon
et al
2004), published patent applications (including US 2005-0152878 and 2005-
0053575, both
by Solomon et al 2005), and articles (e.g., Frenkel et al 2002 and 2004). The
hope and goal
is that such phages, if used as vaccines, may be able to stimulate the
production of
antibodies or other immune responses that may be able to disassemble and
dissolve beta-
amyloid plaques, in the brains of Alzheimer patients. That is a noble goal,
and some day, it
may succeed. However, a clinical trial on a beta-amyloid vaccine (designated
as AN-1792,
by Elan Pharmaceuticals) had to be halted in January 2002, because patients
who received
the vaccine began to suffer from cerebral inflammation (an analysis is
available in Schenk
et al 2004). The cerebral inflammation may have been due to either or both of
two factors:
(1) an autoimmune disorder may have arisen, when the immune systems of those
patients
began attacking a protein that occurs naturally in the blood and brain; and/or
(2)
beta-amyloid plaques may function as a type of "plumber's putty", which
controls and
reduces the leakage of blood out of capillary walls that have become thin and
fragile, in the
brains of elderly people; accordingly, if the sticky deposits that control
that type of leakage
are dissolved, blood leakage into the brain tissue may increase, leading to
edema,
inflammation, and other problems.
[00028 ] In addition to clonal (or monoclonal) phage preparations that can be
used as
vaccines or research, a different type of phage preparation that needs
attention is called a
phage display library (also called a phage library). This type of preparation
is a mixture
containing millions or billions of phages that carry different foreign
polypeptide
sequences. These phage libraries enable "screening" tests that allow certain
specific phages
to be selected and isolated, using one or more cellular or physiological
processes. The
phages that respond in a certain way, in a screening test, can be isolated,
reproduced, and
analyzed, to determine the foreign DNA and/or polypeptide sequence that caused
those
particular phages to behave in a certain way, in that particular screening
test.
[00029 ] One important type of phage display library, which has been
extensively
developed by a company called Cambridge Antibody Technology
(www.cambridgeantibody.com), contains billions of differing human gene
sequences,
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obtained from human populations that included a wide variety of races and
ancestries. The
foreign gene and protein sequences, in the Cambridge Antibody phage display
library,
were derived from the "single-chain variable fraction" (scFv) portions of
human
antibodies. Because of how the foreign gene inserts were isolated, this phage
library is
highly useful in immunological research, and it was used in certain screening
tests
described below. This type of phage library is described in more detail in
articles such as
Pini et al 2000, and on the Cambridge Antibody Technology website.
[00030 ] Other known types of phage display libraries contain foreign DNA
sequences with
either random or controlled nucleotide sequences, which will be expressed into
random or
controlled variations in the coat proteins of phages.
[000311 By using cell culture tests to screen large numbers of phages from
either type of
phage library, the particular phages which happen to carry foreign DNA and
peptide
sequences that trigger certain types of cellular or physiological reactions
can be isolated.
Subsequently, after a "best performing phage" has been identified by an early
round of
screening, its foreign gene sequence can be used as the starting point for
additional rounds
of mutation and screening, using either random or controlled mutagenesis.
[00032 ] Due to various factors (including very fast replication, optimal
particle sizes, and
certain protein "display" traits, which includes the fact that inserted
polypeptide sequences
will be exposed and accessible on the outsides of the phage particles, rather
than hidden
inside the phages), various authors and researchers have recognized and
reported that
bacteriophages may someday be useful as vaccines. Articles that propose such
use include
Miedzybrodzki et al 2005 ("using natural agents such as bacteriophages as a
weapon
against pathogenic viruses could be an attractive and cost-efficient
alternative, and further
studies are urgently needed to test this possibility"), Koch et al 2004
(subtitled, "alternative
and experimental approaches"), and Clark et al 2004 ("work has shown that
whole phage
particles can be used to deliver vaccines in the form of immunogenic peptides
attached to
modified phage coat proteins or as delivery vehicles for DNA vaccines, by
incorporating a
eukaryotic promoter-driven vaccine gene within their genome. While both
approaches are
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promising by themselves, in the future there is also the exciting possibility
of creating a
hybrid phage combining both components to create phage that are cheap, easy
and rapid to
produce and that deliver both protein and DNA vaccines via the oral route in
the same
construct").
[00033 1 However, despite their apparent potential, there has been very little
commercial or
industrial interest in using phages as actual vaccines, in human medicine. The
reasons for
the apparent lack of commercial and industrial interest are numerous, and
include: (i) the
unwillingness of large pharmaceutical companies (which presumably are the only
companies that could afford a major research project that might be able to
create a
completely new and different class of vaccines) to risk hundreds of millions
or even
billions of dollars, on a project that may never succeed; (ii) the reluctance
of large and well
funded companies to even try to begin all of the necessary testing that
presumably would
be needed, to prove very high levels of safety, for vaccines intended for
human use; (iii)
the presence of enough speculative and suggestive proposals concerning
possible phage
vaccines, in the research literature of the 1980's and 1990's, to clutter and
confuse an
assessment of the prior art, making it difficult to reliably predict whether
any strong patent
protection could be gained if a company did invest large amounts of money and
managed
to succeed, by means of an approach that arguably used various suggested
methods and
components; (iv) the reluctance of large pharmaceutical companies to try to
develop new
and different products that might end up cannibalizing the sales and profits
they already
enjoy from their existing vaccines or pharmaceuticals; and, (v) the additional
investment
and profit risks that would arise if a highly effective vaccine can be
manufactured rapidly,
in huge quantities and at very low cost, in numerous countries where patent
rights or other
intellectual property protections cannot be enforced in a practical and
economic manner.
[000341 Despite those problems, researchers in a position to do so have an
obligation to try
their best, to develop better vaccines. Despite various improvements, most
vaccine
production today uses technology that is decades old, and the old technology
has failed,
quite seriously, to accomplish several hugely important goals. As hugely
important
examples, no adequate vaccines have been created for AIDS, malaria,
tuberculosis, or
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several other major diseases. As another example, there appears to be a
general consensus
among scientists and governments that a recently-emergent form of "bird flu"
will
someday mutate into a form that will be transmitted from humans to humans, and
when
that happens, the stage will be set for a worldwide pandemic that likely will
kill many
millions of people. However, a strong presumption arises that old
manufacturing
technologies, which mainly use bird eggs as incubators, is not likely to work
well, for
making vaccines against a virus that aggressively kills birds.
[00035 ] Accordingly, attention needs to turn to a different set of recent
advances and
discoveries, from a completely different line of medical research, which may
allow certain
methods from a different realm of art to be imported into the field of vaccine
development,
manufacturing, and use. That other field of research is briefly summarized
below.
DELIVERING POLYPEPTIDES THROUGH THE BLOOD-BRAIN BARRIER,
USING LIGANDS THAT ENABLE NEURONAL ENDOCYTOSIS AND
TRANSPORT
[00036 ] The inventor herein, Dr. Ian Ferguson, is the first-named inventor of
a separate
patent application, published in April 2003 as Patent Cooperation Treaty (PCT)
application
WO 2003/091387. Its title is, "Non-Invasive Delivery of Polypeptides Through
the Blood-
Brain Barrier, and In Vivo Selection of Endocytotic Ligands". The contents of
that
application are incorporated by reference, as though fully set forth herein.
[00037 ] That invention can be regarded as comprising two main components,
which
interact with each other to provide a complete operative embodiment. The first
portion can
be briefly summarized as follows:
1. Using specialized gene vectors (such as certain types of disarmed viruses
that
carry inserted "passenger" genes), it is possible to transfect certain types
of neurons that
"straddle" the blood-brain barrier (BBB). Such BBB-straddling neurons include,
for
example, olfactory receptor neurons (which have tips that are accessible in
the nasal
sinuses), and various neurons with tips that can be reached by liquids
injected into the
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tongue, or into various muscles.
2. If the gene vectors have certain types of properly-selected proteins on
their
surfaces (proper selection involves the second part of the invention,
described below), they
will be taken into the accessible tips of the BBB-straddling neurons. This
type of transport,
into the tips of neurons, uses "endocytotic" receptors on the exposed and
accessible tips of
the neurons. Endocytosis is a well-known process, and a number of endocytotic
receptors
that appear on the surfaces of certain types of BBB-straddling neurons have
been identified
and fully sequenced.
3. After such genetic vectors have been taken into the accessible tips of the
neuronal fibers, they can be transported from the neuronal tip, to the main
cell body, by a
natural process called "retrograde transport".
4. The trick to activating and driving both: (i) entry into the neuronal tips,
using
endocytotic receptors, and (ii) internal transport to reach the main cell
bodies of the
neurons, is to find and use an effective "transport" protein (which can also
be called a
delivery, carrier, or locomotive protein, or similar terms) that will activate
and drive two
different processes, which are endocytotic uptake into the neuron, and
retrograde transport
within the neuron. A method for screening and identifying such proteins, using
a phage
display library that provided billions of candidate protein sequences, and
that used a
screening test to select, identify, and isolate a few such proteins which
actually function in
the desired manner, is described below, in the discussion of the second major
component
of this invention.
5. After a genetic vector has entered a neuronal fiber and has been
transported to
the main cell body, the gene carried by the vector can be expressed into
proteins, and the
proteins can be provided with a "leader sequence", which can enable two
functions (i)
transport of the protein molecules by neuronal fibers that travel deeper into
the brain; and.
(ii) secretion and release of the proteins, at the "innermost" synapses of the
neuronal fibers.
[00038 ] This approach, using specialized genetic vectors described in more
detail in PCT
patent application WO 2003/091387, provides an effective non-invasive method
for
delivering diagnostic or therapeutic proteins into BBB-protected brain or
spinal tissue.
Although confirmatory data were not available when that patent application was
filed, it
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has been confirmed that this method of delivery functions effectively in
animals, and
efforts are being made to move it closer to human clinical trials.
[00039 ] That comprises the first major component of the invention. As
mentioned above,
in order for it to work, the "transport" or "locomotive" proteins that are
exposed on the
surfaces of the BBB-penetrating genetic vectors must be able to activate and
drive two
different cellular processes, which are: (1) uptake into neuronal fibers,
followed by (2)
transport through the neuronal fibers, toward the main cell bodies of the
neurons. To
identify "transport protein" sequences that can accomplish both of those two
objectives,
the Inventor developed an in vivo screening process, which initially used the
sciatic nerve
bundle, and which later was extended to olfactory neurons.
[00040 ] In mammals, the sciatic nerve is the main nerve bundle that travels
from the base
of the spinal cord, through the hip and the leg, to the foot. To carry out an
in vivo screening
procedure which uses that long nerve bundle in rats, a "ligature" was created,
by surgically
placing and then tightening a loop made from a suture strand, around the
sciatic nerve
bundle near the knee of a rat. This created a form of stress which caused the
affected
neurons to "upregulate" (i.e., increase the expression and placement of)
certain types of
cell surface receptors, including the so-called "p75" receptor, which attempts
to help nerve
cells recover from injuries.
[00041 ] One week later, that ligature near the knee of the rat was removed,
and the sciatic
nerve bundle was completely severed at that location. The exposed end of the
nerve bundle
was packed inside a sleeve that held a gel material containing a phage display
library, with
millions of candidate phage particles carrying different foreign protein
sequences in their
coat proteins.
[00042 ] During that same operation, a different ligature loop was placed and
tightened
around the same sciatic nerve bundle in an "upstream" location, near the hip.
That ligature
was tightened, to block the retrograde travel of molecules that otherwise
would naturally
flow through the nerve fibers, toward the spinal cord.
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[00043 ] A suitable period of time (about 18 hours) was allowed to pass, to
enable phages
that happened to be carrying efficient "locomotive" protein sequences (in the
coat proteins
of the phages) to be both: (i) taken inside the neurons, and (ii) transported
in a retrograde
direction, through the thigh, toward the spinal cord. After that period of
time passed, the
animal was sacrificed, and a short segment of the sciatic nerve bundle,
immediately below
the ligature loop near the hip, was harvested.
[00044 ] Since the phage harvesting site (near the hip) was more than a
centimeter away
from the placement site (near the knee), the only phages that could reach the
harvesting
site were phages that had been taken inside a neuronal cell fiber, and that
had been
transported toward the spinal cord. Since those phages, traveling inside the
nerve bundle,
could not cross the constriction created by the tightened loop of suture
thread near the hip,
the fluid that was attempting to flow through the nerve bundle, toward the
spinal cord,
caused the phages to cluster and crowd into the nerve region immediately
"distal" to the
ligature (i.e., on the "downstream" side of the ligature, distant from the
spinal cord). This
allowed the phages of interest to be harvested, by isolating and removing a
short segment
of the sciatic nerve bundle, immediately downstream from the ligature at the
hip. The
phages were removed from the neuronal fiber segments, by using a buffer and
enzyme
mixture to digest the cell membranes (which are made of lipids) but not the
virus particles
(which are covered by proteins). The phages were then replicated in bacterial
cells, to
provide an "amplified" population.
[00045 ] The resulting mixture of phages from one round of screening was then
screened
again, using the same process. The screening procedure can be repeated any
number of
times, using an "enriched" phage population from a prior screening cycle as
the starting
material for a subsequent screening cycle.
[00046 ] Out of the many millions of candidate phages that were present in a
phage library
that was screened in that manner, a few phages were selected and isolated. The
selection
process ensured that those particular phages contained foreign polypeptide
sequences that
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activated and drove both: (i) endocytosis (i.e., uptake and entry) of the
phages into the
neuronal fibers; and, (ii) retrograde transport inside the neuronal fibers
(i.e., transport from
a neuronal fiber tip, toward the main cell body of the neuron).
[00047 J The above-cited PCT application WO 2003/091387 stated that similar
techniques
could be used to screen phage display libraries, to select phages that: (1)
would enter the
tips of olfactory receptor neurons, which are accessible on the surfaces of
the nasal sinuses
(i.e., the neuronal tips are not protected by the blood-brain barrier); and,
(2) would be
transported through the neuronal fibers, into brain tissue that is protected
by the BBB.
[00048 ] Those comments were supplemented and confirmed by additional work
that was
disclosed in PCT application PCT/IB2005/04077, published as WO 2006/070290, by
the
same inventor herein. Briefly, it was realized that the rates of uptake of
phages into the tips
of olfactory receptor neurons, and the transport of the phages into brain
tissue by the
neuronal fibers, could be used to diagnose, measure, and quantify the health
and vitality of
neurons and brain tissues in people suffering from neurodegenerative diseases,
such as
Alzheimer's disease. This arises from the fact that endocytotic uptake and
axonal transport
are important processes in olfactory or other adjacent neurons, and if a
certain set of
neurons is losing or has lost the ability to carry out those processes, those
losses can reveal
the health and condition of those neurons, and of the neuronal networks they
participate in.
[00049 1 To demonstrate that approach in rats or mice, fluorescently-labeled
phages were
used. Fluorescent labels can be identified and tracked visually, when thin
slices of tissue
from a sacrificed animal are analyzed under ultraviolet or similar light.
However, in human
use, tissue sections will not be available, so other types of labels must be
used, such as (for
example) short-lived isotopes that will show up in non-invasive imaging
methods, such as
single positron emission computerized tomography (SPECT) scans, computerized
axial
tomography (CAT) scans, magnetic resonance imaging, etc.
[00050 ] The results of that research made it clear that a number of phages
had indeed been
selected and taken in by neuronal fiber tips in the nasal sinuses, and had
been transported
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through the nasal sinuses, toward and into the brain.
[000511 During the course of that work, which involved olfactory receptor
neurons, it was
realized by the Inventor herein that an entirely different type of use might
become
available, which could lead to the development of vaccines rather than
diagnosis and
treatment of brain disorders, if certain changes were made in the approach.
[00052 ] Understanding those changes, and how nasally-administered vaccines as
described
herein can be made to work, requires an entirely different set of background
art to be
reviewed and analyzed. Therefore, attention must now turn to a specialized
type of tissue
(called NALT tissue) that belongs to the immune system, and to differences
between the
"innate" immune system (which does not generate antibodies), and the
"adaptive" immune
system (which generates antibodies).
NALT CELLS AND TISSUES
[00053 ] The acronym "NALT" refers to "nasopharyngeal-associated lymphoid
tissue".
The terms "nasopharyngeal" and "lymphoid" both need to be understood.
[00054 ] "Naso-" refers to the nose, including the nasal sinuses. "Pharyngeal"
refers to the
pharynx, which is a transitional region in the back of the throat, below or
behind the
mouth, but above the esophagus. Therefore, "nano-pharyngeal tissue" includes
tissues
located in the nasal sinuses, in the rear portion of the mouth, and/or in the
upper throat
region.
[00055 ] "Lymph" refers to watery fluids and cells that permeate out of blood
vessels and
then travel through the soft tissues. Instead of containing cells packed
closely or tightly
together, soft tissues contain a substantial fraction of extracellular water
(typically about
1/6, by volume), in a "gel" matrix held together by proteins. The
extracellular fluid in
tissue gel allows nutrition to reach cells that are not directly adjacent to a
blood vessel, and
it also allows certain types of immune cells to permeate and travel through
soft tissue, as
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described in more detail below. Those immune cells slowly travel to lymph
nodes, which
are highly important in the immune system. Accordingly, the term "lymphoid"
has two
distinct but heavily connected and interrelated meanings. One meaning refers
to the watery
fluids that slowly pass and permeate through soft tissues; the other meaning
refers to
specialized immune cells and tissues that use, handle, or travel in lymph
fluids.
[00056 1 By combining "nasopharyngeal" with "lymphoid", the phrase
"nasopharyngeal-
associated lymphoid tissue" (NALT) refers to and includes several specialized
sets of
tissues in the nose, mouth, and throat region, which form important components
of a
mammalian immune system. Because the last word is "tissue", the acronym can be
used as
a noun; however, it is also commonly used as an adjective, so "NALT cells" is
correct
usage, and "NALT tissue", although not truly proper, should be politely
tolerated. "NALT"
is sometimes referred to as nose or nasal tissue, but that definition might
exclude the
tonsils or adenoids, which are NALT cells, so it is not used herein.
[000571 Two similar acronyms that are often used are GALT (gut-associated
lymphoid
tissue, which includes specialized intestinal tissues called "Peyer's
patches") and MALT
(mucosa-associated lymphoid tissue, which includes both GALT and NALT cells).
Both
NALT and GALT tissue structures involve a layer of epithelial membrane cells
(often
referred to as M cells; the "M" initially referred to "membrane", since they
have
specialized structures that appear only on the outermost surfaces of the
mucosal layers, but
M cells are sometimes also referred to as mucosal cells and/or as microvilli
or microfold
cells). A layer of M cells rests on top of an underlying cluster or nodule of
tissue called a
"subepithelial lymphoid follicle". The M cells contain finger-like and/or
heavily folded
protrusions, often called microvilli or microfolds, which increase their
surface areas and
their ability to contact and interact with molecules that are being inhaled,
or that are
passing through the digestive tract. They began to receive serious attention
in the 1980's,
and over a hundred review articles describe their structures, activities, and
roles in the
immune system, as well as various efforts to exploit and use them for vaccines
or other
research or medical purposes. Such reviews include Kuper et al 1992, Ermak et
al 1998,
Kozlowski et al 2002, Man et al 2004, Jepson et al 2004, Holmgren et al 2005,
Brayden et
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al 2005, and des Rieux et al 2006.
[00058 ] As discussed herein, M cells are regarded as a component of NALT or
GALT
tissues, and as NALT or GALT cells. Because of certain embryological factors,
which
focus on the origins of cells rather than their functions, some authors may
regard or refer to
M cells as being a cooperating but distinct layer, which is not a part of the
actual immune
system. Such distinctions are merely semantic, so long as a reader understands
that M cells
provide a surface layer of cells that have active and crucially important
sampling and
transport roles, resting on top of an underlying layer, cluster, or follicle
of immunoactive
cells.
[00059 ] NALT cells and tissues are highly important to the immune and
allergic systems,
since many pathogens, allergens, and other compounds are inhaled, and their
first contact
in a mammalian body occurs in the nasal cavities. Therefore, mammals evolved
with
certain types of specialized cells that are exposed on the surfaces of the
mucous
membranes in the nasal sinuses and mouths, which are active components of the
immune
systems. When these cells encounter a protein and recognize it as foreign,
they effectively
"grab" the protein and help deliver it to a lymph node, so that other cells of
the immune
system can process it, and can create antibodies that will help the body
defend against the
foreign antigen, if appropriate.
[00060 ] In accordance with the present invention, it is proposed that NALT
tissues offers a
potential for transport activities triggered by foreign proteins using immune
cells that can
travel in the fluid drainage system provided by lymph. That is a very
different mechanism,
compared to neurons having long fibers that will transport nerve growth factor
or other
therapeutic proteins into brain tissues, by means of endocytosis followed by
retrograde
flow within a neuronal axon.
[000611 Accordingly, to adequately explain this offshoot, which moved away
from
treating neurodegenerative diseases and which focused on immune systems and
vaccine
development, some background information needs to be provided on certain
components
and processes of the immune system. The following three subsections, all
within the
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Background section, are an effort to provide that information. These are
intended as
introductory summaries only, and more detailed information is available in
various
reference works, including various books and review articles cited above.
INNATE VS. ADAPTIVE IMMUNE RESPONSES
[00062 ] A complete mammalian immune system actually comprises two different
subsystems, which are called the innate immune system, and the adaptive immune
system.
The innate system is "hard-wired" and works very rapidly, while the adaptive
system takes
much more time to generate a response. This is analogous to the way a nervous
system
enables both: (1) hard-wired and very rapid "reflex" responses, such as
withdrawing a
finger immediately, when a hot surface is touched; and, (2) learning, which
takes longer,
but which can accomplish much more complex and sophisticated tasks.
[00063 ] The adaptive system requires and uses antibodies, which require
participation by
several different types of white blood cells, including B cells, T cells,
helper T cells, killer
cells, etc. This response takes several days to complete, and any delay
lasting that long
would allow most microbes (which can reproduce many times faster than
mammalian
cells) to generate huge numbers of invading microbes, before a complete
antibody
response can move into action. That delay period explains why vaccines, if
prepared and
administered in ways that allow an animal's immune system to partially get
ready, in
advance, before an infection even begins, can make a huge difference in how
severe a
disease or infection will become.
[00064 ] That delay period (i.e., the fact that several days will pass, before
an animal's
adaptive immune system can respond fully) also explains why most multi-
cellular
organisms evolved with an additional set of specialized proteins and cell
types that form an
"innate" immune system. This "innate" system can respond almost immediately,
to help
the body fight and slow down a set of invading microbes, while reinforcements
(which are
analogous to heavy artillery, including antibodies, B cells, T cells, killer
cells, etc.) are
being prepared, produced, and moved into position. Accordingly, the innate
immune
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system responds and acts rapidly, as a first line of defense, without having
to wait for days;
then, while the innate immune system is slowing down the invaders, an adaptive
(antibody)
response is planned, organized, and made ready.
[00065 ] The innate immune system also is regarded and sometimes referred to
as a
"primitive" immune system. Since most invertebrate animals do not have full
immune
systems with antibodies, their only line of defense against microbes is their
innate immune
system.
[00066 ]As life on earth evolved into larger and more complex forms, innate
(primitive)
immune systems were already up and running, by the time adaptive immune
systems
began to evolve. Therefore, adaptive immune systems evolved in ways that use
the innate
immune system as a "springboard". This was accomplished, at least in part,
through the
use and evolution of "pattern recognition receptors" (PRRs), which evolved in
vertebrate
animals as a response to "pathogen-associated molecular patterns" (PAMPs) in
pathogenic
microbes.
[00067 ] Most of the important groups of pathogenic microbes share certain
types of
"highly conserved" amino acid sequences, in certain domains of certain
proteins. These
highly-conserved domains of important proteins are driven by basic biological
and
biochemical needs and constraints. This prevents microbes from being able to
rapidly
mutate away from the patterns that work well for them, since deviations away
from crucial
and well-designed systems are much more likely to be detrimental, than
favorable.
Therefore, PAMPs are embodied by general patterns, rather than specific
structures.
[00068 ] For example, animal cells do not have hardened cell walls; instead,
they are
enclosed within flexible outer membranes. By contrast, many types of microbes
have cell
walls, which allow those microbes to withstand fluctuations in their
surroundings that
would kill animal cells. Therefore, a number of molecules that are used to
build microbial
cell walls (such as certain types of lipoproteins, lipopolysaccharides, and
peptidoglycans)
came to be recognized as pathogen-associated molecular patterns (PAMPs) by the
innate
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immune systems of small invertebrate animals (and, eventually, by vertebrate
animals as
well).
[00069 ] As a second example, animal cells do not have or use certain types of
protein
complexes that are highly conserved in "flagella", which are the moving whip-
like strands
that E. coli and various other types of bacteria use to move about, in a
liquid. Therefore,
flagellar proteins from bacteria became another pathogen-associated molecular
pattern.
[00070 ] As a third example, the genes of vertebrates evolved in ways that
have two
general traits: (i) methyl groups are gradually bonded to cytosine
nucleotides, as an animal
ages; and, (ii) there tend to be relatively few cytosine and guanidine
nucleotides positioned
immediately next to each other, in a DNA strand of a higher animal. Therefore,
strands of
DNA having unusually high numbers of unmethylated cytosine and guanidine
nucleotides,
adjacent to each other, in the body of an animal larger than a rodent,
indicates that a
microbial invasion probably is occurring. Therefore, DNA strands having large
numbers of
unmethylated cytosine-guanine dinucleotide pairs (this pattern is referred to
as a "CpG
motif') can activate an important class of "toll receptors", discussed below.
[00071 ] The patterns listed above are just a few examples of pathogen-
associated
molecular patterns (PAMPs). These pathogenic microbial patterns led to the
evolution of
corresponding "pattern recognition receptors" (PRRs), initially in
invertebrate animals, as
part of their innate immune systems. These "receptors" are not limited to the
standard
types of cell-surface receptors found in mammals; instead, they also include
other types of
molecules that can effectively latch onto (or otherwise respond to) one or
more pathogen-
associated molecular patterns that are present in various important classes of
pathogenic
microbes. This leads to a discussion of "toll receptors".
TOLL RECEPTORS
[00072 ] An important subclass of "pattern recognition receptors" in animals
is called "toll
receptors". These were first seen in Drosophila (i.e., small fruit flies that
are widely used
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in genetic research). They were called "toll" receptors not because of any
particular
function, but because "toll" is a German word for "amazing". Since their
definitions and
boundaries are not always clear, especially as the DNA and amino acid
sequences for toll
receptors in mice are used to search for "homologous" sequences in humans,
they are often
referred to as "toll-like receptors", using the acronym "TLR" followed by a
number, such
as TLR4 or TLR9.
[00073 ] At least 11 types of toll-like receptors have been identified in
humans; in addition,
two other types are believed to exist in mice, and there is heavy overlap
between human
and mice TLR's. Each toll-like receptor type is associated with at least one
type of
microbial "ligand" that will activate that subclass of TLRs. The known TLR
types, and the
ligands which activate them, are listed and described in literature that can
be downloaded
at no charge from Imgenex (www.imgenex.com), a company that sells ligands,
antibodies,
and other compounds used in research on toll receptors. Illustrated summary
pages are
available at http://imgenex.com/view-data_page.php?id=27 and
http://imgenex.com/Toll-likeReceptors.php, and a 34-page brochure entitled
"Toll-like
Receptor Research Tools" can be downloaded via
http://imgenex.com/dfiles/DownloadN-32.pdf. Review articles that describe toll
receptors
and their ligands include, for example, Hemmi et al 2005, Alexopoulou et al
2005, and
Pasare et al 2005.
[00074 ] Not all TLRs function in the same way, and they can be grouped into
two main
classes, in terms of location. In one class of toll receptors, which includes
TLR1, TLR2,,
TLR4, TLR5, TLR6, and TLR10, the receptors straddle the outer membrane of an
animal
cell, with an extra-cellular portion, and an intra-cellular portion. If the
extra-cellular
portion binds to a microbial pathogen (or to an artificially-administered PAMP
ligand),
changes are triggered in the intra-cellular domain.
[00075 ] In the other class of toll receptors, which includes TLR3, TLR7,
TLR8, and
TLR9, the receptors are associated with membrane-enclosed organelles (also
called
vesicles) located inside a cell. TLR9 receptors are of particular interest
herein, since they
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are located inside macrophage cells (described below), and since they have
been studied
extensively. TLR9 ligands are fairly well understood, and are described in
articles such as
Krieg 2002 and Klinman et al 2004.
[00076 ] Regardless of which subclass of toll receptor is involved, activation
of a toll
receptor will trigger a series of reactions that will lead to movement
(translocation) of
certain types of DNA transcription factors. These initial responses lead to
genes being
expressed that will generate and/or trigger the activation or release of
certain cytokines,
which will then trigger a cellular or multicellular reaction, such as an
inflammatory
10- response, or cellular release of various antimicrobial agents.
[00077 ] In summary, toll receptors recently have been recognized as important
"gate-
keepers", with functions that can be regarded as comparable to sensors. They
can play
major roles in determining whether: (i) a complete antibed immune response
will be
launched, in response to an apparent invasion by a pathogenic microbe; or,
(ii) only a
localized and/or allergic or tolerance reaction will be commenced.
[00078 ]However, because of their complexity, and in view of the number of
different toll
receptors that are known to exist, they cannot be triggered randomly or
indiscriminately,
without posing major risks of provoking autoimmune or other potentially
serious disorders.
Therefore, what is needed (and what is provided by the invention herein) is a
process, and
a set of triggering or activating reagents, which can selectively activate
only certain
targeted types of toll receptors. Those processes and reagents are described
below.
[00079 ] It should also be noted that various adjuvants used in the prior art
(as
accompanying agents that can help trigger a full-blown immune response, when
coinjected
along with a particular antigen) are actually compounds that trigger and
activate toll
receptors. As examples, proteins from bacterial flagella, and polypeptide
segments derived
from a heat-labile endotoxin protein from E. coli, have been used for decades
as adjuvants,
in vaccine formulations. After the role of toll receptors was recognized, it
became clear
that those adjuvants were activating certain classes of toll receptors, in
ways that helped
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activate full-scale immune responses.
[00080 ] As described in more detail in the Detailed Description section
below, one of the
most important aspects of this invention is that phage vectors as disclosed
herein can be
modified so that they will specifically target certain types of toll receptors
that are located
entirely inside immune cells, such as TLR9 receptors. This can be very
advantageous,
since it reduces and minimizes the risks of triggering system-wide shock, a
"cytokine
storm", or other undesired responses that would be more likely to occur if
toll receptors on
the external surfaces of macrophages were being targeted. By using a screening
process
that will select for "locomotive" proteins that can carry out a complex two-
step process
(i.e., entry into a macrophage cell, followed by activation of TLR9 or other
toll receptors
located inside those cells), a higher level of safe and selective targeting
can be provided.
This is discussed in more detail below.
TYPES OF IMMUNE RESPONSES AND CELLS; MONOCYTES AND
MACROPHAGES
[000811 Different types of immune reactions can be created in response to
various types of
triggering agents. As one example, a vaccine might generate either an
undesired allergic
reaction, or a desired antibody-forming response. As another example, while
the vast
majority of vaccines (including essentially all vaccines used in preventive
inoculations) are
designed to provoke antibody formation, some vaccines used to treat specific
patients
suffering from cancer or other diseases are designed to provoke "cell-mediated
immune
responses", which involved specialized cytokines (signaling molecules) and
activated T
cells, without involving B cells or creating antibodies.
[00082 ] Therefore, an important goal in vaccine development is to identify
and use
vaccine components and formulations that will maximize the likelihood of a
desired
response (such as antibody formation), while minimizing the risk of unwanted
responses
(such as allergic reactions).
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[000831 Some of the cell types and components that determine which pathway an
immune
system will take, in response to foreign substances such as microbes, pollen,
or vaccines,
include:
(1) there are two different classes of "major histocompatibility complex"
(MHC)
receptors; these are known as MHC-1 receptors (present on nearly all cell
types) and
MHC-2 receptors (present only on certain types of immune cells);
(2) there also are two different classes of T cells, known as "cytotoxic T
cells"
(which will directly kill animal cells that harbor pathogenic microbes), and
"helper T cells"
(which secrete messenger molecules such as lymphokines, interleukins, or
cytokines,
which activate other cells in the immune system);
(3) helper T cells are further subdivided into two important classes, which
are
called TH 1 cells (which secrete interleukin-2 and gamma-interferon), and TH2
cells
(which secrete interleukin-4 and interleukin-5);
(4) there are five different classes of immunoglobulin molecules, which are:
(1)
IgM globulins, which are the initial forms of globulins sometimes bound to the
cell
membranes; (2) IgG molecules, which are the classic Y-shaped antibodies that
are secreted
by B cells to fight invading microbes; (3) IgA globulins, which are secreted
by mucous
membranes, and which latch onto airborne or other arriving pathogens in an
effort to
inactivate them and deliver them to the stomach or secrete them from the body;
(4) IgD
globulins, which are membrane-bound antibodies produced by B cells early in
their life
cycle; and, (5) IgE globulins, which are involved in allergic reactions.
[000841 In general, MHC1 and Th1 components work together to promote "cell-
mediated"
responses (also called cytotoxic responses), which involve cytokines and other
signaling
molecules and helper T cells, without requiring antibodies. By contrast, MHC2
and Th2
components work together to promote antibody formation. Those two types of
responses
are not mutually exclusive, and they can reinforce and supplement each other,
to produce
exceptionally strong and powerful immune responses. Accordingly, if a single
vaccine
preparation can provoke both type of responses, it may be able to provide
better protection
against various types of diseases. However, issues of timing may also be
important, in such
matters. The establishment of an "anamnestic" (non-forgetting) antibody-
producing
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capability (which can be held quietly in reserve until needed, and then
activated quickly) is
highly beneficial, even if it occurs years before an infection. However, it is
not always
desirable to increase cytokine activities or helper T-cell functions, unless
and until an
actual infection is occurring. Such matters, which are understood by those
skilled in the art,
need to be taken into account in designing and administering different types
of vaccines to
healthy people, or to patients who are already sick.
[00085 ] Another possible option, for an immune system that detects a foreign
antigenic
substance, is to do nothing of significance, and not mount a substantial
response. This type
of nonaction, usually referred to "tolerance", plays crucial roles in avoiding
autoimmune
diseases, allergic overreactions, and other problems. It is governed by
complex regulation,
rather than inattention. Holmgren et al 2005 offers a good overview of
tolerance and how it
may be manipulated in controlled ways to help treat autoimmune diseases,
allergies, etc.
Nevertheless, tolerance is an adverse result, when referring to vaccines,
since it means that
a vaccine failed to create a desired result.
[00086 ] Because the large majority of vaccines are designed to trigger
antibody responses
(rather than cell mediated MHl/Thl responses involving cytokines and T cells),
the
discussion below focuses on antibody formation as a desired response, and on
allergic
reactions as an undesired response.
[00087 1 The fact that some vaccines can provoke either an antibody response
or an allergic
reaction arises largely from the fact that immune cells called "macrophages"
must commit
to triggering either a desired systemic response, or an undesired localized
response, soon
after the macrophages encounter and bind to what appears to be a foreign
invader. As a
brief overview of how this happens, white blood cells called "monocytes"
(which were
given that name because they have a single clear nucleus, unlike similar cells
called
"neutrophils" that have their chromosomes divided into several clusters)
circulate in the
blood, inside blood vessels. While in circulating blood, monocytes are
relatively dense and
compact. They have surface molecules that grip the interior walls of
capillaries and then
permeate through the capillary walls, causing monocytes to leave the
circulating blood and
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enter the tissue itself. After they leave a blood vessel, the monocytes swell
to a larger size.
When that occurs, the enlarged cells become macrophages; as described below,
they are
also called phagocytes or phagocytic cells, because of how they surround and
engulf small
particles.
[000881 Macrophages travel slowly through soft tissues, within the lymph
fluid. As
mentioned above, soft tissues contain extracellular watery fluid (typically
about 1/6 by
volume) in a "gel" form that is held together by thick bundles of collagen,
and by protein
filaments coated with sugar groups, called proteoglycans. The slow travel of
macrophages,
through lymph fluid, is analogous to a policeman walking a beat rather than
riding in a car;
instead of being in a hurry to get somewhere, the policeman is mostly just
looking around,
to see if anything is present that should not be there.
[00089 1 Neutrophil cells should also be mentioned briefly. They act in a
manner similar to
monocytes, but important differences exist. Neutrophils normally remain in
circulating
blood until they receive a signal (such as from a cytokine, lymphokine, or
other signaling
molecule) indicating that an infection is occurring in a certain location. In
response to such
signals, neutrophils undergo a transformation, pass through a capillary wall,
and enter the
lymph fluid in the infected area of tissue. The neutrophils then swell to a
larger size, and
begin attacking the foreign particles. In some cases, they can engage in the
classical form
of phagocytosis (as described below), but more commonly, a neutrophil will
trigger an
"oxidative burst" when it senses that it has reached and contacted an invading
pathogen.
This "oxidative burst" creates and releases large number of "free radicals",
which are
molecules having unstable and aggressively reactive oxygen atoms with unpaired
electrons. Those radicals will attack and help destroy invading microbes, and
they often
kill the neutrophil as well. As a result, the whitish material called pus, in
infected tissues,
often consists largely of neutrophil cell remains.
[00090 1 That defensive process, by neutrophils, can be referred to as
phagocytosis, but not
all authors include or refer to it as actual phagocytosis, which focuses upon
engulfing a
foreign particle and taking it inside a defending cell. By way of analogy, if
a man is using a
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knife and fork to cut apart a piece of meat, some people would refer to that
preparatory
action as eating, while others would say that eating does not actually begin
until the person
puts the food into his mouth. Those types of semantic distinctions are not
important, so
long as one understands the overall process.
[000911 When a true macrophage (rather than a neutrophil) encounters and
recognizes a
foreign cell or other particle (regardless of whether it is a virus, bacteria,
pollen, etc.), it
engulfs the foreign cell. This process is phagocytosis; as mentioned above
under the
definition of bacteriophages, "phago" is derived from the Greek word for
"eat". In some
cases, a macrophage can engulf and swallow dozens of bacteria or viruses.
Since viruses
are particles rather than cells, the word "phagocytosis" has come to refer to
cellular
ingestion of any type of relatively large particle (including, for example,
particles made of
starch or plastic, which can be fluorescently labeled in ways that enable the
rates of
phagocytosis to be easily measured, using automated machines such as flow
cytometers).
Phagocytosis is distinct from a similar process called "pinocytosis", which
refers to the
ingestion of very small particles and/or liquids. In general, phagocytosis is
regarded as the
cellular equivalent of eating, while pinocytosis is the cellular equivalent of
drinking.
[00092 ] The process of phagocytosis is aided by several factors. For example,
most living
mammalian cells (including macrophages) have negative electrical charges on
their
surfaces. Therefore, the presence of negative charges on cells helps prevent
macrophages
from attacking living cells of the host. However, that electrical charge
dissipates after a
cell dies, enabling macrophages to "eat" dead cells so that their building
blocks can be
reused. This aids the gradual turnover and replacement of cells, in ways that
keep soft
tissues (muscles, organs, etc.) flexible and viable.
[00093 ] Importantly, many pathogens have positive charges on their surfaces.
This helps
them rapidly grasp and infect a mammalian cell, and that type of rapid action
is hugely
important for pathogenic microbes. However, the same positive surface charges
that help
pathogenic microbes attach to and infect mammalian cells, also helps
macrophages
recognize and destroy the foreign invaders.
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[00094 ] It should also be noted that in addition to macrophages and
neutrophils, certain
other cell types also can perform phagocytosis, including Schwann cells,
certain types of
glial cells, and various classes of dendritic cells. Indeed, in the process of
apoptosis (which
all soft tissues use, to continuously replace aging cells with new cells),
there is evidence
that after a cell has died, adjacent tissue cells begin participating in
phagocytosis, which
can accelerate their ability to begin taking in nutrients and building blocks
from a dead
neighboring cell that is being recycled.
[00095 ] Accordingly, although the description herein focuses on macrophages,
terms such
as phagocytosis, phagocytic, and phagocyte can also apply to other cell types.
[00096 ]In addition, terms such as "phagocyte" and "phagocytic" are also used
inconsistently for another reason. Some people use those terms to refer to
macrophages
and other phagocytic cells at all times, while others use those terms only
when cells are
actively engaged in phagocytosis.
[00097 J Similarly, the terms "professional" and "nonprofessional" phagocytic
cells
apparently are not always used consistently. Some people refer to all
macrophages and
neutrophils as "professional" phagocytes, while others limit any references to
"professional" phagocytosis as involving the processes that lead to antigen
presentation.
Since phagocytosis involved in apoptosis (i.e., the replacement of aging cells
in soft
tissues) does not lead to antigen presentation, even though macrophages are
involved,
potential conflicts between those different uses of the same term should be
noted.
[00098 J As mentioned above, when a macrophage encounters a foreign object, it
changes
shape, by extending projections (which can also be called fingers,
"pseudopods", or other
terms) out from the main cell body. These projections begin extending around a
foreign
particle, in a way that enables the projections to meet and merge on the far
side of the
particle, effectively engulfing the particle and taking it inside the
macrophage. This type of
shape-changing is similar in several respects to the motions of amoeba and
certain other
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types of microbes, when they encounter and engulf smaller microbes. In its
earliest forms,
phagocytosis enabled single-cell microbes such as amoebae to obtain nutrition.
Subsequently, it became a crucial part of the "innate" immune system of very
small
animals, and still later, it expanded into a major part of the "adaptive"
immune systems of
larger animals. This evolutionary process, and the detailed cellular steps
involved in
phagocytosis, are described in reviews such Jutras et al 2005 (entitled,
"Phagocytosis: At
the Crossroads of Innate and Adaptive Immunity"), and Brumell et al 2003.
[00099 ] When a particle-engulfing process begins, a macrophage is sometimes
called a
dendrite, or a dendritic cell. Those terms are derived from a Greek word for
branching, or
treelike. Accordingly, the three cell types that are commonly referred to, in
the literature,
as "professional" phagocytes are macrophages, neutrophils, and dendrites. The
terms
"dendrite" and "dendritic cells" are not preferred herein, since they are more
commonly
used in medicine to refer to branching structures that occur among neurons,
which use
fibers to establish connections with other neurons.
[000100 ] When carried out by macrophages, the phagocytic engulfing process is
activated by cell-surface receptors, usually called "phagocytic receptors" or
"phagosome
receptors". These receptors are important to this invention, and they are
discussed in more
detail below, since phage vaccine cassettes have been identified herein that
have been
selected for their ability to activate and drive the process of phagocytosis,
by reacting with
phagocytic receptors. A number of phagocytic receptor types are known,
including
various subclasses of lectin receptors (e.g., McGreal et al 2004), Fc
receptors (e.g.,
Swanson et a! 2004), and complement receptors (e.g., Ishibashi et al 1990).
When used
with phage display libraries, the methods disclosed herein offer powerful
tools for
identifying other phagocytic receptor types as well.
[0001011 When a phagocytic receptor has been activated by a microbe, or by a
phage
vaccine particle as described herein, the cell commences a series of steps, in
which the
engulfing cell extends two or more finger-like projections, around the sides
of the particle
that has become bound to the phagocytic receptor. Other cellular "organelles"
that have
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their own membranes (including endosomes, and endoplasmic reticulum) are
recruited to
assist, and they begin contributing all or parts of their own membranes, to
the rapidly-
growing membrane "pocket" that is being formed around the particle that is
being engulfed
by the cell. Once the newly-formed membrane pocket completely surrounds the
particle, it
detaches from the outer membrane of the cell, thereby creating a separate
bubble-like
component, often called a "vesicle", with its own membrane, floating inside
the
cytoplasmic liquid inside the cell that has engulfed the particle. After that
vesicle detaches
from the cell's outer membrane, it is called aphagosome.
[000102 ] A phagosome will then merge and interact with another class of
cellular vesicles,
called lysosomes. After a phagosome merges with one or more lysosomes, the
combined
vesicle is still called a phagosome, or it can be called a phagolysosome.
Lysosomes are the
main digestive components in eukaryotic cells; their internal fluids are very
acidic, and
they contain strong digestive enzymes, usually called lysozymes, hydrolyzing
enzymes, or
hydrolases. Accordingly, lysosomes contribute acids and digestive enzymes to
phagosomes, and the resulting mixture kills and digests the foreign particle.
[000103 ] When a phagosome contains an ingested microbe, then (in at least
some cases)
the digestive process will not completely digest the microbe (i.e., it will
not break apart the
microbe all the way to the level of single amino acids, nucleotides, and other
building
blocks, which would thereby become nutrients for the cell). Instead,
macrophages partially
digest invading microbes, in ways that lead to presentation of antigenic
polypeptide
fragments (from a microbe, or from a vaccine particle) on the "tips" of
specialized types of
cellular "fingers" created by the macrophage. Those fingers, carrying exposed
partial
protein fragments from a microbe or a vaccine particle (the foreign protein
fragments are
"mounted on" MHC-I or MHC-II molecules, as mentioned above), will be
"presented" to
other types of immune cells, including B cells and T cells. Those B and T
cells will then
carry out the next set of steps, in the process of creating antibodies that
will bind to the
protein fragments that have been "presented" by the activated macrophages.
[000104 1 Accordingly, efficient binding of vaccine particles to phagocytic
receptors on the
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surfaces of macrophages, and efficient entry of the vaccine particles into
functional
phagosomes inside the activated macrophages, are crucial steps in the overall
process that
leads to "antigen presentation" by macrophage cells.
[000105 1 That is important because, as described below, this invention has
enabled phage
libraries to be screened in ways have identified certain particular phages
(from among a
huge number of candidate phages, in a phage display library) which happen to
be carrying
surface polypeptide sequences that can potently activate and drive not just
one or two
important steps, but an entire sequence and series of steps that will potently
activate a
desired type of immune response. The necessary transport and delivery steps
(all of which
preferably should be promoted and driven by a single phage "cassette"
particle, which can
be used to carry, transport, and deliver a selected antigenic polypeptide
sequence that is
encoded by a foreign DNA insert, inserted into the cassette) include each and
all of the
following:
(1) cellular uptake (or entry, intake, ingestion, endocytosis, or similar
terms) of
the phage vaccine "cassette" particles, into NALT tissues that are exposed
and/or accessible inside the nasal, mouth, and throat membranes (this
includes both an outer layer of M cells, and NALT cells positioned beneath
that surface layer);
(2) transport (or delivery, presentation, or similar terms) of the phage
vaccine
"cassette" particles, to the surfaces of macrophages formed from
monocytes;
(3) binding of the phage cassette particles to one or more phagocytic
receptors
on the surfaces of the macrophage cells;
(4) activation of the phagocytic receptors, in a manner that leads to
efficient
intake of the phage vaccine cassette particles into properly functioning
phagosomes, inside the macrophage cells.
[000106 ] Each and all of those steps, in that entire sequence and series of
steps, have
indeed been completed and accomplished, by a single particular phage that was
very
effective and efficient in driving each and all of those steps, in mice.
Therefore, the use of
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multi-step screening methods that are disclosed herein, and that have been
shown to be
fully capable of identifying specific phage particles (from a large phage
display library)
that will efficiently drive that entire sequence and series of steps, can now
be carried out
for any other type of mammal (including rodents, primates, humans, livestock,
companion
animals, etc.), and for other vertebrate species having immune systems that
create and use
antibodies (which includes poultry and other types of birds).
ANTIGEN-PRESENTING CELLS (APCs)
[000107 ] For completeness, it should be noted that several different types of
phagocytosis
occur. One important class involves a type of "cellular eating" that plays a
major role in a
process called "apoptosis". In that process, dying or dead cells are engulfed,
broken apart,
and digested by macrophages, leading to the release and recycling of their
building blocks,
which are used to form new cells. Apoptosis enables soft tissues to
continually replace old
cells with new cells.
[000108 ] That type of phagocytosis is unrelated to how the immune system
responds to
microbes or vaccines, and to antibody formation. Accordingly, to avoid
confusing this
invention (involving vaccines and immune responses) with apoptosis and cell
replacement
(which is not relevant herein), the term "macrophage" as used herein is
limited to cells that
can be converted, under proper triggering conditions, into "antigen-presenting
cells"
(abbreviated as APC, with the plural forms APCs or APC's). Monocytes (i.e.,
pre-macrophage cells that have not yet left the circulating blood),
macrophages that can be
converted into APCs, and dendrites or dendritic cells that have commenced the
process of
engulfing and swallowing a microbe or vaccine, all fall within the definition
of APCs, if
they are involved in the immune system. More information on antigen-presenting
cells can
be found in numerous reviews, such as Trombetta et al 2005.
[000109 ] Similarly, terms such as "phagocytosis" and "phagocytes", as used
herein, are
limited to the types of "particle swallowing" processes that lead to antigen
presentation,
rather than to the killing and recycling of aging cells that occurs in
apoptosis and tissue
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renewal.
DIFFERENT POTENTIAL PATHWAYS FOR MACROPHAGES
[000110 ] As mentioned above, a macrophage that has engulfed a foreign
particle generally
will respond to one or more signals that will lead it down one of several
diverging
pathways. Accordingly, a macrophage that has encountered a microbe or vaccine
particle
must effectively choose between several options.
[000111 ] One set of options involves a choice as to whether it will stay at
the site and
engulf more particles (for example, some macrophages reportedly have engulfed
as many
as 100 bacteria), or whether it will begin traveling rapidly toward a lymph
node, to alert
other cells in the immune system that a problem has been detected so that a
full response
can be prepared as rapidly as possible.
[000112 ] A second set of options involves whether the macrophage will commit
to
generating either: (1) an antibody reaction, also called a "humeral" response;
or, (2) a
cell-mediated immune response, which will involved specialized activated T
cells without
involving B cells or antibodies.
[000113 ] If a macrophage commits to helping form a systemic response (which
can be
either an IgG antibody response, or a cell-mediated response), it will begin
moving rapidly
toward a lymph node, as though responding to signals indicating that an
emergency has
arisen, and it must hurry, before more invaders can multiply and cause more
problems.
During this process, the macrophage begins converting (or "maturing") into an
"antigen-presenting cell" (APC). In this form, using phago-lysosomes as
mentioned above,
it will semi-digest the particle it has ingested, and it will transport a
partially-digested
protein fragment, mounted on either an MHC-I or MHC-II complex, to the tip of
one or
more of its extended projections. This presents the antigen to other cells of
the immune
system, including B cells, T cells, and "helper T cells", which will begin
performing the
next steps in the systemic response. In addition, a macrophage that has been
activated in
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this manner will secrete certain types of messenger molecules, such as
interleukin- 1. These
molecules, also called "co-stimulatory" molecules, help. activate other types
of immune
cells.
[000114 ]Accordingly, macrophages occupy and perform a gatekeeper role, at a
crossroads
where the adaptive immune system (in vertebrates) branches out from the innate
immune
system (which originally formed in primitive animals, and which is the only
immune
system most invertebrates have). Most invertebrates have cells that can
effectively act as
macrophages, which will engulf and eat foreign invading pathogens. However,
invertebrates do not have any additional components (including antibodies, B
cells, and T
cells) that can provide more sophisticated adaptive immune responses.
[000115 ] Because of how they evolved, macrophages in vertebrate animals stand
at a point
where two different pathways split and went in different directions. When a
macrophage
encounters a foreign invader, it must either commit to a primitive-type
localized response
(mainly involving phagocytosis of foreign or damaged cells and debris), or it
must commit
to a more complex, sophisticated, and time-consuming response to recruit and
train an
entire team of cells that will defend the organism.
[000116 ] Therefore, when a vaccine is used to create an antibody response,
steps should be
taken to steer and guide macrophages toward that desired response, rather than
toward an
allergic reaction, or a passive "tolerance" response. That type of "steering"
is what
well-selected and properly targeted adjuvants can help accomplish, as
discussed elsewhere
in this Background section, and it also can be promoted by the methods and
reagents of
this invention.
[000117 ] Except as specifically noted, any remarks in this application about
vaccines refer
to the types of vaccines that will help an animal develop a strong IgG-type
and/or IgA-type
antibody (or "humeral") and/or a cell-mediated (or "cytotoxic") immune
response (or a
combination both types of responses). Most such vaccines are designed to help
humans or
animals resist infections by pathogens; however, a number of vaccines are
being tested in
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the hope that they will be able to help patients fight non-infective diseases,
such as cancer,
Alzheimer's disease, etc. Those types of vaccines are discussed in more detail
below, and
more information on them is available in numerous other sources. Similarly,
various types
of vaccines are being tested and developed that involve peptide hormones
and/or hormone
receptors, for purposes such as birth control, stimulating growth, and
fighting certain
diseases that are aggravated (or "fueled") by hormones. As will be recognized
by those
skilled in the art, the teachings herein can be adapted for use in any such
vaccines that
involve antibody-forming and/or cell-mediated (cytotoxic) immune responses.
[000118 ] Accordingly, in view of all Background information summarized above
and
known to those who specialize in immunology, one object of this invention is
to disclose a
new approach to developing and manufacturing vaccines, using nonpathogenic
bacteriophages that contain foreign polypeptide sequences that have been
screened and
shown to actively trigger NALT uptake, association, or other processing.
[000119 ] Another object of this invention is to disclose a new approach to
developing and
manufacturing vaccines, using nonpathogenic bacteriophages containing genes
and coat
protein polypeptides that can promote delivery of the vaccines (and/or
antigenic proteins
carried by the vaccines) to antigen-presenting cells, and/or to phagosome
components
within antigen-presenting cells.
[000120 ]Another object of this invention is to disclose new types of vaccines
(which can
use bacteriophages, other types of viral particles such as glycosylated
viruses that infect
eukaryotic cells, or cellular microbes) that have been enhanced by
incorporating into them
a "targeting-and-delivery" polypeptide sequence that will potently trigger and
drive (i)
intake into NALT and/or GALT cells and tissues, followed by (ii) phagocytic
intake and
processing by macrophages or other antigen-presenting cells. In addition to
containing
enhanced transport polypeptide sequences, such vaccines also will carry at
least one
antigenic sequence that lead to antibody formation, to help an inoculated
animal or patient
resist an invading pathogen, fight cancer cells or other disorders, etc.
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[0001211 Another object of this invention is to disclose new types of vaccine
"cassettes"
that can be used to insert any desired antigenic polypeptide sequences into a
highly
efficient delivery system that already contains: (i) a NALT-targeting, APC-
targeting,
and/or phagosome-targeting peptide sequence which will help ensure delivery of
completed "cassette" vaccines into specific targeted cells or sites of the
immune system;
and, (ii) a toll receptor ligand that will help such vaccines reliably provoke
desired immune
responses, rather than allergic, tolerance or localized reactions, such as by
activating one or
more toll-like receptors.
[000122 ] Another object of this invention is to disclose new types of
particulate phage
vaccines, with at least one a NALT-targeting, APC-targeting, and/or phagosome-
targeting
peptide sequence and at least one selected antigen sequence, that can provoke
strong and
rapid antibody and other immune responses of the type that can subsequently
help a host
animal resist a pathogenic infection.
[000123 ] Another object of this invention is to disclose new types of
particulate phage
vaccines, with at least one component (which may comprise, for example, a DNA
strand
that contains CpG motifs) that functions as a toll receptor ligand, to help
provoke strong
systemic immune responses in inoculated animals and patients.
[000124 ] Another object of this invention is to disclose new methods to
identify and
isolate, from phage display libraries, ligands that will potently activate
phagosome
receptors.
[000125 ] Another object of this invention is to disclose uses for NALT-
targeting and/or
phagosome-targeting phage particles, in targeting the delivery of diagnostic
and/or
therapeutic payloads (such as DNA gene expression plasmids, tracking and/or
imaging
reagents, etc.) into selected phagocytic cells.
[000126 ] Another object of this invention is to disclose new types of
vaccines that have
been modified in ways that enable the phage particles to provide potent
adjuvant activity
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while carrying antigenic protein sequences and/or nucleotide segments that
function as toll
receptor ligands.
[000127 ] Another object of this invention is to disclose new types of
vaccines that can be
manufactured very rapidly, in large quantities, and at low cost, using
bacteria (or other
cells that can be grown in stirred cell culture) as the host cells for
incubation.
[000128 ] These and other objects of the invention will become more apparent
through the
following summary, drawings, and description.
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SUMMARY
[000129 ] Methods and reagents are disclosed for using bacteriophages (i.e.,
nonpathogenic
viruses that infect bacteria such as E. coli) to manufacture mucosal vaccines,
which can be
administered without requiring needles, such as via nasal sprays. The coat
proteins of
phage vaccines for nasal usage must contain foreign polypeptide sequences that
will cause
the phage particles to bind to and activate nasopharyngeal-associated lymphoid
tissue
(NALT) cells. Such phages have been and can be identified and isolated from a
phage
display library containing billions of candidate phages, by means of an in
vivo screening
process in which a phage display library is administered nasally to a lab
animal, which is
later sacrificed so that tissue samples can be harvested and treated, to
extract phages that
were taken in and transported by NALT cells. Additional screening tests on
such
"enriched" NALT-targeted phage populations have been and can be screened by
additional
screening tests, to identify subsets of any such NALT-targeting phages that
will also
potently drive phagocytic intake and processing, by macrophages or other
antigen-
presenting cells (APCs).
[000130 ] After a polypeptide sequence that potently triggers and drives both
NALT intake
and APC intake has been identified by screening of a phage display library,
the DNA
sequence which encodes that polypeptide can be used to prepare a "cassette"
vector, which
can receive and hold any additional foreign gene sequence, inserted into one
or more
surface proteins (such as one or more coat proteins, if filamentous phages are
used as the
vaccine particles). In one embodiment, the completed vector will contain, in
an exposed
and accessible location, one or more antigen sequences that will provoke an
antibody
response that will help animals fight an invading pathogen, such as viruses or
other
pathogens. In other types of vaccines, selected antigens can help a patient's
immune
system attack and destroy cancer cells, beta-amyloid plaques in Alzheimer
patients, or
other cells or materials that cause or aggravate noninfective and/or
nonmicrobial disorders.
[000131 ] The resulting vaccines will contain a combination of useful
components and
traits, including the following:
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1. they will incorporate and use "targeting" polypeptides that have been
screened
and selected for high levels of binding to, and transport through, specialized
epithelial cells
(often referred to as "M" cells that provide the surfaces of "nasopharyngeal-
associated
lymphoid tissue" (NALT) or similar mucous membrane surfaces;
2. the NALT-targeting polypeptides that will enable and drive the first step
in the
desired transport sequence will also be selected and screened to ensure that
they will
stimulate active phagocytosis by macrophage cells (which also can be called
dendrite cells
once they begin the process of phagocytosis), thereby driving and promoting a
second
crucial step that will create a desired immune (rather than allergic or
tolerance) response;
3. the "cassette" system provided by the NALT-active engineered phages can
also
be used to provide one or more components (such as CpG motifs) that will
actively
stimulate one or more targeted toll-like receptors (which can be located
entirely within the
interiors of targeted immune cells, such as TLR9 receptors), in ways that will
further
promote a desired immune response rather than an unwanted allergic, tolerance
or
localized response;
4. the phage particles will contain both (i) toll receptor activating and/or
other
adjuvant-type components, and (ii) a selected antigenic polypeptide sequence,
preferably
in integral and unitary particles that will hold together, rather than in
emulsions or other
mixtures that may become separated in ways that can render them less
effective;
5. these "cassette"-type phage vaccines will be in particulate form, and will
have
sizes that are ideal for stimulating phagocytosis by immune cells, which is
another
important step that can promote desired immune responses rather than unwanted
allergic or
tolerance reactions; and,
6. by manipulating and using both of two different coat proteins, these
cassette
phages can incorporate a first foreign protein sequence that has either a
small or moderate
size, and a second protein sequence having a substantially larger size if
needed; either
sequence can provide the NALT-active transport sequence that will initiate
uptake,
transport and processing by NALT cells, while the other sequence can provide
an antigenic
sequence that will trigger a desired antibody formation response.
[000132 1 In addition to those factors, which come into play after a vaccine
has been
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administered, other important benefits are also provided by this approach,
including:
(a) this type of phage system can enable extremely rapid manufacture of huge
numbers of phage particles, since the bacterial host cells are not killed as
they continuously
secrete very large numbers of filamentous phages through their cell membranes;
(b) this system of vaccine "cassettes" will enable the development of greatly
improved methods for identifying, testing, and developing different antigenic
protein
sequences for different candidate viruses, since the entire "cassette" system
can remain
constant and predictable, gradually accumulating a consistent and predictable
body of
knowledge around it, with only specific and limited antigen sequences being
changed to
create different vaccines for different pathogens;
(c) a consistent and predictable "cassette" mechanism, which will change only
in
the particular antigen sequence inserted into it, can enable greatly
accelerated development
(including any necessary safety and efficacy testing) of new vaccines each
time a new
microbial threat appears (such as at the start of the flu season, each year);
(d) by proper use and manipulation of coat protein sequences, this cassette
system
enables the creation and use of divalent, trivalent, or multivalent vaccines,
such as flu
vaccines having several different antigenic sequences from different strains
of flu;
alternately, mixtures of different phage vaccines, each one carrying a
specific antigenic
sequence (these types of vaccine preparations are often called "subunit"
vaccines), can be
administered in a vaccine mixture;
(e) chemical treatments also can enable these phage vaccines to carry, on
their
surfaces, additional antigens or adjuvants, such as oligonucleotides or longer
DNA strands,
labeling agents, etc.;
(f) since they will be administered nasally, these type of vaccines will be
well-
suited for inoculating poultry, livestock, and other animals, such as by using
a "fogging"
device to emit large quantities of the phages in a mist form, into the air
being breathed by
animals in an enclosure; and,
(g) the bacteriophages being used to provide the cassette/delivery mechanism
have
a long history of study and use, and are nonpathogenic to animals or plants,
since they
cannot infect any known types of eukaryotic cells.
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[000133 ] Accordingly, the vaccines and vaccine cassettes disclosed herein can
provide
optimized delivery and adjuvant activities, and can be used with any antigenic
polypeptide
sequence to provide potent and effective vaccines that can be administered via
nasal sprays
or similar means.
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BRIEF DESCRIPTION OF THE DRAWINGS
[000134 ] FIGURE 1 is a flowchart that shows the major steps for screening a
display
library containing a very large number of phages, in a manner that will select
and isolate
those particular phages which demonstrate: (i) efficient binding to NALT cells
that are
accessible in the nasal sinuses, and (ii) efficient intake and transport of
those particular
phages, by NALT cells in the nasal sinuses.
[000135 ]FIGURE 2 shows the time-dependent passage of two different
populations of
phages into olfactory bulbs in the forebrains of mice, after administration by
nasal spray.
The open circles indicate that "wild-type" phages (with no foreign DNA
sequences from a
display library) passed through the olfactory bulbs with a single early peak.
The dark
circles indicate olfactory bulb concentrations of phages that contained
polypeptide
sequences from single-chain variable fractions (scFv) of human antibodies. A
double peak
was observed. The early peak indicates that some phages passed through
quickly, like
wild-type control phages. The subsequent peak indicated that other phages
passed through
more slowly, suggesting that the phage scFv sequences in those phages had some
level of
binding affinity for either NALT tissue, or olfactory bulb tissue.
[000136 ] FIGURES 3A and 3B show the time-dependent passage of two different
populations of phages into circulating blood, in mice. FIG. 3A shows the
transport of. (1)
wild-type phages without any foreign scFv sequences ("single-chain variable
fraction"
sequences, isolated from human antibodies), indicated by open circles; and (2)
phages
containing scFv sequences, indicated by black triangles. FIG. 3B (which uses a
vertical
scale that is flattened by a factor of 10x) compares the same types of wild-
type phages,
against phages carrying scFv peptide sequences that had been selected by four
successive
rounds of in vivo nasal-to-blood transport screening.
[000137 ] FIGURE 4 shows a screening test for selecting NALT-targeting phages
from a
phage display library containing random 15-mer foreign polypeptide sequences
in a coat
protein. In the first stage, phages that lingered in the olfactory bulb
(thereby showing some
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affinity for olfactory bulb tissue) were harvested, grown in bacterial hosts,
and used as the
starting population for second-round screening. In the second round, phages
were
harvested from NALT cells in an animal's windpipe. In the third stage, phages
selected by
the two prior rounds were incubated with mammalian white blood cells, such as
peripheral
blood mononuclear cells (PBMCs), to identify phages that bind to the immune
cells. In the
fourth stage, phages selected by the third round were isolated from the
"phagosomes" of
mammalian PBMCs, to identify and isolate phages carrying peptide sequences
that drove
efficient "phagocytosis" (i.e., intake and processing) by the immune cells. In
the final
stage, the phages that were selected by the successive screening steps listed
above are
analyzed, to determine the gene and amino acid sequences of the polypeptide
fragments
carried by those particular phages. Those genes and polypeptide sequences can
efficiently
drive both: (i) intake and transport by NALT cells, and (ii) phagocytosis and
antibody-
related processing by white blood cells.
[000138 ]FIGURES 5-10 are photographs or color drawings that will not
reproduce well in
a published patent application. Therefore, the corresponding pictures have
been posted and
and publicly available on a website, at www.tetraheed.net/ferguson.
[000139 ] FIGURE 5 is a photomicrograph showing fluorescent-labeled phages
that were
transported into NALT tissues in the nasal regions of mice. These tissue
samples were
taken 30 minutes after nasal administration.
[000140 ] FIGURE 6 is a photomicrograph showing fluorescent-labeled NALT-
targeting
phages that were transported into immune tissues in lymph nodes in the neck,
and into
antigen-presenting cells (APCs), two hours after nasal administration.
[0001411 FIGURE 7 is a photomicrograph showing fluorescent-labeled NALT-
targeting
phages that intensely labeled a minority of the cells in a preparation of
human
lymphocytes.
[000142 ] FIGURE 8 contains panels 8A, 8B, and 8C, which display the results
of
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fluorescent-activated cell sorting of human lymphocyte cells that had been
incubated with
NALT-targeting phages that had been selected in prior screening tests.
[000143 ] FIGURE 9 contains panels 9A, 9B, and 9C, which are photomicrographs
created
by using fluorescent-labeled antibodies that bind to MHC-1 protein sequences,
showing
that screened and selected NALT-targeting phages efficiently triggered the
clustering of
MHC-1 proteins on human lymphocytes. This result indicates that the cells have
committed to a desirable cross-presentation of antigen, rather than triggering
an undesired
allergic, tolerance or localized response.
[000144 ] FIGURE 10 is a photograph of the results of a gel-shift assay
showing a
reduction in electrophoretic mobility, when plasmid DNA was coated onto the
surfaces of
cationised phage. Since DNA is negatively charged, it clung to the cationised
phages (i.e.,
phage particles that had been treated to create positive electrical charges on
their surfaces),
creating neutralized DNA/phage complexes with little if any net electrical
charge. Since
the DNA/phage complexes had little if any electrical charge, they were not
driven through
the gel by an applied voltage.
[000145 ] FIGURE 11 depicts the results of a purification method that used
ceramic
hydroxyapatite in an affinity column to purify phages. When the concentration
of sodium
biphosphate was increased, during a series of elution steps, phages emerged in
purified
form, in the peak that includes elution fractions 61 through 71.
[000146 ] FIGURE 12 is a drawing depicting a phage particle that carries a
NALT-targeting polypeptide in one type of coat protein, and an antigen
sequence derived
from a microbial pathogen (or from cancerous cells) in a different coat
protein. Exposed
DNA strands with a CpG motif (a known type of "pathogen-associated molecular
pattern"
(PAMP) that can activate TLR9 toll-like receptors) cling to the surface of the
cationised
phage particles, to help ensure a desired immune response rather than an
undesired allergic
or tolerance reaction.
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[0001471 Amino acid sequence data also is disclosed herein, for polypeptide
sequences
that were demonstrated by screening tests to potently drive both: (i) NALT-
targeting,
intake, and transport activity; and (ii) phagocytic intake and processing by
antigen-
presenting cells (APCs).
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DETAILED DESCRIPTION
[000148 ] As summarized above, this invention discloses methods for developing
and using
modified virus particles (which includes phages as well as eukaryotic viruses)
having
specialized "transport" polypeptide sequences (also called targeting-and-
delivery
sequences, as described below), as potent vaccines that can be administered to
mucosal
surfaces of animals, such as in the nasal sinuses and/or the mouth and throat,
without
requiring needles.
[000149 ] As used herein, "animals" includes humans, and can refer to either
an individual
or a population, and the term "inoculated" refers to any animal or human (or
any
population) that has received a vaccine, regardless of the route or mode of
administration.
To satisfy the claims, a vaccine preparation must be suited for use in at
least one animal
species, and does not require activity in all species (for example, a vaccine
intended for
humans does not require activity or efficacy in any other species, and a
vaccine intended
for chickens does not require activity or efficacy in any other species). The
phrase, "A
vaccine preparation for delivering antigens to an immune system of at least
one type of
animal" includes and covers both: (i) a batch of vaccine material in bulk,
such as in a bulk
container being transported from a manufacturing site to a packaging,
distribution, and/or
inoculation site, and (ii) a batch or aliquot of vaccine material that is
packaged in some
type of single-dosage or multiple-dosage form.
[000150 ] The invention also discloses three closely-related compositions of
matter, all of
which can be created by using the methods disclosed herein. The first
composition of
matter comprises vaccine "cassettes", which have been optimized so that the
viral genome
is ready to have a foreign DNA or RNA sequence inserted into a target
insertion site, as
described below. This type of "vaccine cassette" particle or preparation does
not yet carry
an antigenic DNA or RNA sequence that will trigger the production of
antibodies; instead,
a "cassette" is designed and optimized to receive and handle any antigenic
sequence that a
vaccine-manufacturing company chooses to insert into a cassette.
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[000151 ] In addition to vaccine cassettes, this invention also covers
complete vaccine
particles, in which a foreign (or heterologous, exogenous, passenger, payload,
etc.) DNA
or RNA sequence has been spliced into a target (or insertion) site in the
viral genome. The
foreign DNA or RNA sequence will encode an antigenic polypeptide sequence,
such as a
sequence derived from a surface protein of a pathogenic microbe. When complete
vaccine
particles are reproduced, by culturing them in host cells, the foreign antigen
sequence will
appear in an exposed and accessible location, in copies of a surface protein.
As used
herein, "surface protein" refers to microbial proteins that are exposed and
accessible
(usually in multiple copies) on one or more surfaces of a virus or other
microbe. "Coat
proteins" of phages (and of various other types of viruses) are a class of
surface proteins;
other types of viruses are surrounded by membrane-type envelopes (usually made
of lipid
bilayers, taken from one or more membranes of a host cell), and the surface
proteins in
such viruses are embedded in, or otherwise affixed to, the envelope.
[000152 ] The third composition of matter disclosed herein comprises vaccines
that contain
"targeting-and-delivery" polypeptide sequences (referred to in the claims as
"transport"
sequences) that were identified by screening of a phage display library,
regardless of
whether the vaccine particles are, or are not, phage particles. After a potent
targeting-and-
delivery polypeptide has been identified (such as the transport polypeptide
disclosed herein
with sequence data), it can be inserted into various types of viruses or other
vaccine
components other than bacteriophages, to make enhanced vaccines. For example,
a potent
"target-and-deliver" polypeptide sequence can be inserted into surface
proteins of vaccine
viruses that are manufactured by culturing them in eukaryotic cells, such as
in bird eggs,
caterpillars, insects, mammalian cells, etc. This approach can enable a
transport sequence
to be incorporated into "glycosylated" viruses and other types of viruses that
are formed
and/or processed by eukaryotic cells, in ways that bacteria cannot accomplish.
This
approach is discussed in more detail below, under the heading, "NALT-Targeting
Polypeptides In Eukaryotic Viruses".
[000153 ] As another option, the types of "target-and-deliver" polypeptide
sequences
disclosed herein also can be inserted into surface proteins in vaccines made
from cells,
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such as killed or disarmed pathogenic microbes that are used to create various
types of
vaccines. This option also is discussed in more detail below.
[000154 ] Because of how they function, and because of how transport sequences
as
discussed are identified, isolated, and reproduced, a vaccine cassette (and a
vaccine
cassette preparation) becomes useful, valuable, and lawful for use as a
vaccine, only after a
"clonal" isolate has been identified, isolated, and sequenced, using screening
tests such as
described below, and has been proved and demonstrated to carry a polypeptide
sequence
that will actively promote the specialized "target-and-deliver" functions
described herein.
Accordingly, selection and use of suitable screening and isolation steps,
leading to
identification and purification of clonal isolates that will deliver antigen
sequences in
vaccines to targeted immune cells, is crucial to this invention.
[000155 ] As used herein, the terms "clonal" and "monoclonal" are used
interchangeably,
and refer to a population of viruses (which can include eukaryotic viruses,
bacteriophages,
virions, etc.) that have descended from a single ancestor virus, with all
members of the
population presumably being genetically identical. Clonal (or monoclonal)
colonies of
viruses (and of virus-infected host cells) can be obtained by known methods.
Typically, a
dilute preparation of host cells, briefly incubated with phages or other
viruses, are
inoculated at moderate density onto one edge of a solid (agar or gel) nutrient
that contains
an antibiotic, in a shallow dish. The inoculated area is then "streaked" at
high speed
(creating low density) across the remaining area of the plate, using a tiny
wire loop. Since
the only cells that can grow on the nutrient in the culture plate are cells
that contain an
antibiotic-resistance gene carried by the viruses, clonal colonies (isolated
from each other,
and typically having small round shapes that grow larger as time passes) will
arise. A small
sample of virus-carrying cells from a clonal colony can be harvested from the
culture plate,
and those cells can be grown (in very large numbers, but still in clonal form)
in liquid or
other media. These and other methods for isolating clonal populations are well-
known and
conventional.
[000156 1 In order to identify clonal phages containing specific polypeptide
sequences that
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will perform the highly specialized "target and deliver" functions disclosed
herein, the
clonal phage isolates disclosed herein were screened and selected from "phage
display
libraries" that contained millions or billions of candidate phages, all having
different
"inserts" containing foreign polypeptide sequences. Any such phage display
library may
contain several (or possibly dozens or even hundreds) of phages that may
function, with
varying levels of moderate, good, or excellent potency, as a vaccine cassette
as disclosed
herein. However, it would be grossly unsafe, illegal, and unethical to use, as
a vaccine, an
unscreened or even partially-enriched library, which will contain millions or
billions of
unhelpful and unwanted phages that could provoke potentially severe and
dangerous
allergic or immune reactions. A safe and effective targeting-and-delivery
system for a
vaccine can be created only by identifying, isolating, and reproducing one or
more specific
phages which happen to carry specific foreign polypeptide sequences that will
activate and
drive a series of desired immune cell responses, as described herein.
Accordingly, the
claims below contain phrases referring to, for example, compositions of matter
such as "a
clonal virus (or phage) preparation", which is distinct and very different
from a phage
display library.
[000157 ] Terms such as "purified" and "vaccine preparation", as used in the
claims,
require attention. They do not require that a purified preparation must
contain only certain
phages or other viruses, and nothing else. Instead, formulated vaccine
preparations are
controlled mixtures, having an overall level of purity that renders such a
mixture suited for
medical or veterinary use, in which a "purified preparation" of clonal phages
(or other
particles with antigenic protein sequences) is one component. Other components
may
include carriers and/or diluents (which can be either a liquid or a powder);
microbicides or
other preservatives or stabilizers; an acid, alkali, or salt; adjuvant-type
additives; and, if a
vaccine is to be administered to a mucous membrane, one or more mucoadherents.
In
addition, some viral vaccines contain mixtures of two or three different types
of vaccine
particles (these are often referred to as divalent or trivalent vaccines), and
some bacterial
vaccines have even more substituents (for example, two vaccines against
pneumonia are
referred to as 7-valent and 23-valent).
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[000158 ] Accordingly, a vaccine preparation (or a clonal population of
phages, other
viruses, or cellular microbes, or an intermediate preparation that is created
during a
manufacturing process) is deemed to be "purified" if the vaccine particles
have been
processed by one or more purification steps of a type used in vaccine
manufacture. Such
purification methods, used in manufacturing vaccines and other pharmaceutical
or
biochemical compounds, are well-known, and include (as non-limiting examples)
filtering
(which can remove a population of viruses from a population of host cells),
centrifugation
and other physical methods, methods that use affinity binding, and methods
that exploit
differing travel rates, settling positions, or other factors that can be
exploited by using gels,
matrices, or other semi-permeable media (often used in conjunction with
voltages, ionic
gradients, etc.).
[000159 ] Similarly, "vaccine preparations" are not limited to final and
complete
formulations. Instead, a "raw" or unfinished preparation containing clonal
virus particles is
a vaccine preparation, if it will be passed through one or more purification,
processing,
blending, or other manufacturing steps that will render it suited for use as a
vaccine.
[000160 ] It should also be noted that at least some preparations disclosed
herein are likely
to not need refrigeration or other special handling. This can greatly improve
their potential
for use in less-developed countries. For example, most bacteriophages and
other viruses
that are enclosed in coat proteins, rather than lipid membranes, do not
contain water as an
essential component. Therefore, they can be manufactured and packaged in
lyophilized,
dessicated, or other dehydrated form, which can endure in exceptionally stable
form for
long periods of time, even in hot climate conditions. This factor also takes
advantage of the
fact that "infective viability" of virus particles is not essential, to
provide potency and
efficacy as vaccines (as evidenced by the fac that many vaccines are
specifically made
from heat-inactivated or otherwise "killed" viruses).
[000161 ] Certain other terms in the claims also merit attention. For example,
certain
claims specify, "a polypeptide sequence that is foreign to said viruses (or
bacteriophages),
and that has been demonstrated by screening tests to promote: (i) uptake of
particles
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carrying said polypeptide sequence into at least one class of immune cells on
at least one
type of mucosal surface, and (ii) entry into phagosomes of antigen-presenting
cells..." In
that claim language, the phrase, "foreign to said viruses" refers to
polypeptide sequences
that are "foreign" with respect to wild-type viruses. Unless otherwise
modified, all
descendants of a clonal phage line will contain the same "foreign" insert. The
fact that the
progeny phages will inherit and contain a "foreign" insert from their ancestor
does not
make an insert "native" or less foreign, with respect to those viruses;
instead, if a
polypeptide sequence is "foreign" to wild-type phages, then it also is foreign
to any
progeny in a clonal population.
[000162 ] As used in the claims, the term "promote", when applied to
polypeptide-driven
uptake (or intake, transport, delivery, or similar terms) of particles by NALT
cells and/or
phagocytic antigen-presenting cells (which includes macrophages), requires a
level of
activity (or efficacy, potency, or similar terms) that allows otherwise
identical particles
(such as bacteriophages having an assortment of different foreign polypeptide
inserts) to be
cleanly and clearly separated, isolated, and identified, by means of actual
performance in
screening tests that rely upon intake of such particles into NALT cells and/or
into the
phagosomes of antigen-presenting cells. Such screening tests provide a
straightforward and
functional method that will enable anyone skilled in this art to determine
whether (and how
strongly) a certain polypeptide sequence can and will promote the intake of
such particles,
into such cells. The results of such tests can be quantified, if desired, by
various methods,
such as competitive screening tests (comparable to competitive binding
assays), in which a
polypeptide sequence of interest can be pitted and tested against another
polypeptide
sequence, such as in a test that can, if desired, use a mixture of two
different phages, at
identical concentrations, tested in a single animal or cell culture. The other
polypeptide
sequence (usually called a comparison or control sample, or similar terms) can
be provided
by, for example: (i) a random and unsorted population of phages, such as in a
phage
display library; or, (ii) the polypeptide sequence disclosed herein.
[000163 ] In theory, questions might arise over whether low-level promotion of
cellular
uptake is actually occurring, if the rates of uptake are only slightly higher
than
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"background" levels. However, in this context, questions about weak or
borderline
transport-promoting activity will not be significant, and will not need
attention, since any
vaccine manufacturing or supply company will obliged to disclose information
to
governmental reviewing agencies that will support claims that a proposed
vaccine is
potent, and will offer widespread and useful protection in a large majority of
a treated
population. That requirement, for proven and potent performance, will oblige
companies to
select and use polypeptide sequences that will strongly and undeniably promote
transport
of their vaccine particles into NALT cells and/or macrophage cells.
[000164 ]For purposes of quantification, since numerical measuring standards
are nearly
always useful in research, an arbitrary "benchmark" level of potency is hereby
established,
at a level of at least 50% of the cell-intake efficacy of the polypeptide
sequence disclosed
herein, when measured using NALT-related cells or tissues (which may include
measurements of "downstream" tissues in animals), or in monocyte, macrophage,
or other
appropriate cell cultures. This type of "benchmark" standard can be measured
by
inoculating a population of animals (preferably with at least six mice, rats,
or chickens per
sample, to obtain statistically-significant results) or cells, with a 50:50
mixture that
contains both: (i) a first phage preparation carrying the foreign polypeptide
sequence
disclosed herein, and (ii) a second phage preparation carrying a second
candidate foreign
polypeptide sequence that is being tested and measured. If the number of cells
or
phagosomes that contain phages having the second candidate foreign polypeptide
sequence
is equal to 50% or more of the number of cells or phagosomes that contain
phages having
the polypeptide sequence disclosed herein, then the second candidate foreign
polypeptide
sequence should be regarded as being capable of promoting cellular intake into
NALT
cells and/or antigen-presenting cells.
[000165 ] Clearly, the ultimate goal of such tests should be to identify
polypeptide
sequences that can perform even more potently and efficiently than the
sequence disclosed
herein, to enable the development and use of improved nasally-administered
vaccines that
are even more potent and effective than vaccines which carry and use the
polypeptide
sequence disclosed herein. However, since the sequence disclosed herein is
believed to be
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highly and even extremely potent in promoting the specific types of targeting-
and-delivery
(transport) activities that are of interest in vaccines, that polypeptide
sequence, disclosed
herein, provides a useful and convenient "benchmark" level, and any other
polypeptide
sequence that can promote the same types of transport, at a comparative level
of 50% or
greater, should be regarded and classified as potent and efficacious, even if
it is not as
potent as the sequence disclosed herein.
PHAGE CASSETTE DESIGN; DNA INSERTION SITES
[000166 ] The reference herein to "cassette" systems (or vaccine cassettes, or
similar
terms) uses a term that is well-known in genetic engineering. Such "cassettes"
usually
involve plasmids, phages, or other vectors that have been manipulated in ways
that allow
them to be readily and conveniently modified, by inserting additional strands
or segments
of DNA or RNA into one or more predetermined locations in such vectors. The
term
"cassette" arose in the pre-digital era of music, when a cassette player could
play a tape
containing any musical selection the owner happened to have. Cassettes had
standard sizes
and mechanisms (several types became popular, initially for audio recordings,
and
subsequently for video recordings). To use a cassette, an owner merely
inserted a tape
containing the desired music or video (or a tape that was ready to be recorded
onto), into
an accommodating machine. Furthermore, the cassettes themselves could be
loaded with
any audio or video content that the owner or operator chose.
[000167 ] In genetic engineering, "cassette" systems are somewhat different
but
well-understood. Their principal feature is that they are designed to receive,
accommodate,
and work with essentially any DNA or RNA sequence that is inserted into them,
so long as
the insert has a compatible format, as will be understood by those skilled in
the art.
[000168 ] While RNA vectors and cassettes sometimes are used, and are widely
discussed
in the prior art, DNA vectors generally are preferred, for a number of
reasons. One
important set of reasons arise from two facts: (i) DNA is more chemically
stable than
RNA; and, (ii) all cell types have mechanisms that actively and continuously
degrade
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strands of RNA, as part of the continuous process of making, using, and
recycling
messenger RNA (mRNA), which occurs constantly in all active cells. Therefore,
DNA
vectors are more stable and reliable, since the risk of unwanted chemical
and/or enzymatic
degradation of DNA strands is lower than for RNA strands.
[000169 ] Another major factor that leads to a preference for DNA vectors and
cassettes
arises from that fact that whenever an RNA vector is used, some type of
"reverse
transcription" step (i.e., creation of DNA, from the RNA strand carried by the
vector) is
almost always required, to create a lasting and inheritable transformation. If
an additional
required step must be inserted into an already-complex process, it creates
another set of
potential problems, and reduces the likelihoods and rates of desired outcomes.
Therefore, it
usually is easier and more reliable to use DNA vectors, unless strong reasons
indicate that
an RNA vector is better suited for some particular task. The filamentous
phages described
herein carry DNA, rather than RNA, and that approach is usually preferred, for
uses such
as described herein.
[000170 ] As a third important factor, DNA vectors tend to be easier to work
with than
RNA vectors, because there is a broader (and more adaptable and useful)
assortment of
"restriction endonuclease" enzymes that can be used to manipulate DNA vectors,
compared to RNA vectors. As described below, restriction endonucleases that
can cleave
double-stranded DNA are very useful, in genetic vectors, since they enable a
cassette to be
provided with several unique insertion sites, allowing foreign DNA sequences
to be
inserted into specific targeted locations without disrupting any important
genes in a vector.
[0001711 Nevertheless, some types of viruses (often called "retroviruses")
carry RNA
rather than DNA, in their genomes; examples include the human immunodeficiency
viruses (HIV) that cause AIDS, and the coronaviruses that cause severe acute
respiratory
syndrome (SARS). Accordingly, although the discussion herein focuses mainly on
DNA
vectors, for convenience, the teachings herein also can be adapted and applied
to vaccines
that use retroviruses, and to other types of vectors that carry RNA rather
than DNA.
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[000172 ] Most "cassette" vectors used in genetic engineering are provided
with a genome
that has been manipulated to allow "foreign" DNA sequences (which also can be
called
exogenous DNA, heterologous DNA, passenger DNA, inserted DNA, payload DNA,
cargo
DNA, or similar terms) to be inserted into one or more specific known target
sites. This is
accomplished by providing a vector with at least one (and preferably several)
unique
"restriction" sites, which can be cleaved by "endonuclease" enzymes (such as
EcoRl,
BamHI, Hindlll, etc.). These types of enzymes will cleave a DNA strand only if
a certain
sequence of nucleotides is encountered. Restriction sites usually require four
to six
nucleotides, in an exact sequence; as one example, the restriction sequence
for BamH1 is
G/GATC/C, where the slash marks indicate the cleavage locations on the double-
helix
strands of the DNA. The BamHl sequence is symmetric, since the sequence of
bases on
the other strand of the double helix is the reverse, CCTAGG. The BamH1 enzyme
will
leave a 4-base "sticky end" on each of the two resulting "cut ends" of DNA,
when it
cleaves double-stranded DNA. A foreign DNA segment can be given accommodating
"sticky ends", to promote insertion of the foreign DNA sequence into the
cleaved vector.
[000173 ] Cassette vectors usually are designed to have several unique
restriction sites
(such as one site that can be cleaved by EcoRl, another site cleaved by BamHl,
and a third
site cleaved by HindIII), clustered together in a location that enables a
foreign DNA
sequence to be inserted into a targeted location without disrupting any genes
or other
sequences that are important to functioning of the vector. If several such
cleavage sites are
available, at least one endonuclease almost always will be available that will
not
inadvertently cleave a foreign DNA sequence being inserted into the cassette.
If all of the
desired restriction sequences are present in a foreign DNA sequence, the
foreign DNA
sequence usually can be altered, by replacing one or more 3-letter codons in
the foreign
DNA sequence with other codons that encode the same amino acid residue (this
is enabled
by the redundancy of the genetic code, which uses 64 different codons to
encode only 20
different amino acids). In vaccines of the type disclosed herein, a typical
antigen sequence
usually is only about 15 amino acids long, and rarely (if ever) exceeds 30
amino acids.
Therefore, "codon swapping" is simple and easy, and a foreign DNA insert that
will
encode a desired antigen sequence (with any desired selection of codons) can
be made
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rapidly, using automated DNA synthesis machines.
[000174 ]In addition, any gene (or other sequence of interest) in a cassette
vector can be
flanked by one or more unique restriction sites, to enable the gene to be
removed from the
cassette vector at an appropriate time. For example, viral vectors usually
contain an
antibiotic resistance gene (such as a gene that encodes an enzyme that will
inactivate
ampicillin or tetracycline) or some other type of "selectable marker" gene, to
make it easier
to isolate and reproduce the vector in bacterial cells. To avoid any questions
or concerns
about allergic reactions, unwanted antibiotic resistance, or other
complications, any such
marker gene can be removed, after the basic research has been completed and a
vaccine
candidate is approaching final testing and actual use.
[000175 ] These and.other components and methods that make genetic cassettes
easy to
handle and use are well-known, and they can be adapted for use in vaccine
cassettes as
disclosed herein.
[000176 ] When filamentous phages are used as vaccine cassettes, at least one
and
preferably several candidate DNA insertion sites preferably should be located
in the coding
portion of a gene that encodes "coat protein 3" (also referred to as pIII,
cpIII, or cp3),
which is present (in several copies) at one end of each phage, as illustrated
in FIG. 11. This
will cause the foreign polypeptide sequence to be inserted into (or added to
one end of) the
amino acid sequence of coat protein 3, which can carry relatively large
foreign protein
sequences.
[000177 ] In another preferred embodiment, the phage cassettes also should
contain
insertion sites in the gene that encodes "coat protein 8" (also referred to as
pVIII, cpVIII,
or cp8). Over 2000 copies of that coat protein are present in the cylindrical
outer shells of
filamentous phages.
[000178 ] In yet another preferred embodiment, two different genes that encode
coat
protein 8 (cp8) can be present in the viral genome, and only one of those two
genes, under
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the control of a relatively weak and/or inducible promoter, will be provided
with DNA
insertion sites. This can create phage particles with a few hundred copies of
a foreign
protein sequence, while most of the coat protein 8 subunits have unmodified
sequences.
This will reduce the "burden" (or passenger load, payload bulk or weight, or
similar terms)
that the resulting vaccine particles must carry. If all copies of the cp8
subunit in a modified
phage vector carry foreign polypeptide sequences, the foreign sequence often
must be
limited to less than about 10 altered amino acid residues, to avoid hindering
reproduction
of the phages. By contrast, if only a minority of the copies of the cp8 coat
protein carry a
foreign insert, the insert often can be substantially longer, such as up to
about 15 to 20
amino acids, and possibly more. In either case, the exact length of a
tolerable insert will
vary, depending on the specific amino acid sequence of the insert.
TARGETING-AND-DELIVERY COMPONENTS AND ASPECTS
[000179 ] The phage vaccine cassettes disclosed herein can be regarded as
comprising
a targeting and/or delivery system. Because of the nature of its functions and
components,
it also can be referred to by other terms that imply an intentionally-designed
delivery
system (such as, for example, a transport, transfer, carrier, vehicle, ferry,
uptake, intake,
conveyance, or homing system).
[000180 ] A DNA sequence that is inserted into the genome of a phage cassette,
and
the foreign polypeptide sequence that will be encoded by the foreign DNA
(which will
appear in one of the coat proteins of the modified phage) also can be referred
to by various
terms. For example, such DNA sequences (and the polypeptide sequences they
encode)
can be referred to as passenger or payload sequences (or components), or as
antigen or
antigenic sequences. Inserts for vaccines must be antigenic, because of the
nature of
vaccines; however, non-antigenic sequences can be inserted into a phage
cassette for other
uses, if desired. For example, a foreign DNA sequence encoding a polypeptide
sequence
which is not antigenic, but which binds tightly to a known monoclonal antibody
preparation or to a particular surface molecule on certain types of cells, can
be useful in
research or in diagnostic, imaging, or similar work.
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[000181 ] If an inserted DNA and/or amino acid sequence is referred to as
foreign,
heterologous, exogenous, or similar terms, such terms imply that the inserted
sequence is
not present in the phage, regardless of whether the "foreign" sequence might
be present in
an animal or human treated by a vaccine. For example, a polypeptide sequence
that is
"foreign" to a phage cassette can contain a polypeptide sequence that appears
on the
surfaces of cancer cells, in cancer patients who will be treated by the
vaccine, in a vaccine
designed to trigger the formation of antibodies (and/or the activation of
cytotoxic T cells)
that will kill the cancer cells.
[000182 ] As mentioned above, specialized immune cells are present on several
different
mucosal surfaces, including vaginal and rectal surfaces. Accordingly, vaccine
cassettes as
disclosed herein can be identified and isolated for any such set of mucosal
immune cells,
merely by screening for cells that are actively taken into any such cluster or
class of
immune cells.
[000183 ] However, it has been reported (Kozlowski et al 2002) that in humans,
nasally-
administered antigens can stimulate stronger antibody responses than vaginal,
rectal, or
other candidate mucosal routes. In addition, nasal administration can be
enhanced and
speeded up, merely by inhaling a nasal spray; it does not require any removal
of clothes or
other time-consuming preparatory actions; and, if a major pandemic emerges, a
long line
of people can be treated very rapidly (without even requiring any delays for
creating or
keeping records, if local public health officials deem it prudent to speed up
mass
distribution as much as possible).
[000184 ] For those and other reasons (combined with the fact that nasal
routes can
eliminate needles and the problems that accompany needles), the NALT tissues
in the
nasal sinuses and pharynx area appear to offer an ideal site for administering
vaccines as
disclosed herein. Accordingly, any references herein to "NALT-targeting"
activity are used
for convenience, and are intended to be exemplary rather than limiting, to
refer to activity
in triggering uptake (also referred to as intake, entry into, etc.) by one or
more types of
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specialized immune cells that are exposed and accessible on one or more types
of mucosal
surfaces.
[000185 ]If desired, specialized bactericidal nozzles for nasal sprays can be
used. For
example, the surfaces of certain metals (such as silver) can increase the
microbicidal
potency of alcohol and certain other disinfectants. Accordingly, nasal-spray
nozzles coated
with silver or other metals can be rapidly and efficiently disinfected by
wiping them with
alcohol, between uses. Alternately or additionally, a spray nozzle (coupled to
a supply tube
from a trigger-operated dispensing unit) can be designed with a placement
component that
would be pressed against the epidermal skin, above the upper lip and below the
nose. This
would enable two thin tubes at the upper tip of the spray nozzle, spaced
roughly a
centimeter apart, to inject a small pressurized jet or spray of liquid into
both nostrils,
without touching anything inside the nostrils.
[000186 ] As mentioned in the Background section, prior versions of intranasal
vaccines
have not lived up to their potential, because of various limitations and
shortcomings.
Among other factors, adverse reactions (such as inflammation of the olfactory
bulbs, after
intranasal administration) have been observed in some cases (Van Ginkel et al
2000); and,
better intranasal adjuvants are needed (Lang 2001; Levine 2003). There has
been too much
variability, among test animals in populations that have been treated, making
it difficult or
impossible to determine reliable parameters such as optimal dosing regimens.
In addition,
lab animals sometimes respond to nasally-administered vaccines in ways that
are different
from human responses, leading to still more unwanted variables and
complications.
[000187 ]The specialized targeting-and-delivery components and aspects of the
mucosal
vaccines disclosed herein are believed to overcome those problems, and offer a
greatly
improved class of mucosal vaccines, in which a combination of useful
components will
work together synergistically to provide improved vaccines that can outperform
any known
mucosal vaccines in the prior art.
[000188 ] Furthermore, the cassette approach of this invention can enable the
screening and
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development of various different sets of phage cassettes, with each set being
optimized for
a particular species. For example, the first set of NALT-intake screening
tests described
herein used mice. If desired, a second set of phage cassettes can be screened
and isolated in
rats, using the same or similar screening approaches; a third set of phage
cassettes,
screened and isolated in chickens, for use in chickens; and so on. Those types
of screening
tests can be repeated in any species of interest (such as in species that are
important in
farming or food supply, in veterinary or medical use, etc.), up to and
including humans.
Similarly, these types of phage cassettes can be optimized for various wild
species that are
being endangered by viral or other epidemics.
[000189 ] Based on factors of evolution, homology, species similarities, and
cross-reactivity, it is highly likely that phages isolated in one species will
also function
effectively in closely-related species. As examples, phage cassettes that are
screened and
isolated in mice are likely to function efficiently in rats, rabbits, and
other rodents; phage
cassettes that are isolated in chickens are likely to function efficiently in
turkeys or other
poultry; and phage cassettes that are isolated in monkeys or chimpanzees are
likely to
function efficiently in humans and other primates.
[000190 ] It also should be recognized that the immune systems of all mammals
face very
similar pressures, demands, and needs, since all mammals must be able to fight
off heavily
overlapping types of microbial pathogens. As a result, there are unusually
high levels of
similarity, homology, and cross-reactivity among some components of mammalian
immune systems, even among widely different classes of animals (such as
rodents, and
primates). This is shown by, for example, the extensive homology that exists
among toll-
like receptors in species as different as mice and humans. Accordingly, phage
cassettes
such as disclosed herein, and adaptations of the screening methods disclosed
herein, will
provide immunology researchers with powerful tools for analyzing and
quantifying various
types and degrees of overlap and cross-reactivity, between various components
and cell
types of the immune systems of different types of mammals. During such
research, it is
likely that some particular phage cassettes will show high levels of vaccine-
type potency
among different classes of mammals (such as between mice and humans), while
other
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phage cassettes will show lower levels of cross-reactivity.
[000191 ]Accordingly, whenever desired, isolation of a preferred phage
cassette for use in
a particular species of interest (and, in some cases, in a particular strain,
race, or other
group of interest, such as in specific strains of mice or rats that are widely
used in
immunological research, or in humans who live on continents or islands, where
different
types of pathogenic microbes pose the most severe threats to health) may be
able to
provide even more potent and efficient carrier phages, for use in a particular
species or
other ancestral group. While some phage cassettes are likely to emerge that
can provide
good vaccine potency among all humans, other more specialized phage cassettes
may be
able to provide even higher levels of potency among people whose ancestors
lived and
evolved among a semi-localized and particularized set of pathogens.
[000192 ] For practical reasons, since solid tissues can be harvested easily
from mice, the
screening tests described herein initially used mice, to screen for phage
intake into NALT
tissues, and for transfer from NALT tissues into other tissue types (such as
olfactory bulb
tissue). In subsequent screening tests involving monocyte cells (white blood
cells which
are the precursors of macrophage cells), human cells were used, partly because
it is easier
to obtain large quantities of white blood cells from humans than from mice,
and also
because the goal of this research is to move rapidly toward vaccines that can
and will be
used in humans.
[000193 ] With regard to isolating phage cassettes that are optimized for
human medical
use, two points should be noted. First, in many situations, collecting phage
isolates that
have passed a screening test will not require solid tissue samples; instead,
isolates can be
collected by using blood and/or lymph, by means that only require aspiration
(removal of
liquid) using a hypodermic needle. Second, if a compelling need ever arises
for obtaining
solid tissue samples from humans, such samples can be obtained from people who
have
been declared brain-dead after a major trauma (such as a fatal automobile
accident, cardiac
arrest, massive stroke, etc.), or who have lapsed into a terminal coma at the
end-stage of a
disease such as cancer. Fatally-injured people who are "brain-dead" are often
kept alive for
hours, days, or even weeks, using respirators and intravenous feeding, to
enable the
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harvesting of organs or other tissues for transplant purposes. That is an
accepted and
respected medical practice, and it provides grieving relatives with a sense
that a terrible
loss of a loved one was able to save other lives. Accordingly, similar
practices can used to
enable harvesting of NALT or other tissues, from humans, if a compelling need
ever arises.
USEFUL STEPS; COMPLETE AND OPTIMIZED DELIVERY
[000194 ] Two different and potentially conflicting concepts must be
understood and kept
in balance, to understand this invention.
[000195 ] The first concept is this: if a properly screened and isolated
vaccine cassette can
accomplish even a single crucial targeting-and-delivery step, it can provide
an important
and useful advance over the prior art. Accordingly, any such advance merits
recognition
and coverage for what it has accomplished, and some of the claims below focus
on phage
vaccine cassettes that were screened and isolated because they were able to
accomplish a
single specific step that is useful or crucial in provoking a desired immune
response to a
vaccine.
[000196 ] The second concept, which points in a different direction but which
must also be
taken into account, is this: if a single targeting-and-delivery vaccine
cassette can
accomplish not just one or two steps, but an entire series of steps, all of
which will increase
the likelihoods and rates of desired immune responses in the largest possible
number of
animals or people among a treated population, then that type of enhanced multi-
step
targeting-and-delivery system provides an even more important and useful
advance,
compared to a targeting-and-delivery system that was selected by only one or
possibly two
screening steps.
[000197 ] This factor arises from how vaccine preparations are used, in the
real world. To
prevent the spread of disease, they must be administered in large numbers, to
large
populations. Because of the stochastic, probability-dependent, bell-curve
nature of large
biological populations, and no one can predict with certainty how any
particular person or
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animal will respond. Therefore, any company, agency, institution, physician,
or other
health care provider that distributes or administers vaccines to animals or
people must
accept and assume the responsibility to develop, procure, and use the best,
most potent,
most effective candidate vaccine(s) that are available, to provide optimal
benefits.
[000198 ] This obligation is especially important, since most diseases pose
the greatest
risks to people who are not entirely healthy, and not in optimal condition to
fight a disease.
As examples, very young children do not have fully-functioning immune systems;
the
immune systems of elderly people gradually grow weaker, less agile, and less
effective;
and, the immune systems of many adults have been compromised and weakened by
various injuries, infections, and diseases, and in many cases by smoking,
suboptimal diet,
excess weight, alcohol or drug abuse, and other problems.
[000199 ] Accordingly, targeting-and-delivery phage vaccines should (and
ultimately must)
be identified and isolated, not just by using a single round of screening, but
by using a
succession of several different screening tests, where the starting population
for each
screening test is obtained from candidates that performed well in other types
of screening
tests. This has been accomplished by the methods disclosed herein, and if
desired, it can be
repeated in screening tests that are limited to one particular species (such
as humans, for
example), to isolate phage vaccine cassettes that will be truly optimized for
that particular
species.
[000200 ] The sequence of screening steps that were used in the initial
demonstration of
this invention, and the immune cell responses that were screened for, by those
particular
tests, can be summarized as set forth below. Because of the nature of these
tests, in some
cases a single screening test (which culminated in harvesting and isolating
phages from a
specific type of tissue or cell) required that several distinct cellular steps
had to be
completed successfully, in order for phage isolates to be isolated from the
targeted cells or
cell compartments.
1. The first screening test isolated phages that had been taken into NALT
tissues
(which line the nasal sinuses and upper throat area, and which are present on
the surfaces
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of breathing passageways in rodents), and which were then transported to one
or more
types of "downstream" (or "second-stage") tissues or cells. Accordingly, this
test identified
and isolated phages that successfully completed each and all of the following
steps: (i)
intake into NALT cells, presumably via endocytotic or other cell-surface
receptors; (ii)
release of the phage by the receptor, after a phage/receptor complex had
entered a NALT
cell; (iii) secretion of the phages by the NALT cells, into some type of
cellular junction
that delivered the phages to one or more types of "downstream" cells; and,
(iv) intake of
phages that had passed through NALT cells, into one or more types of "second
stage" cells
or tissues.
To complete this screening test, the "second stage" tissue was harvested, and
the
membranes of the cells were dissolved, using a detergent-type "lysis buffer"
that dissolves
lipid membranes of cells, without damaging the coat proteins of viruses. That
step released
the contents of the cells, allowing the phages to be extracted. The isolated
phages were
then reproduced in E. coli bacteria, to provide a starting population for
subsequent
screening tests.
2. A second screening test (which used "enriched" populations of phages that
had
been screened for NALT intake and transport, as described above) was used to
isolate
phages that triggered binding to (and/or intake into) macrophage cells. As
mentioned in the
Background section, the term "macrophage cells" as used herein includes
monocyte cells,
which are precursor cells that become macrophage cells after they pass through
a capillary
wall, leave the circulating blood, and enter the lymph fluid in soft tissue.
This screening test began with sampled human blood. The red blood cells were
removed, and the semi-purified white blood cells were processed by a surface-
binding
step, to isolate monocyte (macrophage) cells, which have unusual surface-
adhering
molecules that enable monocytes to grip a capillary wall and permeate through
the
capillary wall, to reach the lymph fluid. The purified monocyte (macrophage)
cells were
incubated with phages that had been fluorescently labeled, and the cell/phage
mixture was
processed by "cell sorting", using a machine called a flow cytometer, to
isolate monocyte
(macrophage) cells that were strongly labeled by fluorescent phages. This was
done by
setting the controls of the machine so that only the top 3% of the cell
population was
isolated, based on strength and intensity of the fluorescent signal from a
cell/phage
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complex.
The membranes of the isolated highly-fluorescent monocyte (macrophage) cells
were dissolved, viable phages were extracted, and the harvested phages were
reproduced in
E. coli, to provide starting populations for subsequent screening tests.
3. The third screening test (which used phages that had already been selected
by the
screening tests described above) isolated phages that triggered phagocytosis
(i.e., entry into
macrophage cells). This test was carried out by using several stages of
centrifugation, to
isolate phagosomes (i.e., intra-cellular compartments enclosed within their
own
membranes) from macrophage cells, obtained from human blood by the surface-
binding
selection process mentioned above. The cells were incubated with phages that
had passed
the prior screening tests, and were then processed using a mechanical
homogenizer. The
homogenizer broke apart cells, without breaking the phagosomes (which are much
smaller
than cells). Intact phagosomes were isolated by (i) a first mild
centrifugation, which
formed a pellet of intact cells and nuclei, which were discarded; and, (ii) a
second stronger
centrifugation, which pelletized the phagosomes. The phagosome pellet was
resuspended
in liquid, then the membranes were dissolved by a lysis buffer, and phages
contained
within the phagosomes were harvested, and reproduced in E. coli. Accordingly,
to be
present in intact and functional phagosome compartments, the isolated phages
had to
activate and participate properly in three sequential cellular processes: (i)
binding (as a
"ligand") to a phagocytic receptor, on a surface of a macrophage cell; (ii)
intake of the
bound receptor/ligand complex into the macrophage; and (iii) separation of a
completed
and functional phagosome, from the outer cell membrane.
ENHANCEMENTS AND ADJUVANT ACTIVITIES
[000201 ] In a preferred embodiment, the phage cassettes disclosed herein can
include
(and/or can be supplemented by) enhancing components that can promote
additional
desired responses. Such cellular responses might include, for example: (i)
activation of one
or more types of toll-like receptors (TLR's), to help ensure that an immune
response leads
to desired immune response, rather than an undesired allergic or tolerance
reaction; (ii)
activation of a desired Thl and/or Th2 response; and, (iii) activation of a
desired MHC-1
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and/or MHC-2 response. These types of enhancements can enable vaccines to be
optimized
for either of two different usages: (1) inducing an antibody-producing
"humeral" response,
for fighting off microbial pathogens; or, (2) inducing other cell-mediated
responses, such
as for killing cancer cells, or for removing other types of cells or materials
(such as, for
example, beta-amyloid plaques in the brains of people suffering from
Alzheimer's
disease).
[000202 1 Some of those enhancements and/or adjuvant features, activities, and
advantages
of these vaccines arise from the inherent properties of the phage cassettes.
As one example,
these phages appear to have an ideal size for triggering intake into NALT
cells, and into
phagosomes within macrophages.
[000203 J Other enhancements, which can include components that can be
regarded as
adjuvants (or as having adjuvant-like activity), can be provided in any of
several ways,
such as by using one or more of the following approaches: (i) adjuvant,
adjuvant-like, or
other enhancing components can designed and incorporated into phage cassettes,
so that
such components will be integral and inseparable features of all vaccine
particles; (ii)
adjuvant or other enhancing components can be covalently or non-covalently
bonded to
phage particles (which presumably will occur after final assembly of completed
vaccine
particles that carry inserted foreign DNA sequences and antigenic polypeptide
sequences),
so that the adjuvant or enhancing components will remain securely bonded to
the vaccine
particles, until phagocytic or other cellular or enzymatic processes take over
and begin
dismantling a vaccine particle; and/or, (iii) adjuvant or other enhancing
components can be
coupled to the phage cassettes by other means, such as by ionic or hydrogen
bonding.
[000204 ]As used herein, terms such as adjuvant, adjuvant-like activity, or
enhancing
components are intended to refer to any component, activity, or other trait or
feature that
will either: (i) increase the likelihood (or rates, probabilities, yields,
etc.) of triggering
desirable immune responses, in members of a large treated population; and/or,
(ii) reduce
the amount of a vaccine preparation that must be administered, to provoke
desired immune
responses in some target fraction or percentage of a treated population. In
interpreting such
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phrases, it should be kept in mind that some of the vaccines disclosed herein
will be
designed to help an organism defend itself against an infection, while other
vaccines will
be designed to help fight cancer or kill other unwanted cells, or to dissolve
or neutralize
plaques or other unwanted materials. Accordingly, components or traits that
can activate
one or more types of toll-like receptors (TLRs), or that can preferentially
promote MH1 or
MH2, Thl or Th2, or other types of responses, are included within terms such
as adjuvants
or enhancing components, as used herein.
[000205 1 The ability to incorporate adjuvant components into the vaccine
particles
themselves, or to use covalent, hydrogen, or ionic bonding to couple
components to
vaccine particles, can eliminate or minimize a number of problems that have
plagued the
prior use of adjuvants that are merely stirred into a liquid or slurry
mixture. In any such
mixture, vaccine particles can separate from adjuvant additives, and the two
types of
components can migrate in different directions, inside a body, in ways that
can reduce the
efficacy of the adjuvant, and therefore of the vaccine. Accordingly, by
developing and
optimizing phage particles into unitary vaccines, which have adjuvant as well
as antigen
components incorporated into or bonded to the same particles, a set of
problems that has
plagued and limited vaccines in the past can be avoided.
[000206 ] These approaches also enable improvements in quality control, which
poses a
major concern in vaccine manufacture and safety, for a number of reasons.
"Manufacturing
tolerances" must be extremely tight when human lives are at stake, especially
in countries
where potential lawsuits and liability are major factors that often control
business decisions
(and that typically impede and hinder major innovations). Quality control
problems
increase whenever biological (rather than purely chemical or mechanical)
manufacturing is
used, and quality control problems increase even more when time pressure is
important, as
in vaccine manufacture (for example, every year, when flu season approaches,
public
health officials would prefer to wait and delay, for as long as possible, to
study what is
happening among various candidate flu strains in various populations, before
finally
selecting the strains that will be incorporated into a flu vaccine that will
be used that year).
Quality control will become even more difficult yet critical, if a need arises
to manufacture
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hundreds of millions (or even billions) of dosages very rapidly, as may be
required if a
"bird flu" strain emerges that can be transmitted human-to-human, or if a
strain of
HIV/AIDS emerges that can be transmitted by insects.
[000207 ] The use of phage vaccines that can be manufactured in huge
quantities in a
matter of hours, using in vitro culture of bacterial cells (or other types of
eukaryotic,
glycosylating, or other cells that can be grown in cell culture), can make
quality control
issues and problems simpler and more manageable, compared to prior
manufacturing
methods, such as incubating vaccines in bird eggs (which normally must
incubate for
weeks).
[0002081 Accordingly, the coat proteins of phage vaccines as disclosed herein
can be
chemically treated (or modified, enhanced, or similar terms), to enable them
to carry or
deliver additional antigens, adjuvants, or other useful molecules. For
example, if phage
particles are "cationised" (i.e., treated with agents that will impart a
positive electrical
charge to their surfaces), then relatively short strands of DNA, called oligo-
nucleotides or
oligo-deoxy-nucleotides (abbreviated as ODN's) will cling to the phages (since
DNA
strands are negatively charged). This will allow DNA segments having (for
example)
"CpG motif' sequences to be affixed to the surfaces of the phages. As
described in the
Background section, in DNA strands with CpG motifs, large numbers of
unmethylated
cytosine residues are positioned adjacent to guanidine residues. These are
recognized by
mammalian immune systems as a "pathogen-associated molecular pattern" (PAMP),
which
can activate toll-like receptors, to promote immune response rather than
allergic or
tolerance responses. Accordingly, DNA strands having CpG motifs can be
synthesized and
affixed to the surfaces of "cationised " or otherwise treated phages, using
ionic and/or
hydrogen bonding, to increase the efficacy and potency of the resulting
vaccine particles.
[000209 1 Alternately or additionally, certain amino acid residues in proteins
can be
chemically treated, by known reagents, in ways that will covalently crosslink
other
compounds to the proteins. As an example, the side chain of each lysine
residue in a
protein has a reactive primary amine group, at the end of a four-carbon chain.
The
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accessibility and hydrophilic (water-soluble) nature of such amine groups
allows certain
known reagents (such as isothiocyanate, or bis(sulfosuccinimidyl)suberate) to
react with
lysine residues, in ways that will create covalent crosslinking bonds with
other compounds.
Since covalent bonding generally is stronger than ionic and/or hydrogen
bonding, such
reagents can be used to covalently bond adjuvants, secondary antigens, or
other potentially
useful compounds to the surfaces of phage particles in vaccines, if desired.
[000210 1 Using such means, phage vaccines as described herein can be used to
carry, into
the phagosomes of targeted antigen-presenting cells, molecules or substances
(which can
be referred to by terms such as payloads, passengers, supplements, enhancers,
adjuncts,
etc.) that normally would not be efficiently internalized by such cells, or
that normally
would not potently activate a desired immune response in a large fraction of a
treated
population. Examples of such molecules or substances include: (1) small
antigenic epitope
molecules; (2) labeling or imaging molecules (also called trackers, tracers,
or similar
terms), which can be useful in research, diagnostic medicine, and other
situations; (3)
small and/or soluble antigen molecules that are only weakly immunogenic unless
attached
to a larger molecule; (4) adjuvant molecules, such as short DNA strands having
CpG
motifs, or other agents that can activate toll-like receptors; and, (5)
plasmid DNA, which in
some cases may be able to provoke gene expression, leading to useful
polypeptides within
(or possibly secreted by) targeted cells. Those are non-limiting examples of
compounds
that can be useful if incorporated into, or affixed to, phage vaccines having
targeting-and-
delivery capabilities as disclosed herein.
SCREENING FOR POLYPEPTIDE SEQUENCES THAT DRIVE NALT INTAKE
[0002111 As described in the Background section, published Patent Cooperation
Treaty
patent application WO 2003/091387 described a method for carrying out an in
vivo
screening process, using the long sciatic nerve bundle in a rat leg, to
identify, select, and
isolate certain particular phages (out of millions or billions of candidate
phages, in a phage
display library) that will be efficiently taken into nerve fibers (by
endocytotic receptors on
the neuronal surfaces), and that will be transported within the nerve fibers,
toward the main
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cell bodies of the nerves. This screening method required that any phages that
were
actually selected, in the screening test that was used, had to actually and
effectively
perform both of those two different and distinct functions, by emplacing
candidate phages
at a first location near the knee, and by harvesting phages from a different
location near the
hip.
[000212 ] Subsequently, the inventor herein realized that an analogous type of
in vivo
screening test, using a phage display library administered by nasal spray, can
be used to
identify, select, and isolate certain particular phages (out of millions or
billions of
candidate phages, in a display library) that will efficiently target and enter
NALT cells in
the nasal and throat region.
[000213 ] That task has been completed successfully, and DNA and amino acid
sequence
data, for the best-performing isolated clonal phage that was identified and
isolated by such
screening tests in mice, is provided herein. Accordingly, the work done to
date in mice
demonstrate the efficacy of this approach, and similar screening tests, using
the same or
similar types of phage display libraries, can be done with any other species
of interest, by
using the same or similar steps.
[000214 ] The particular steps used in the mice tests are not claimed or
asserted to be
optimal; instead, certain steps were taken because the inventor herein had
done similar
work for other purposes previously in his career, and therefore was already
familiar with
certain types of tests. Nevertheless, the complete set of steps that emerged
and evolved,
during the course of this research, were shown to work effectively. Now that
this approach
have been disclosed, experts and researchers who study these disclosures can
adapt and
improve these particular screening steps, for use with various species.
Accordingly,
provided that the goals and results remain essentially the same (i.e., to
identify phages that
will drive NALT uptake and phagosomal intake into macrophages, leading to
strong
immune response, for use in vaccines or related immunological research), the
exact details
of similar screening tests that may be developed or used in the future are not
essential to
this invention.
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[000215 ] Furthermore, since the goal of such screening tests is to use a
series of tests to
identify phages that happen to carry foreign polypeptide sequences that will
activate and
drive several different steps, the sequence of such screening tests is not
crucial. For
example, to minimize the number of research animals that must be used, any in
vitro
screening tests that only require white blood cells can be completed first,
and enriched
populations of phages identified by the in vitro tests can then be screened by
in vivo tests,
using intact animals.
[000216 ] It also must be recognized that once a high-performance phage that
can function
as a vaccine cassette has been identified, either for any particular species
or for some range
of species (such as a first phage cassette that has been optimized for
rodents, a second
phage cassette that has been optimized for humans and primates, a third phage
cassette that
has been optimized for birds, etc.), additional screening tests will no longer
be necessary,
and the selected phage cassette(s) can simply be reproduced (and can be
"tweaked" and
further improved and adapted, if desired).
[000217 ] Accordingly, the screening tests developed and used for in vivo
testing in mice
used the following steps:
1. During prior research that had occurred over a span of years, before this
invention arose, the inventor had identified various reagents and methods that
helped this
work proceed more rapidly and efficiently. Two such reagents are worth noting.
First, the
inventor used a compound called FITC, which uses fluorescein as a label and
isothiocyanate as a linker, which will form a covalent bond that links a
fluorescein label to
an amine group (such as on a lysine residue) on a protein. The FITC labeling
agent enabled
rapid analysis of tissue slices under a fluorescent microscope (i.e., a
microscope that uses
ultraviolet or other short-wavelength light as a light source). It also
eliminated the need for
expenses and delays that would have occurred if other types of labeling had
been used that
required incubation with antibodies, specialized enzymes, or other reagents.
[000218 1 Second, the inventor used a mixture of parabenzoquinone and
paraformaldehyde,
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as a tissue fixative. This allowed the exposure time for the fixative reagent
to be reduced,
which helped preserve the structure of various molecules, and avoided the
creation of
excessive crosslinking bonds, which otherwise can interfere with analysis.
2. Since the inventor, a medical school professor who specializes in CNS
physiology in mammals, had previously done other types of research that
involves tracking
the travel of various things through brain cells and tissues, he was already
familiar with
various problems and obstacles that can arise in such research. Therefore, he
decided to do
a series of preliminary tests, to find out: (i) where a diverse phage library
would go, if
administered nasally to mice, and (ii) how rapidly phages would appear in (and
be
removed or cleared from) various types of tissues within the brain. Since he
had seen prior
results and reports (involving other, earlier research) indicating that some
types of
compounds, when inhaled by mice, will be taken into the olfactory bulbs, and
will pass
through that portion of the brain before being taken elsewhere, he decided to
include
olfactory bulbs as one of the tissue types that were tested for phage
concentrations, at
various time intervals after nasal administration.
3. The initial tracking and timing tests were performed using fluorescent-
labeled
"wild type" phages with no foreign gene sequences. The results, indicated by
the open
circles in the graph in FIG. 2, indicated a peak of high phage concentration
in olfactory
bulb tissue at the earliest testing time, in mice sacrificed 5 minutes after
phage
administration. That peak passed fairly quickly, as indicated in FIG. 2.
4. A second series of tests using olfactory bulb tissues was carried out using
a
phage display library (prepared by Cambridge Antibody Technology) containing
scFv
gene sequences from human antibody genes, inserted into coat protein 3 (which
is present
in several copies at one end of a phage particle). The results, indicated by
the dark circles
in FIG. 2, indicated an early first peak, which coincided in time with the
single early peak
of the wild-type phages. Unexpectedly, a second peak was seen at 2 hours after
phage
administration, which had not been present at 1 hour or at earlier test times.
[000219 1 After considering the possible causes for a second peak, the
inventor concluded
that it most likely reflected some type of low or moderate affinity binding,
between: (i) the
foreign antibody-derived sequences carried by some of the phages in the phage
library, and
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(ii) the tissues in the mouse brain. These types of binding reactions are
occunng as part of
a complex and ever-changing series of interactions between several types of
fluids and
several types of membranes, within and around the olfactory region of a mouse
or rat
brain. While a detailed analysis of those tissue and fluid types is not
necessary for
understanding the current invention, it is important to realize that after
carefully
considering all relevant factors, the inventor realized that the types of low
or moderate
affinity binding reactions he had observed could be exploited and utilized, by
means of
time-sensitive in vivo screening tests that could identify phages with NALT-
uptake
activity.
5. A first round of in vivo screening tests was carried out, using nasal
administration of a phage library containing a diverse set of inserted DNA
sequences
encoding random 15-mer polypeptide sequences in coat protein 8 (which is
present in over
a thousand copies, in the cylindrical shells of the phages). Either 30 minutes
or 60 minutes
after administration, animals were sacrificed (n = 10 total), and about 20 to
30 ml of saline
solution was injected into the aorta, to rinse unattached phages out of the
vasculature.
Tissue was harvested from the olfactory bulb portion of the brain, and the
cells were
incubated with a lysis buffer, which dissolved the cell membranes and released
viable
phages. The harvested phages were reproduced in bacteria, with the help of a
tetracycline-resistance gene carried elsewhere in the phage genome (the scFv
phage library
similarly contains an ampicillin resistance gene). Samples from both sets of
animals were
pooled together, to provide an enriched starting population for the second
round of
screening.
6. In the second round of screening, the enriched phage population (containing
15-mer inserts in coat protein 8) were administered. After 45 minutes, the
mice (n=10)
were sacrificed and perfused with saline solution. NALT tissues (which are
visible
elongated lumps, which flank both sides of the midline along the bottom of the
windpipe)
were harvested. The cell membranes were dissolved by lysis buffer, and
harvested phages
from the NALT cells were replicated in bacteria cells.
7. The NALT screening process was repeated, using phages selected in the first
round of NALT screening as a starting population, to further enrich for NALT-
targeting
phages.
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[0002201 The ability of those screened and selected phages to actually target
and bind to
NALT cells, and subsequently to be transported by the immune system into lymph
nodes,
is confirmed by the photomicrographs in FIGS. 5 and 6. Phage preparations that
were
screened and isolated as described above were replicated, and fluorescently
labeled using
FITC. The labeled phages were then administered to mice, in nasal sprays.
After 30
minutes or 2 hours, some of the mice were sacrificed, and perfused with a
mixture of
parabenzoquinone and paraformaldehyde. Tissue sections were obtained, and
photographed with a fluorescent microscope. The large picture in FIG. 5
indicates the
region of tissue that was photographed. The two smaller pictures (which show
up more
clearly in color photographs posted on a website, www.tetraheed.net/ferguson)
clearly
indicated the presence of various clusters of fluorescent-labeled phages that
had been
transported to NALT tissues.
[0002211 In mice that were sacrificed after 2 hours, the lymph nodes that
service the
NALT tissues in the windpipes of the mice were harvested and photographed. The
photographs in FIG. 6 clearly indicate that the phages selected by NALT
screening (as
described above) were indeed transported to those lymph nodes.
SCREENING FOR MACROPHAGE BINDING AND/OR INTAKE
[000222 1 Additional screening tests, which began with 15-mer phage
populations that
already had been selected by NALT screening tests in mice (as described
above), were
used to identify phages that were actively taken into, and processed by,
macrophages.
Human macrophages were used, for two reasons: (i) sufficient quantities of
macrophages
can be obtained more easily from human blood, than from mouse blood; and, (ii)
the goal
was to identify phages that can lead to actual vaccines for human medicine,
rather than
merely creating research tools for use in rodents.
[000223 1 A crucial processing step that is carried out by macrophage cells,
when
antibodies are generated in response to a pathogen or vaccine, involves
phagocytosis (i.e.,
the intake of a solid particle into a cell). As described in the Background
section (and in
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numerous textbooks and review articles), phagocytosis involves four sequential
steps, all
of which must occur for the complete process to succeed.
[000224 1 In the first step, a pathogen or vaccine must bind to (and activate)
a phagocytic
receptor, on the surface of a macrophage. Using a standard term that applies
to cell surface
receptors, any molecule that will bind to and activate such a receptor is
called a "ligand".
Accordingly, the in vitro screening steps described herein were used to
identify specific
phages, from among a large library of candidate phages, which happen to carry
polypeptide sequences that act as "phagocytic receptor ligand" sequences.
[000225 ] In the second step, the receptor-binding reaction must activate a
membrane-
altering process, to trigger formation of finger-like membrane extensions by
the cell. These
membrane extensions will flank and then surround the receptor/ligand complex,
enabling
the receptor/ligand complex to be taken into the cell.
[000226 1 In the third step, the membrane "pocket" containing a
receptor/ligand complex
grows larger, and is drawn deeper into the cell, with the aid of additional
"organelles"
having their own membranes (such as endosomes and lysosomes). The membranes of
those organelles merge with the membrane of the phagosomal pocket that is
being formed,
to enlarge the phagosomal membrane and speed up the process.
[000227 1 In the fourth step, the phagosome disengages from the outer membrane
of the
cell, to form a cxomplete and intact phagosome (i.e., a discrete compartment
with its own
membrane), which encloses a particle such as a microbe or a vaccine.
[000228 1 Once the phagosome has been formed, the macrophage uses specialized
processes and enzymes to partially digest the microbe or vaccine, in a way
that creates
relatively short polypeptide sequences, typically about 15 amino acids long.
Those short
polypeptide sequences, from a partially-digested microbe or vaccine, are
"mounted" on
either MHC 1 or MHC2 proteins, to form an antigen/MHC complex, which is
transported
to the external surface of the macrophage cell. When that occurs, the
macrophage becomes
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an "antigen-presenting cell" (APC). It will deliver its surface-mounted
antigen polypeptide
(from the microbe or vaccine particle) to a "B cell", which will perform the
next steps in
creating antibodies that will bind to microbes having the same antigenic
polypeptide
sequence.
[000229 ] Since phagocytosis is a multi-step process, two different screening
tests were
used, to ensure that the screened and selected phages would be highly potent
and efficient,
in initiating and enabling all of the steps in the process. If desired, the
first phagocytosis-
related screening test might be eliminated, since: (i) the second screening
test will screen
for the desired final result, and (ii) successful completion of a desired
final result implies
and even requires that any necessary earlier steps also must have been
completed,
successfully.
[000230 ] However, the initial screening test is not difficult; it merely
requires an initial
incubation, followed by flow cytometry using an automated machine. In
addition, since
vaccines and immune responses involve "stochastic" (probability-dependent)
events that
will occur in large and varied populations, steps should be taken to ensure
that the most
potent and efficient candidate phage(s), from a large phage library, will
indeed be
identified and selected. Therefore, if an additional screening test can
increase the
likelihood that the most potent and efficient targeting-and-delivery phage
(for use in
vaccine cassettes) will be identified and isolated, then that screening test
should be
regarded as useful and desirable, even if not strictly necessary.
[000231 ] Accordingly, a "first phagocytotic" screening test was performed,
using a phage
population that already had been screened and selected for NALT-targeting
activity, as
described above. To perform this screening test, human blood was sampled and
loaded into
tubes, on top of a buffer solution called "Lymphoprep" (sold by Nycomed
Pharma, Oslo,
Norway). When centrifuged using the manufacturer's instructions, RBCs will
pass through
the buffer and can be removed and discarded, while WBCs will remain on top of
it. The
white cells were harvested, washed twice in Dulbecco's phosphate-buffered
saline (PBS),
and placed in plastic tissue culture flasks.
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[000232 1 As mentioned in the Background section, the white blood cells of
interest are
called "monocytes" when they circulate in blood. They have special surface-
adhering
molecules that cause them to grip and cling to the internal surfaces of
capillary walls. That
clinging process is crucial, in enabling these cells to pass through a
capillary wall and enter
the lymph fluid (the watery fluid that moves slowly through soft tissues).
After a monocyte
cell leaves the circulating blood and enters the lymph, it swells to a larger
size, and is
called a macrophage.
[000233 1 Therefore, by exploiting the unusual "surface-adhering" activity of
monocytes, it
is possible to isolate monocytes from a much larger population of mixed white
blood cells.
To perform that process, the semi-purified WBC population was placed in
plastic tissue
culture flasks. After 30 minutes of incubation, any cells that did not cling
to the plastic
surfaces of the culture flasks were rinsed away, and discarded. The adhering
cells were
gently scraped off, using a rubber spatula, and were resuspended in a buffer
solution. They
were then incubated with fluorescent-labeled phages that already had been
screened for
NALT-targeting activity as mentioned above.
[0002341 After incubation (to provide time for phages to bind to phagocytic
receptors on
the surfaces of the monocyte cells), which can be followed if desired by
stirring, shaking,
or similar processing to rinse away and remove any unbound phages, the
monocyte/phage
mixture was processed by "fluorescent-activated cell sorting" (FACS). In this
process, the
cells (suspended in a clear liquid) pass rapidly through a very thin glass
tube, in a machine
that uses an ultraviolet or similar light beam to activate the fluorescent
labels bonded to the
phages. A photo-detector (which detected the emission wavelength of the FITC
label on
the phages) was used to activate a tiny jet of liquid, injected into the flow
passageway,
each time a highly fluorescent cell passed through the glass tube. That
control mechanism
caused highly fluorescent cells to be sent to a special collection chamber,
while cells with
lower levels of fluorescence were sent to a discard bin. The controls on the
flow cytometer
were adjusted so that only 3% of the monocytes were selected; this is
indicated by the
rectangle on the right side of FIG. 8A.
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[000235 ] In this "first phagocytotic" screening test, it did not matter
whether phages
entered the cell interiors, or merely clung to the cell surfaces. However, the
test did
required substantial binding to occur. Therefore, this screening test selected
phages that
happened to be carrying polypeptide sequences that function as ligands that
bind tightly
(with high affinity) to phagocytotic receptors on the surfaces of monocyte
(macrophage)
cells.
[000236 ] The selected monocytes (and their associated phages) were treated by
lysis
buffer, to dissolve the cell membranes and release the phages. The phages were
replicated
in bacteria, and were used as the starting population in yet another round of
screening, as
described below.
[000237 ] FIG. 5 (which includes FIGS. 5A and 5B) provides visual confirmation
that the
flow cytometry screening test performed as intended. To obtain those
photographs, phages
selected by the flow cytometry screening were incubated with a preparation of
white blood
cells. The white cell population contained a relatively small number of
monocytes and
macrophages, mixed with other non-macrophage lymphocytes. FIG. 5A shows large
numbers of white blood cells, under normal visible light (the smaller cells
are mainly
platelets). FIG. 5B is a photograph of the same cells in the same pattern and
position, at a
fluorescent wavelength. It shows that fluorescent-labeled phages became bound
to only a
few of the large cells, which presumably and apparently are monocytes and/or
macrophages, while the labeled phages did not bind to the other types of
lymphocytes.
These results confirm that the isolated phages were selective, and bind with
high affinity
only to certain targeted types of white blood cells. The "grayscale"
photographs submitted
with this patent application are not as clear and vivid as the color
photographs, which are
available at www.tetraheed.net/ferguson.
SCREENING FOR INTAKE INTO PHAGOSOMES
[000238 ] Since the flow cytometry screening test (described above) tested
only for binding
of phages to macrophage cells, a final screening test was performed, to
isolate phages that
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were efficiently taken into phagosomes (i.e., discrete organelles with their
own
membranes) inside macrophage cells. This test was deemed necessary after prior
tests that
screened for neuronal transport (as described in PCT application WO
2003/091387)
indicated that phages which became very tightly bound to endocytotic
receptors, on the
surfaces of neuronal fibers, apparently were not released properly by the
receptors, and
were not transported efficiently within the neuronal fibers.
[000239 ] To carry out the phagosomal screening test, another set of PBMC's
that adhered
to the plastic walls of culture flasks was prepared, as described above. These
cells were
contacted by a population of phages that already had been screened for NALT
uptake, and
for macrophage surface binding. After incubation with the monocyte cells,
unbound
phages were washed off and removed, and a liquid that contained trypsin (a
protein-digesting enzyme) and ethylamine-diamine-tetra-acetate (EDTA, which
enhances
trypsin activity) was added, to detach the cells from the plastic surfaces of
the flasks. This
.15 trypsin treatment step also served to digest and render non-infective any
phage adhering to
the outer surfaces of the cell, thereby reducing any risk that phages isolated
from
phagosomes might be contaminated by phages that did not promote phagocytosis.
The
harvested cells were suspended in fresh media, centrifuged, and resuspended.
[000240 ] The cell/phage preparation was then homogenized, using 10 strokes of
a
mechanical plunger device known as a Dounce glass-glass tissue homogenizer.
Breakage
of the cells by the homogenizer ruptured the outer membranes of the cells, in
a way that
did not destroy the phagosomes and other organelles (which are much smaller)
contained
inside the cells. The homogenate was centrifuged under conditions that
pelletized the cell
nuclei and unbroken cells, while leaving the phagosomes (with any uptaken
phages) in the
supernatant. The supernatant was then centrifuged at a higher speed for a
longer time, to
pelletize the phagosomes. The supernatant was discarded, and pelleted
phagosomes (with
internalized phages still contained inside them) were resuspended in fresh
buffer. Lysis
buffer was used to dissolve the phagosomal membranes without damaging the
phages. This
released phages, which were replicated in E. coli cells.
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[0002411 Taking all of the foregoing screening stages into account, a combined
synergistic
screening process, as listed above, is summarized in the flowchart of FIG. 1.
Several clonal
phage colonies containing 15-mer foreign polypeptide sequences were isolated
by that
process, and those phages were sequenced, to determine both: (1) the sequences
of the
45-base foreign DNA sequences carried by those phages, and, (2) the amino acid
sequences of the 15-mer foreign polypeptide sequences phage carried by those
phages. The
DNA and polypeptide sequence data are contained and provided within this
application.
[000242 ) It also should be noted that screening and selection of a phage
display library,
using methods such as described herein, can become a very useful tool in
studying,
analysing, and utilizing various aspects of phagocytosis, including
identification of new
and additional classes of phagocytic receptors. While a number of classes of
phagocytic
receptors are already known (such as lectin receptors, Fc receptors, and
complement
receptors, as mentioned in the Background section and as reviewed in articles
such as
Aderem and Underhill 1999, Jutras and Desjardins 2005, and Blander 2007),
other classes
and types are likely to exist, and merit attention. Accordingly, the methods
described
herein can be used to screen phage display libraries (including phage display
libraries that
may not have been previously screened at all, or that may have been screened
using
methods other then the NALT-related screening described herein), to identify
additional
receptor types of receptors that can trigger phagocytosis.
[000243 1 Once this research pathway becomes better recognized and understood,
identification of phagosome receptor ligands for specialized cell types may be
used to
target efficient delivery of payloads to particular cells, or to stimulate or
otherwise
modulate various types of phagocytic processes that are important in
development, disease,
or other processes or problems. For example, stimulation of enhanced
phagocytosis of
beta-amyloid peptides by microglial cells, in brain tissue, may be able to
provide or
improve various types of research and/or therapy, in Alzheimer's disease; this
concept is
discussed in more detail in articles such as Gelinas et al 2004. Similarly, in
a process called
"Wallarian degeneration", Schwann cells phagocytose the myelin proteins that
form the
sheaths of neuronal fibers, (e.g., Hirata et al 2002); and, olfactory-
ensheathing glial cells
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phagocytose the axons of olfactory cells that have been replaced (e.g.,
Wewetzer et al
2005). Accordingly, those processes can be better studied and understood, and
likely
exploited for diagnostic or treatment purposes, by screening phage display
libraries by
methods such as disclosed above, to identify and isolate specific phages and
polypeptide
sequences that will bind to and activate specific phagocytotic receptors that
are not yet
adequately understood, but that are likely to be present on the surfaces of
various types of
specialized cell types.
METHODS AND COMPONENTS FOR TOLL RECEPTOR ACTIVATION
[0002441 As briefly discussed in the Background section, if a vaccine
preparation can
activate one or more types of "toll receptors" (also referred to
interchangeably as toll-like
receptors, abbreviated as TLR's), on or in macrophage cells, the vaccine can
be much more
efficient in provoking a desired immune response. Accordingly, the phage
cassette
vaccines disclosed herein can be designed in ways that will strongly activate
one or more
types of toll receptors.
[000245 1 In particular, phage cassette vaccines can be designed and assembled
in ways
that will activate specific toll receptors that are present inside macrophage
cells, rather than
on the cell surfaces. This can help increase the safety of vaccines derived
from such
vectors, since it can avoid unintended activation of toll receptors on the
surfaces of
macrophage cells, which otherwise might increase the risks of triggering or
aggravating
allergic or other unwanted responses in some recipients.
[000246 1 Cell-internal toll receptors that are known at the present time are
believed to
include TLR3, TLR7, TLR8, and TLR9. A large and rapidly growing body of
published
reports have identified DNA sequences containing CpG motifs (mentioned above)
as
among the agents that are known to activate internal TLR9 class of toll
receptors.
Accordingly, DNA sequences contain CpG motifs can be "woven into" the single-
stranded
DNA genome of phage cassettes as described herein. Alternately or
additionally, relatively
short DNA segments (called oligo-deoxy-nucleotides, or ODN's) can be affixed
to the
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surfaces of phage particles, using either covalent or ionic/hydrogen bonding
as described
above, to trigger the activation of TLR9 receptors.
[000247 1 In addition, this type of controllable targeting of specific types
of toll receptors is
believed to be capable of creating vectors that can drive and steer the immune
system
toward creating either: (i) a "humeral" response, involving antibodies and B
cells; or, (ii) a
"cell-mediated" response, involving activated T cells without any substantial
involvement
by antibodies or B cells. It is believed and anticipated that this type of
"guiding" system
that targets certain types of toll receptors can be used, in phage cassette
systems as
disclosed herein, to create two different but important classes of vaccines:
(1) vaccines that will provoke antibody-producing humeral responses, for
fighting
off microbial pathogens; or,
(2) vaccines that will provoke specifically targeted cell-mediated responses,
for
killing cancerous cells and for dissolving or otherwise removing or moderating
certain
other types of harmful or dangerous cells or materials, such as plaques or
other deposits.
[000248 ]FIG. 12 is a schematic depiction of a cassette-type phage particle
40, with
various elements indicated by callout numbers. Callout number 41 indicates a
NALT-targeting peptide that will stimulate phagocytosis by human monocytes;
callout
number 41 indicates another attached molecule that has a different desired
function, such
as a fluorescent label, a compound that, will provoke toll receptor
activation, etc. Callout
number 43 is a DNA strand carried by the phage, which can contain (for
example) a strong
CpG motif, to activate toll receptor 9. Callout number 44 depicts an antigenic
protein that
has been incorporated into coat protein 3 (CP3) of the phage; several copies
of the CP3
proteins are present at the end of a phage filament, and at least one (and
presumably all) of
those copies have been modified to include a relatively large foreign
polypeptide sequence.
[0002491 With regard to toll receptors, it should be noted that the use of CpG
motifs, in
DNA strands carried inside filamentous phages, is believed to represent an
advance in
methods and reagents for exploiting toll receptor activation. When a
phagocytic cell
internalises and processes a bacteriophage, DNA sequences that were carried
and hidden
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inside the phage will be exposed, and can begin to stimulate TRL9 receptors,
in ways that
can initiate and/or increase an immunostimulatory cascade that will lead to an
enhanced
immune and antibody response. Filamentous phages provide a different
technology
platform for developing and delivering CpG sequences, as immune adjuvants,
compared to
other candidate delivery vehicles. In other settings, ODNs typically must be
synthesized to
be resistant to DNAase activity by the host cells. In the phage cassettes as
disclosed herein,
immunostimulatory CpG motifs carried within the phage filament will be
shielded and
protected from DNAase inactivation, by the phage coat proteins. However, those
phage
coat proteins will be removed, after a phage vector has entered a macrophage
or other
phagocytic cell.
[000250 1 Furthermore, in contrast to synthetic ODNs, which become
increasingly
expensive to manufacture and purify as the number of base pairs in the
oligonucleotide
increases, filamentous phage DNA is not constrained by size. Therefore, it
becomes
entirely practical to incorporate CpG or similar immunostimulatory DNA
sequences
having lengths greater than 200 base pairs, into phage genomes. This will
allow, for
example, repeating CpG motifs, as well as permutations that will combine CpG
sequences
from different microbial species and/or that will have several different
recognized patterns
combined in ways that can be conveniently produced by phage cassette systems.
For
example, Klinman et al 2004 describes three distinct subclasses of CpG motifs,
designated
as D-type, K-type, and C-type motifs. Accordingly, phage cassettes as
disclosed herein
will enable researchers to determine whether the presence of two or more such
patterns can
lead to additive or even synergistic levels of potency and efficacy, when
incorporated into
vaccines.
[000251 1 In summary, by considering the components and factors described
above, and by
using gene and polypeptide sequences that have been identified (by screening
tests) as
having strong levels of NALT-targeting activity, experts who are skilled in
the art, after
studying and considering the teachings herein, will be able to understand how
to create
NALT-targeting phage cassettes and vaccines, and how to enhance the adjuvant
properties
of such cassettes and vaccines in desirable ways, such as by modifying the
cassette
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genomes to include stimulatory or inhibitory CpG motifs, or other components
of interest.
These cassette systems will be able to receive and incorporate selected
foreign DNA
sequences (such as, but not limited to, sequences that encode antigenic
proteins from
pathogenic microbes), and they will be able to deliver the foreign DNA
sequences to
specific targeted classes of phagocytic cells, in ways that have not been
possible under the
prior art.
COLLECTION, ASSEMBLY, AND COORDINATION OF COMPONENTS
[000252 ] In understanding the types of phage cassette vaccines disclosed
herein, it is
important to understand how each of the component parts contribute, and
interact with
each other. These components and their roles can be summarized as follows:
(1) the phage cassette will provide components that can be referred to as
carrier,
vehicle, transport, targeting, or delivery components, or by similar terms. To
function in
this manner, the cassettes must provide, in an exposed surface-accessible
location, at least
one polypeptide sequence that has been shown to provide at least one (and
preferably all)
of the following activities:
a. targeting-and-delivery activity that will promote intake of vaccine
particles into
specialized cells that are exposed and accessible on one or more mucosal
surfaces. As
mentioned above, because of several factors, NALT cells (in the nasal and
throat region)
offer a convenient, rapid, efficient, and preferred route for mucosal
administration.
However, other mucosal routes (involving vaginal, rectal, or other surfaces)
can be used if
desired. Accordingly, references herein to "NALT-targeting" activity are used
for
convenience, and are exemplary rather than limiting, and refer to activity in
triggering
uptake by one or more types of specialized immune cells that are exposed and
accessible
on one or more types of mucosal surfaces.
b. targeting-and-delivery activity that will promote the intake of vaccine
particles
into phagosomes, inside macrophage cells. As described above, phagocytic
processing by
macrophage cells (which is a multi-step process) is a crucial step in
generating a desired
antibody response to a vaccine. Therefore, a coat protein of a phage cassette
preferably
should contain one or more exposed and accessible polypeptide sequences that
will
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actively trigger binding to (and activation of) phagocytic receptors, on the
surfaces of
macrophage cells. Such polypeptide sequences can be referred to as phagocytic
(or
phagocytotic) ligands.
(2) In addition to the targeting-and-delivery components mentioned above,
other
components (which can be referred to as adjuvant components, enhancing
components, or
similar terms) also be incorporated into such vaccines, if desired, as
optional components
or improvements. As one example, the phage cassettes disclosed herein can
contain DNA
sequences having CpG motifs (either carried within the genomes of the phages,
or affixed
to the surfaces of the phage particles), which can activate targeted toll-like
receptors in
ways that will increase the likelihood of a desired immune response (which
will depend on
the type of vaccine that is being administered).
(3) One or more foreign gene sequences will be inserted into the genome of a
phage
cassette, into targeted insertion sites that are properly positioned within
the coding
sequence for a viral coat protein. These insertion sites will contain unique
sequences that
can be recognized and cleaved by at least one (and preferably several)
restriction
endonuclease(s). In vaccines designed to trigger antibody production to help
fight a
microbial pathogen, such as a virus or bacteria, the inserted foreign DNA
sequence will
encode an antigenic protein sequence that is normally found on the surface of
the
pathogen. By placing such antigenic protein sequences into phage cassettes
that will
specifically target and deliver the antigenic polypeptide sequences to NALT
cells and then
to macrophage cells, increased efficacy and potency for such vaccines can be
achieved,
and nasal administration of these vaccines can offer an effective route of
administration,
not just for humans, but for large numbers of animals as well (notably
including poultry).
[000253 ] Now that this new class of vaccines has been described as set forth
above, a
reader should review the comments in the two itemized lists set forth in the
"Summary of
the Invention" section, above. One list is numbered as items 1 through 6,
while the other
list is numbered as items "a" through "f'. Those skilled in the art will reach
a better
understanding and appreciation of those listed factors and potentials, and
will be able to
create more effective use of those factors and potentials, once they have
studied and
recognized the concepts and components summarized in this "Detailed
Description"
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section.
DNA VACCINES
[000254 ] It has been reported that microparticles can be used to deliver,
into a mammalian
body, DNA strands that can be expressed into foreign polypeptides, by means of
normal
cellular processes. This approach, called "DNA vaccination", is reviewed in
articles such
as Jilek et al 2004. Under the prior art, it has not been highly efficient,
and it has not
become a widely-used method of vaccination.
[000255 ] However, certain aspects of the current invention can be adapted and
utilized in
ways that will substantially increase the efficacy and potency of the "DNA
vaccine"
approach. In particular, single-stranded DNA segments (such as from phages
that carry
ssDNA) or double-stranded DNA segments (such as from bacterial plasmids) can
be
affixed to the surfaces of phage particles as disclosed herein, using either
covalent
bonding, or ionic/hydrogen bonding, as mentioned above. By using such methods,
strands
of ssDNA and/or dsDNA can be affixed to the surfaces of particles that can be
manufactured inexpensively in large quantities, and that can potently and
efficiently
deliver surface-affixed DNA strands into specific targeted cells, such as: (i)
into NALT
cells and macrophages, if the phages that were identified and isolated by
screening tests
such as disclosed herein; or, (ii) into other cell types, if the phages were
identified and
isolated by screening tests that select for uptake into those particular cell
types.
[000256 ] One such treatment pathway that merits special attention involves so-
called
"Thl" responses, as described in more detail in the next section.
VACCINES THAT SELECTIVELY ACTIVATE TH1 OR TH2 RESPONSES
[000257 ] As mentioned in the Background section, "T helper" cells will
respond to
different types of triggering events in ways that will "commit" the cells to
converting into
either of two distinct classes of cells, referred to as Thl or Th2 cells.
There are several
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ways to distinguish between Thl and Th2 cells, depending on how they are
activated, and
what they do after they are activated. Importantly, those known differences
also offer
potential ways to manipulate and control vaccines, in ways that can "steer" or
"direct" T
helper cells in either of those two directions when desired.
[000258 ] The distinctions between Thl cells versus Th2 cells are described in
review
articles such as Moingeon 2002, Knutson et al 2005, and Burrows 2005. While
other
articles such as Rosloniec et al 2002 point out that the distinctions are not
always entirely
clear, and that apparently paradoxical responses are sometimes observed,
relevant reports
generally indicate and agree upon the following:
(1) T helper cells commit to the Thl pathway when a messenger molecule called
interleukin 12 (IL- 12) is present. The ThI cells then begin producing gamma
interferon,
interleukin 2, and tumor necrosis factor alpha.
(2) by contrast, T helper cells commit to the Th2 pathway when IL-4 is
present.
The Th2 cells then begin producing more IL-4, as well as IL-5 and IL- 10.
(3) Thl cells are involved in what are often called "cytotoxic T cell" (CTL)
responses, also called "cell-mediated" responses. These can be important in
fighting
cancer, and in fighting some types of chronic, lingering microbial diseases.
However,
cell-mediated responses also are involved in autoimmune diseases, and can
create severe
problems in some cases.
(4) by contrast, Th2 cells are involved in systemic responses that use B cells
to
create antibodies (these are also called humoral responses).
[000259 ] By taking those differences between Thl versus Th2 pathways into
account, and
by also considering other articles and teachings in this field, it is believed
and anticipated
that steps can be taken that will enable phage cassette vaccines as described
herein to be
manipulated in ways that will trigger either: (1) Th2 responses, when desired,
such as for
creating antibodies that will fight off microbial pathogens that typically
cause short-term
infections, such as flu viruses; or, (2) Thl responses, when desired, such as
in vaccines for
treating cancer or other disorders, or for treating some types of lingering
infections. As
stated in Guy et al 2005, "one can now choose adjuvants able to selectively
induce T
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helper Thl and/or Th2 responses, according to the vaccine target and the
desired immune
response."
[000260 ]As examples, DNA sequences with CpG motifs that will stimulate Thl
("cell-mediated") responses are described in articles such as Krieg et al
1998, while other
DNA sequences with "suppressive" CpG motifs that will steer the immune system
toward
Th2 (antibody-generating) responses are described in articles such as Ho et al
2003 and
Shirota et al 2004. Accordingly, these types of relatively short DNA sequences
will merit
attention, as enhancers and adjuvants that can be incorporated into, or
affixed to the
surfaces of, phage cassette vaccines as described herein.
[0002611 In addition, timed coadministration of selected interleukin molecules
or certain
other cytokine molecules, and/or other types of adjuvants as described in
articles such as
Guy et al 2005, can also be used to help steer an immune response in a desired
Thl or Th2
direction.
[000262 1 A primary reason to vaccinate is to "prime" the immune system, so it
can
recognize a future invasion and respond as rapidly as possible. As part of
this priming
activity, when the immune system launches a Th2 type of response, it generates
"memory"
B cells that effectively "remember" the vaccine antigens. This process
involves a complex
mechanism, wherein a large variety of responsive B cells with reshuffled short
DNA
sequences are generated, which will encode a variety of newly-created variable
fragments
that are incorporated into new types of antibody molecules. Subsequently, the
immune
system identifies particular B cells that happen to be making antibodies that
efficiently
bind to the invading microbe. Those particular B cells are stimulated to
reproduce rapidly,
causing the enlarged population of selected B cells to secrete large numbers
of their
antibodies. Then, after an infection recedes, the number of those particular B
cells drops
off greatly. However, a few of those B cells remain in the system for years or
even
decades, and if a need arises, they can be stimulated to cause them to rapidly
reproduce
again, so that they and their progeny cells can rapidly begin making a renewed
supply of
the antibodies that were effective in helping fight off a particular type of
microbe.
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Accordingly, if the same antigens that were introduced earlier (by a vaccine)
are seen
again, during a genuine microbial invasion, the "memory" B cells that were
generated as
part of the response to the vaccine will be (in effect) "scanned", retrieved
from a "library"
or "archive" of candidate antibody-producing cells, and signaled to begin
multiplying
rapidly. Those B cells will begin secreting large numbers of antibodies, which
will attach
to the antigens on the invading microbe, acting as "flags" to attract and
activate phagocytic
cells, which will engulf and destroy the invading microbes. This process, as
described
above, mainly relates to Th2 responses.
[0002631 By contrast, in a Thl-related response to a vaccine, antigen-specific
memory
CD8(+) T cells are generated. Like the antibody secreting memory B cells
generated by
vaccination, these memory CD8(+) T cells are primed to multiply rapidly, if
the same
antigen subsequently appears in the body. CD8(+) T cells are also known as
"killer" T
cells, because one of their primary roles is to detect and destroy cells that
have become
infected by invading viruses. By responding rapidly, in ways that do not
require antibodies
but that can kill a virus-infected cell before a virus has had enough time to
fully take over a
cell's internal machinery and make more viruses, antigen-specific memory
CD8(+) T cells
can recognize the early signs that a cell has been infected by a virus, and
can destroy
virus-infected cells, to minimize the number of additional viruses being made
by the
infected cells. The importance of Thl responses and antigen-specific CD(+) T
cells, in
protecting against viral infections, is described in reviews such as Wiley et
al 2001, and
Wong and Pamer 2003.
[000264 1 Since a combination of both types of responses (Thl and Th2) can be
highly
useful, in some situations, it would be desirable in such cases to administer
a vaccine
mixture that contains both (1) a first set of phage particles that will
stimulate and drive Thl
responses, and (2) a second set of NALT-targeting phages that will stimulate
and drive Th2
responses. Using influenza viruses as an example, NALT-targeting phages
carrying
TRL-related or other PAMP-related components, selected and designed to trigger
a Th2
response, can carry one or more genes that encode the haemagglutinin (HA)
and/or
neuraminidase (NA) proteins. Those two proteins are exposed on the surfaces of
influenza
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viruses, and they help flu viruses bind to and infect cells; as a result,
those are the influenza
proteins that can be readily bound by antibodies that are created by "memory"
B cells. In
the same vaccine mixture, NALT-targeting phages carrying TRL-related or other
PAMP-related components that will trigger a Thl response can carry one or more
genes
that encode the influenza proteins that take control over a host's cellular
machinery,
because those proteins are more likely to be present and active, in host cells
that have been
infected by the viruses and that need to be destroyed by killer T cells.
[000265 ] In addition to creating a "two-handed" vaccine mixture (which can
also be
referred to as double-barreled, double-pronged, or similar terms) that can be
highly
effective in responding to a particular microbe, another advantage of that
type of approach
should also be noted. By creating a vaccine mixture that can generate both (i)
viral-antigen-specific memory B cells via a Th2 response, and (2) viral-
antigen-specific
memory CD8(+) T cells via a Thl response, the vaccine can reduce the risk and
probability
that a single recombination event or mutational shift, in a pathogenic virus,
would lead to
emergence of a new and virulent strain that cannot be recognized by a
vaccinated
population, and which therefore might spread more rapidly and cause greater
illness,
suffering, and deaths.
OTHER POTENTIAL ENHANCEMENTS
[0002661 After experts who works in this field recognize how the phage vaccine
cassettes
as disclosed herein can be constructed and used, potential enhancements will
begin to be
recognized by such experts, especially when additional articles have been
taken into
account. The identification and creation of such enhancements, after the basic
logic,
methodology, and structure of this new approach to vaccines is learned and
understood,
can be referred to by phrases such as "putting meat on the bones".
[000267 ] As a demonstration, during a period of a few weeks after a meeting
between the
inventor and the patent attorney who drafted this application, while a draft
of the initial
provisional application was being prepared by the patent attorney, the
inventor, with little
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or no assistance, identified the articles mentioned in this subsection,
through a literature
search. Each article cited in this section appears to offer or suggest what
may become an
important enhancement in one or more versions or embodiments of phage cassette
vaccines as described herein. After experts who specialize in immunology have
recognized
how these types of phage cassette vaccines can be created and optimized, they
will
recognize various other, additional enhancements based on articles such as
cited below.
Accordingly, this list merely provides a starting list of potential
improvements that merit
attention, evaluation, and testing.
1. A CpG motif that may be effective for both avian and human vaccines has
been
identified and disclosed. Work with macrophage cells from poultry has shown
that a B/K
type CpG motif designated as "ODN 2006", which is known to have strong
stimulatory
activity in human cells, also strongly stimulates costimulatory molecule
expression in
avian macrophage cells (Xie et al 2003).
2. It has been reported that certain DNA sequence motifs apparently can help
stimulate the transformation and maturation of monocytes and/or macrophages
into
dendritic cells. Gursel et al 2002 reported that D-type ODN sequences could
trigger human
monocytes to mature into functional dendritic cells, while K-type ODN
sequences did not
have the same effect; the reported data also suggested that the cell
maturation effects of the
D-type ODN sequences apparently did not work via TLR9 mechanisms. In addition,
Coban et al 2005 reported that when certain CpG motifs were inserted into
plasmid DNA
that was used for vaccination, PBMC monocyte cells could be stimulated to
develop into
mature dendritic cells.
3. In some situations, the genomes of opportunistic pathogens have evolved in
ways that help those pathogens "fly under the radar" of a host's immune
system. This
usually happens when a mutant strain of a pathogen stumbles across a way to
delete or at
least hide, disguise, or obscure a certain component (such as a pathogen-
associated
molecular pattern, or PAMP, as described in the Background section) that the
pathogen's
hosts use as a way to recognize and identify that pathogen. In a situation of
that nature, it
may be possible to mark and even spotlight that type of pathogen, leading to
efficient
recognition and destruction by vaccinated hosts, by providing a vaccine
component that
provides the component of the pathogen that is missing or obscured.
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[000268 ] As an illustration, since DNA sequences with CpG motifs in a
pathogen's
genome can stimulate active immune responses that will help a vertebrate host
fight off
invading pathogens, there has been selection pressure, on viruses that infect
vertebrate
animals, to reduce the relative frequency of CpG dinucleotides in their
genomes. This has
been described in articles such as Karlin et al 1994 and Kreig et al 1998,
using examples
such as influenza viruses with reduced CpG motifs, as indicated in Table 1 in
Karlin et al
1994. Therefore, as reported in Cooper et al 2004, if conventional flu
vaccines are mixed
with oligonucleotide preparations that contain large numbers of CpG motifs,
the
oligonucleotides will act as adjuvants. The CpG adjuvants call attention to
the presence of
the injected vaccine particles, thereby boosting the immune system and pushing
it into high
gear. The activated immune system will then create an amplified response to
the antigenic
influenza protein sequences carried by the flu vaccines.
[000269 ] Accordingly, if a certain type of "stealth pathogen" has evolved in
a way that
avoids, minimizes, or delays detection by host animals, by a mechanism such as
reducing
the concentration, prominence, or other traits of a molecular pattern that
hosts were using
to identify the pathogen, then that "stealth pathogen" may be highly
susceptible to a
vaccine that specifically includes and utilizes the same type of signaling
agent that the
"stealth pathogen" managed to delete, minimize, or obscure.
4. It should also be noted that a similar concept can be made to work in
reverse, in
a very useful manner, when working with viruses that normally infect only
bacteria and not
mammals. In particular, the dinucleotide content of filamentous bacteriophages
such as
phage M13 evolved in ways that resemble and mimic the dinucleotide content of
E. coli
cells, which are natural hosts for the bacteriophages. That type of phage
evolution allowed
the phages to efficiently use the genetic machinery of their E. coli hosts
(Blaisdell et al
1996). However, certain components of E. coli genomes (including large numbers
of CpG
motifs in their DNA) stimulate immune responses among mammals and other
vertebrate
animals. Therefore, since the genomes of phages that infect E. coli tend to
have similarities
and homologies with E. coli genomes, such phages come already equipped with
CpG
motifs, in their genomes, that will activate the same types of vertebrate
immune responses
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activated by E. coli infections. That is a useful and fortunate factor, which
will help
improve the efficacy of phage cassette vaccines for use in vertebrate animals
(e.g., Frenkel
et al 2004).
5. Phage genomes can be manipulated, in ways that can allow the phage vectors
to
provide an array and assortment of different CpG motifs. Studies have shown
that the
response of monocyte cells, from different human donors, to stimulation by DNA
sequences having CpG motifs, is not always consistent; no single oligo-deoxy-
nucleotide
sequence is maximally stimulatory in all human monocytes (e.g., Klinman and
Currie
2003, and Leifer et a! 2003). Therefore, a mixture of ODN sequences having
different
sequences and activities can be woven into a phage genome. This can be done,
for
example, by exploiting the redundancy of the genetic code, which allows any of
several
different codons to be used to specify a number of amino acids, with no change
in the
amino acid sequence of a resulting protein. The resulting phage cassettes may
be able to
induce the desired immune system activation in the widest possible range of
recipients, in
mixed populations that will have diverse genomes and potentially differing
responses to
such vaccines.
[000270 ] It also should be noted that research into DNA vaccines, which today
mainly
uses relatively short oligo-deoxy-nucleotide sequences that must be chemically
synthesized, to avoid major and costly purification problems, has been stunted
and limited
by the costs of synthesizing the DNA strands used in such vaccines. By
contrast, since
phage cassette vaccines can be manufactured in huge quantities merely by
culturing
phage/bacteria mixtures with a few simple and inexpensive nutrients, the
methods and
approaches disclosed herein may well be able to push research into ODN's, DNA
vaccines,
and CpG motifs, to a much higher level of accelerated and fruitful research.
NALT-TARGETING POLYPEPTIDES IN EUKARYOTIC VIRUSES
[0002711 The third composition of matter disclosed herein comprises vaccines
that contain
targeting-and-delivery polypeptide sequences that were identified by screening
of a phage
display library, regardless of whether the vaccine particles are, or are not,
phage particles.
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If a certain polypeptide sequence, carried by a phage particle that is one out
of millions or
billions of phages in a phage display library, has been discovered and shown
to be highly
potent and effective at triggering both (i) uptake into NALT cells, and (ii)
transport into the
phagosomes of macrophages, then that "target-and-deliver" polypeptide sequence
can be
used in ways that are not limited to vaccine particles made by phages.
[000272 ] For example, a target-and-deliver polypeptide sequence can be
incorporated into
various types of viruses that infect eukaryotic cells, rather than infecting
bacteria. Such
viruses are widely used in vaccines today, in either "disarmed" forms (which
can also be
referred to as attenuated, crippled, etc.) or in "killed" (or inactivated,
nonviable, etc.)
forms. Most "subunit" vaccines, which contain an antigenic polypeptide
sequence from a
pathogen that has been spliced into some other type of carrier-type virus,
also use carrier
viruses that infect eukaryotic cells, rather than bacteria.
[000273 1 These types of vaccines are reproduced in various types of
eukaryotic cells, such
as in bird eggs, insects or caterpillars (or insect cells grown in tissue
culture), or human or
monkey cells grown in cell culture (such as "Vero" cells, a cell line obtained
from vervet
monkeys in the early 1960's, widely used for making vaccines against polio and
certain
other diseases). All of those eukaryotic cell types can "glycosylate" vaccine
particles,
which refers to attaching sugar moieties to protein surfaces. Many pathogens
have sugar
moieties on their surface proteins, since a "cloud of sugar" can help obscure
the nature of a
pathogenic invasion, and give a pathogen a better chance to establish itself
and reproduce
before a victim's immune system can fully respond. Therefore, glycosylated
vaccines can
more closely resemble and mimic the surfaces of a pathogenic microbe that a
vaccine is
designed to defend against. Since bacterial cells normally cannot glycosylate
proteins,
glycosylated vaccines usually must be manufactured in eukaryotic cells.
[0002741 Similarly, bacteria cannot perform some types of protein folding
(i.e., shaping of
a protein strand into a certain three-dimensional shape and conformation), or
other types of
"post-translational processing" performed by eukaryotic cells.
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[000275 ] However, if a vaccine must be cultured and reproduced in eukaryotic
cells rather
than bacteria, this can impede the use of bacteriophage particles as vaccines,
since
bacteriophages normally infect only certain types of bacteria, and cannot
infect animal
cells.
[000276 ] Using genetic engineering methods, it often is possible to find ways
around such
obstacles. For example, eukaryotic cells can be genetically manipulated, to
give them a
foreign gene that will express a new surface protein that will serve as a
binding site (or
docking site, or similar terms) for phages. This can allow phages to enter
eukaryotic cells
which carry those proteins on their surfaces.
[000277 ] Alternately, after a "target-and-deliver" polypeptide sequence has
been identified
by screening a phage display library, that polypeptide sequence can be
incorporated into a
coat protein (or other surface protein) of other types of engineered viruses
that are used to
create vaccines. This approach can be used to create nasally-administered
vaccines, using
engineered viruses that can be cultured and manufactured in eukaryotic host
cells (such as
bird eggs, insects or caterpillars, yeast, mammalian cells, etc.) that will
perform
glycosylation, protein folding, post-translational, or other processing steps
that may be
necessary to give the vaccine a desired activity and potency. When
administered via nasal
spray (which can be a liquid, aerosol, powder, etc.), the "target-and-deliver"
polypeptide
sequence (which initially was discovered in a phage display library, and which
later was
"transplanted" into a virus that infects eukaryotic cells) will cause the
viral vaccine
particles to more readily bind to (and enter) NALT cells in inoculated
animals, which will
then deliver the engineered viral vaccines to macrophages, and possibly to one
or more
other types of phagocytic antigen-presenting cells. Accordingly, a "target-and-
deliver"
polypeptide sequence, when inserted into a type of vaccine virus that can be
manufactured
(and glycosylated or otherwise processed) in eukaryotic cells, can increase
the potency and
efficacy of the resulting vaccine, and can render the vaccine well-suited for
administration
using a nasal spray, rather than a needle.
[000278 1 Accordingly, NALT-targeting polypeptide sequences as disclosed
herein can be
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inserted into vaccines such as influenza vaccines, which are manufactured by
culturing
viruses in eukaryotic cells, such as bird eggs. Flu viruses are among the most
rapidly-
mutating viruses known, and new mutants appear each year that can cause severe
illness
and major epidemics, even among people who have been exposed multiple times to
previous flu infections and/or vaccines. Two major surface proteins of
influenza are
haemagglutinin (HA) and neuraminidase (NA). Therefore, each year, newly-
updated
vaccine strains must be generated, by using genetic engineering to alter a
"master strain"
that is attenuated (i.e., effectively crippled, so that it cannot cause major
problems or
severe illness). Using genetic splicing methods, partial amino acid sequences
from the
haemagglutinin and neuraminidase surface proteins of recently emerged wild-
type strains
that threaten large-scale epidemics are inserted into the attenuated "master
strain". This
creates an attenuated vaccine carrying partial haemagglutinin and
neuraminidase sequences
that are carried by the most threatening wild-type mutant strains.
[0002791 The teachings herein can be adapted for use within that system, by
including (in
the final versions of the influenza vaccine viruses) a "target-and-deliver"
polypeptide
sequence (such as the sequence disclosed herein) that will trigger and promote
the two
immune cell reactions discussed herein, which are: (i) uptake of the vaccine
particles into
NALT cells in the nasal and/or throat region, followed by (ii) transport of
the vaccine
particles into the phagosomes of macrophage cells, which will become antigen-
presenting
cells.
[0002801 The preferred insertion site, for such a NALT-targeting polypeptide
sequence,
can be determined by experts working for a company that manufactures such a
vaccine.
Several companies manufacture such vaccines, and each vaccine is somewhat
different; as
just one example, an annually-updated, nasally-administered flu vaccine is
sold by
Medhnmune, under the trademark NASALMIST.
[0002811 The experts at any company that manufactures a flu vaccine know the
entire
DNA sequence of the genome of the particular "master strain" virus they use as
their
carrier. Based on that genome, they also know the complete amino acid
sequences of all
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surface proteins on their "master strain". From that starting point, it is
within the skill in
the art to select an insertion site, somewhere in the genome of the "master
strain", that will
accommodate a relatively short segment of DNA (roughly 50 base pairs), in a
position that
will cause the altered gene to express a viral surface protein having a
"target-and-deliver"
polypeptide sequence as disclosed herein, in an exposed surface location. In
most cases,
the "target-and-deliver" polypeptide sequence (which typically will comprise a
stretch of
roughly 15 amino acid residues or less) can be used to replace a sequence
having a similar
length, or added as an "epitope tag", presumably near the N-terminus or C-
terminus as
appropriate, in a surface protein of the attenuated master strain, so that the
size of the
modified surface protein will be unaltered, or altered only slightly. This can
avoid creating
surface proteins with substantially altered sizes, which might hinder assembly
of the
modified virus particles.
[000282 J It also should be noted that in any viruses of interest for vaccine
use, dozens or
hundreds of copies of each surface protein are present, on the surface of each
viral particle.
This allows an approach in which two different genes can encode two different
variants of
a single surface protein. One of the two gene variants can be engineered to
create a
polypeptide with a NALT-targeting polypeptide sequence, as disclosed herein.
That
NALT-targeting surface protein (in multiple copies on each virus particle)
will trigger
entry of the vaccine particles into NALT cells, and then into phagosomes in
macrophage
cells. The other gene variant can be used to provide copies of. (i) an
unmodified surface
protein, if desired; or, (ii) a modified protein carrying an antigenic
polypeptide sequence
from a pathogenic microbe, which has been selected because it will trigger the
formation
of antibodies that will enable an inoculated host to mount a rapid immune
response against
the pathogenic microbe.
[000283 ] Other examples of viruses that can infect the upper respiratory
system include
coronaviruses (which includes SARS-CoV, a variant that causes "severe acute
respiratory
syndrome", or SARS), picornaviruses, rhinoviruses, adenoviruses, etc. Most of
these
viruses (other than SARS-CoV) do not cause potentially lethal illnesses, so
most of them
have not received major attention in terms of vaccine development (a notable
exception
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involves adenoviruses, which have been extensively studied and developed for
various
types of gene therapy, largely because they can be used with a class of so-
called "helper
viruses" (also called satellite viruses) that enable various useful techniques
and
safeguards). However, all of the virus types listed above can trigger immune
responses.
Accordingly, any of these types of viruses can be converted into nasally-
administered
attenuated carriers, which can carry exposed surface proteins that will
include: (i) at least
one NALT-targeting polypeptide sequence, which will efficiently deliver
vaccine particles
to antigen-presenting immune cells; and, (ii) at least one antigenic
polypeptide sequence
from a pathogenic microbe, which will be incorporated into the vaccine in
order to trigger
the formation of antibodies that will bind to the pathogen.
[000284 ] Another important class of viruses that can be improved by inclusion
of NALT-
targeting polypeptide sequences are called "nonsegmented negative-strand
viruses"
(NNSV). The term "nonsegmented" means that the entire viral genome is carried
in a
single molecule, of either single-stranded or double-stranded DNA or RNA (by
contrast,
some viruses require two different segments of DNA or RNA to be gathered and
packaged
into each viral particle). The term "negative-strand" (also called anti-sense
strand, or
nonsense strand) means that when a "complementary" copy of the viral DNA or
RNA is
formed by a cell, the "complementary" strand will be the "sense" strand, with
codons that
directly encode a viral protein. In addition, many NNSV viruses are surrounded
by lipid
membranes, usually called "envelopes". These usually are obtained from a host
cell by
means of a "budding" process, in which each virus particle surrounds itself by
an initial
pocket that enlarges into a bubble-type enclosure, made from one or more
membranes of a
host cell.
[000285 1 All of those factors enable NNSV viruses to reproduce more rapidly
than most
other types of viruses that infect mammals. First, a requirement for only a
single molecule
of DNA or RNA, when virus particles are being assembled, leads to greater
speed and
reliability, compared to viruses that must assemble and package two different
strands of
3 0 DNA or RNA. Second, if a virus carries the "negative-strand" of a DNA or
RNA
molecule, then as soon as it "hijacks" the cell machinery and directs it to
make more
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strands of RNA, the newly-formed "positive strand" RNA will have codon
sequences that
will cause the cells to rapidly make more viral proteins. Third, the ability
of a virus to
simply take part of a cell's membrane, rather than needing to make a complete
set of coat
proteins, reduces the number of molecules that must be created and assembled,
to make
more viruses; in addition, viruses that are released by a "membrane budding"
process
usually spare the lives of infected cells, allowing infected cells to make
even more copies
of the virus.
[000286 ] Due to those factors, NNSV viruses tend to pose the most rapid,
acute, and
aggressive pathogenic threats to health. They include, for example, rabies
virus, measles
and mumps viruses, sendai virus, human parainfluenza viruses, vesicular
stomatitis virus,
Newcastle disease virus, human respiratory syncytical and metapneumovirus,
Ebola and
Marburg viruses, and Bora disease virus. NNSV viruses are regarded as posing
the greatest
threats of bioterrorism, and they have received a great deal of attention and
research.
[000287 ]The design and use of NNSV viruses, in vaccines, is reviewed in
articles such as
Bukreyev et al 2006. Since some types of NNSV viruses can efficiently infect
nasal and
throat tissues, those NNSV viruses offer promising vectors for creating
attenuated or
inactivated vaccine vectors, which can be enhanced by inserting NALT-targeting
sequences into one or more of their exposed surface proteins. Such NALT-
targeting
sequences can accelerate and increase the entry of the resulting modified
vaccines into
NALT cells, and then into the phagosomes of macrophage cells.
[000288 ] It should be noted that NNSV vaccines that have been enhanced in
that manner
are not limited to vaccines for preventing NNSV diseases. Instead, NNSV
vaccine vectors
can be modified to include antigenic proteins that will immunize recipients
against
completely different types of pathogens.
[000289 ] In addition, the potency of at least some vaccines created from NNSV
vectors (or
3 0 from coronavirus, picornavirus, rhinovirus, or other viruses) is likely to
be increased even
more, by also incorporating one or more additional components that will
activate one or
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more types of TLR receptors, or that can otherwise help direct and guide an
immune
response toward a desired response, such as either an MHC-1 or MHC-2 response,
or a
TH-1 or TH-2 response.
VACCINES AGAINST CELLULAR PATHOGENS
[000290 1 NALT-targeting polypeptide sequences as disclosed herein also can be
incorporated into vaccines made from bacteria or other cellular (rather than
viral)
microbes. The term "cellular" is used conventionally, to exclude viruses while
including
bacteria, fungi (including yeast cells), mycobacteria, and other microbes that
are regarded
as "cells" by biologists. In general, viruses do not carry the enzymes
necessary to
metabolize nutrients, synthesize DNA or proteins, or make new lipid membranes,
so
viruses must obtain those building blocks from host cells. By contrast, with a
few minor
exceptions (mainly involving "auxotrophic" deficiencies in microbes that grow
only in
environments that supply any missing nutrients), cellular microbes carry the
enzymes
necessary to metabolize nutrients, synthesize DNA and proteins, and make lipid
membranes.
10002911 Because various respiratory diseases involve mucosal tissues in the
nose and
throat, respiratory diseases are likely to be of early interest among
researchers studying the
disclosures herein. Examples of respiratory diseases caused by cellular
microbes include
tuberculosis (caused by Mycobacterium tuberculosis), pneumonia (caused by
Streptococcus pneumoniae, also referred to as pneumococcus), and a disease of
horses
known as "strangles" (caused by Streptococcus equi). Vaccines containing
killed or
attenuated microbes are available for all three diseases, as described in
various articles,
government reports, and websites maintained by vaccine manufacturers. However,
none of
those vaccines are fully optimal, and improved vaccines for any of those
diseases could be
useful and helpful.
[000292 1 The same principle applies to vaccines for other diseases caused by
cellular
pathogens, regardless of whether they infect nasal or throat tissues. No
vaccines are
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perfect, and the disclosures herein can enable improvements in numerous types
of
vaccines.
[000293 ] It also should be noted that two major diseases, malaria and AIDS,
have
frustrated all efforts to develop truly effective vaccines. Accordingly, it is
not asserted or
claimed herein that vaccines developed by the methods disclosed herein can
provide fully
optimal and ideal vaccines for all diseases. Instead, it is asserted that
these methods and
components will enable researchers and companies to create substantially
improved
vaccines for at least some diseases and pathogens. Whether this new approach
can help
create effective vaccines against the two most intractable challenges, malaria
and AIDS,
cannot be predicted as this application is being drafted and filed.
[0002941 Any of several approaches can be used to incorporate NALT-targeting
polypeptide sequences into vaccines against cellular (non-viral) diseases and
pathogens.
Briefly, three major routes include: (1) development of phage particle
vaccines, which will
include NALT-targeting polypeptide sequences identified by screening methods
as
described herein, combined with antigenic sequences derived from cellular (non-
viral)
pathogens; (2) development of viral vaccines derived from viruses (presumably
glycosylated) that normally infect eukaryotic cells, which will include NALT-
targeting
polypeptide sequences, combined with antigenic sequences derived from cellular
(non-viral) pathogens; and, (3) development of killed or attenuated cellular
vaccines,
which will incorporate NALT-targeting polypeptide sequences, and antigenic
sequences
derived from cellular (non-viral) pathogens, into various cell-surface
proteins.
[0002951 Preferably, when researchers attempt to determine the optimal type of
vaccine
against a particular disease caused by a cellular (non-viral) pathogen, all
three approaches
mentioned above should be tried and tested, to prepare different classes of
candidate
vaccines. Each such candidate vaccine class can be tested in animals, to
determine which
class appears to work best, for that particular disease. Candidate vaccines
which show the
most promise in animals can then be tested in humans. Such tests normally
require one or
more rounds of non-pathogen tests, in which no infections, exposures, or
"challenges" by
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pathogens are involved. These tests are used to evaluate a candidate vaccine's
safety, any
unwanted tendencies to provoke allergic, tolerance or other undesired
reactions, and its
ability to trigger the formation of circulating antibodies that will actively
bind to the
pathogen, in in vitro tests. If a candidate vaccine performs well in the
safety tests, it can be
tested for efficacy against actual pathogens, using known procedures (such as
inoculating a
large population of people who are at elevated risk of a certain disease, then
gathering
statistical data on how many inoculated people contracted the disease,
compared to how
many people contracted the disease in an untreated population of the same
size).
VACCINES FOR FIGHTING CANCER AND SIMILAR DISORDERS
[000296 ] As mentioned at several locations above, various types of vaccines
are being
tested and used in the hope that they will be able to help patients fight and
overcome
various nonmicrobial diseases. Cancer vaccines have been extensively
researched, as
described in sources such as Acres et al 2007 and
www.cancer.gov/cancertopics/factsheet/cancervaccine. While some types of
cancer can be
caused by viral infections (such as cervical cancer, caused by papilloma
viruses), most
cancers have no specific microbial cause, and arise from mutations that can
occur when
cells reproduce. In addition, even if a cancer is caused by a virus or other
microbe, the
nature of the disease requires that the cancerous cells must be attacked and
destroyed, and
the initial causative factor has little or no importance after cancerous cells
begin replicating
uncontrollably.
[000297 ] Other nonmicrobial disorders that may someday benefit from vaccine
therapy
include Alzheimer's disease, autoimmune disorders, hormonal or endocrine
disorders, and
other disorders that arise when native cells, body parts, or metabolites go
wrong, rather
than arising from infections by microbes. Information on how vaccines might
benefit such
nonmicrobial disorders can be located easily by searching the U.S. National
Library of
Medicine database, or various Internet databases.
[000298 1 When vaccines for noninfective disorders are involved, the
distinctions between
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MHC1 and MHC2 molecules, and between ThI and Th2 immune responses, will need
and
merit careful attention, because "cell-mediated" Thl responses (rather than
Th2, humeral,
IgG-antibody responses) likely will offer the best routes for treatment. For
vaccines that
are intended to target cancer cells or other native cells (rather than
targeting microbial
invaders), MHC1 receptors and TH1 cells may be usefully involved, and will
deserve close
attention.
[000299 ] It is believed that the methods and reagents of this invention will
enable phage
vaccines to be developed that will be able to specifically target either MHC1-
and TH1-
mediated cells, receptors, and/or processes, or MHC2- and TH2-mediated cells,
receptors,
and/or processes. This will enable the extension of the types of phage
vaccines described
herein, into both: (i) potent and efficient treatments for cancer and other
diseases; and, (ii)
double-pronged approaches that can provided improved defenses against some
types of
microbial diseases.
[000300 ] In general, approaches that can extend NALT-targeting vaccines (as
disclosed
herein) into uses for treating cancer or other nonmicrobial diseases can be
summarized as
follows:
(1) large numbers of phage particles, having a variety of different
polypeptide
sequences in their coat proteins, can be nasally administered, in the form of
a phage
display library suspended in a liquid or powder solution;
(2) the NALT cells in the treated animals will take in, transport, and process
some
of those phages, in certain ways;
(3) those phages that were selected and transported by an animal's NALT cells
can
be isolated and reproduced;
(4) the isolated NALT-targeting phages can be tested, using cell culture
methods, to
determine whether they activated either: (i) MHC1 receptors and TH1 cells, or
(ii) MHC2
receptors and TH2 cells. This can be done, for example, by using assays that
can detect
interleukin-2 or gamma-interferon proteins (which will indicate that TH1 cells
were
stimulated), versus assays that can detect interleukin-4, interleukin-5 or
interleukin-10
proteins (which will indicate that TH2 cells were stimulated).
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[0003011 The foreign DNA and polypeptide sequences carried by NALT-targeting
phages
that activate MHC1 receptors and TH1 cells can then be used as targeting
polypeptides, in
vaccine preparations that use clonal phage particles that are ideally sized
and suited for
such use. These types of anticancer (or similar) vaccines will be used to
deliver selected
antigen protein sequences to T cell types that, when activated, will begin
killing and
destroying cancer or other disease-causing cells.
[000302 1 As mentioned above, those types of cell-mediated immune responses,
by
activated T cells responding to antigenic polypeptides that were presented to
them by
macrophage cells, can be triggered by a sequence of steps such as the
following:
(1) a gene that encodes a known antigenic polypeptide sequence, which will
have
the same amino acid sequence as a cancer-related antigen found in large
numbers on the
surfaces of certain types of cancer cells, is inserted into a NALT-targeting
phage cassette
that activates MHC 1 receptors and TH 1 cells preferentially over MHC2
receptors and TH2
cells;
(2) the resulting anti-cancer vaccine, carrying both (i) a cancer-antigen
protein
sequence, and (ii) a NALT-targeting polypeptide, is administered to an animal
or patient,
via a mucosal mode, such as a nasal spray;
(3) the phage vaccine particles will be transported into NALT tissue, and it
then
will be taken into the phagosomes of macrophage cells, because of the target-
and-deliver
protein sequence inserted into its coat proteins;
(4) at least some of the macrophages will convert into antigen-presenting
cells,
which will travel to lymph nodes and "present" the phage antigens to T cells;
(5) the phage components that were selected and used, in that particular type
of
vaccine cassette, because they activate MHC1 receptors and TH1 cells, will do
their work,
and will steer and guide the immune system into launching a cell-mediated
immune
response, which uses activated T cells, rather than triggering formation of
IgG antibodies
via B cells;
(6) at least some of the activated T cells will bind to cancer cells that
have, on their
cell surfaces, the same cancer-related antigenic protein sequence that was
placed into the
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coat proteins of the genetically-engineered anti-cancer vaccine particles;
(7) after an activated T cell attaches itself to a cancer cell that contains
the same
antigenic protein sequence that was present in the cancer-fighting vaccine,
the activated T
cell will inject perforin into the cancer cell;
(8) the cancer cell's mitochondrial membranes will become permeable, due to
the
action of the perforin from the activated T cells; and,
(9) the induced mitochondrial permeability, inside the cancer cell, will lead
to
mitochondrial release of a signaling molecule called "cytochrome c", which
will trigger a
series of events leading to the apoptotic death and destruction of the cancer
cell.
[000303 ] In summary, by using an appropriate screening test on millions or
billions of
candidate phages in a phage display library, NALT-targeting polypeptides can
be screened
and selected. Particular polypeptide sequences, selected because they
performed efficiently
in those screening tests, can then be incorporated into the coat proteins of
phage vaccines
(or other types of vaccine particles, as described above), by genetic
engineering methods.
This will create "cassette"-type phages, which can then be modified by a final
step to turn
them into cancer-fighting (or similar) vaccines. These "cassette"-type phage
vaccine
vehicles can be supplemented, by inserting into the phage genome an additional
gene
sequence, which will encode (in one of the phage coat proteins) a second
foreign
polypeptide sequence, which will be a known cancer antigen. The resulting anti-
cancer
phage vaccine particles will have: (i) a targeting sequence that makes the
phage particles
efficient in promoting uptake by NALT cells, followed by uptake into the
phagosomes of
macrophages; (ii) one or more components that will steer and guide the
response by the
macrophage cells in a manner that'activates MHC1 receptors and TH1 cells
preferentially,
over MHC2 receptors and TH2 cells; and, (iii) an antigen sequences that will
cause
activated T cells to seek out and destroy cancer cells that have the same
antigen proteins on
their surfaces.
VACCINES AGAINST PEPTIDE HORMONES
[000304 1 Another class of vaccines that can be enabled and/or enhanced by the
disclosures
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herein involve vaccines that will trigger the production of antibodies that
will bind to one
or more types of peptide hormones, or hormone receptors.
[000305 ] In mammals and birds (and likely in many types of reptiles also),
the central
nervous system, the endocrine and paracrine systems, and certain other body
parts use
various types of hormones that are peptides (i.e., formed by linking amino
acids together).
A major reason for the use of peptide hormones is that their concentrations
can be tightly
and reliably controlled, through mechanisms that directly control the
expression levels of
genes. By contrast, nearly all nonpeptide (or "small molecule") hormones (such
as
adrenalin, estrogen, and testosterone, as examples) are made by enzymes that
will
chemically modify any precursor (or "substrate") molecules they encounter.
Because
nearly any such enzyme will convert any and all available precursor molecules
into
product molecules, it is much more difficult to reliably control the quantity
of small-
molecule hormones that are made by enzymes.
[000306 ] In a number of situations, it can be very useful to reduce or
inhibit the activities
of certain peptide hormones. As just one example from human medicine, two
peptide
hormones called "follicle-stimulating hormone" (FSH) and "luteinizing hormone"
(LH)
have been shown to accelerate and worsen brain damage and dementia, caused by
Alzheimer's disease in elderly humans (e.g., US 6,242,421, Bowen 2001).
Therefore, if
vaccines could be developed that would trigger the production of antibodies
that would
bind to (and thereby inactivate) FSH and/or LH (or GnRH, which stimulates the
pituitary
gland to secrete FSH and/or LH), such vaccines might be able to help treat and
minimize
Alzheimer's disease. Similar examples can be provided for various types of
cancer that are
"fueled" by certain types of peptide hormones (including prostate cancer, as
one example).
[000307 ] In animal medicine, several examples merit brief attention. As one
example, in
livestock, a hormone called "gonadotropin" (Gn), which is released under the
control of an
"upstream" hormone called "gonadotropin release hormone" (GnRH), causes
powerful
effects in sexual maturation, and in the cycle of estrus. Therefore, vaccines
have been
developed that trigger the production of antibodies that bind to GnRH. These
vaccines are
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used to control estrus and estrus-related behavior in mares, and as an
alternative to
castration of males (e.g., Thompson 2000; Naz et al 2005; Elhay et al 2007).
[000308 ] As another example, animal growth is moderated and controlled by a
balance
between two offsetting peptide hormones. The term "somatotropin" is a
technical (and
marketing) term for "growth hormone"; the root "somato-" refers to body, and "-
trop"
refers to nutrition, sustenance, and/or growth. The offsetting (growth-
inhibiting) hormone
is called "somatostatin", where "statin" indicates "unchanging" (as in found
in words such
as stay, stable, and stationery). Therefore, if a vaccine could potently
trigger the production
of antibodies that bind to and inactivate somatostatin, animals raised for
food (such as
poultry, hogs, cattle, etc.) might be induced to grow larger, with more muscle
and less fat,
without requiring the injection of any growth hormones. Since injecting food
animals with
growth hormones raises concerns and objections among many consumers, health
care
experts, government agencies, and others, the potential for a simple one-time
vaccine to
produce larger, faster-growing, and more food-efficient livestock merits
attention. Prior
efforts in this field have been described in articles such as Xu 1994;
however, no such
vaccines have yet been developed that are optimal and effective.
[000309 ] Those are just brief examples of how vaccines that would trigger
formation of
antibodies that will bind to peptide hormones might be used, both in human
medicine, and
in agricultural and/or veterinary medicine. Other potential uses are known to
endocrinologists and other specialists.
[000310 ] It also should be noted that vaccines are being developed and tested
for birth and
population control, in humans and in various animals. Such vaccines are
reviewed in Naz
et al 2005, and numerous other sources. For example, using the technology
disclosed
herein, painless nasal administration of a vaccine to dogs or cats may be able
to eliminate
the need for surgical neutering.
[0003111 Another major implication of the hormone-related teachings herein
should also
be noted. Most mammalian hormones (either peptide or nonpeptide) act by
binding to a
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receptor protein that is exposed and accessible on the surfaces of cells. In
some situations,
it may be useful to administer a vaccine that will trigger the production of
antibodies that
will bind to, occupy, and effectively block and inactivate certain types of
hormone
receptors, rather than binding to the hormones. In general, in young patients
who have not
yet reached or passed child-bearing age, those types of vaccines normally
would be used
only in dire circumstances, such as to combat lethal diseases. Among people
who have
passed beyond their child-bearing years, such vaccines might be useful in a
broader range
of therapies.
SCREENING OF PHAGES THAT ENTER BLOOD OR PASS THROUGH
MEMBRANES
[000312 ] The types of screening tests disclosed herein also can be used to
identify
particular phages, from a display library, that will pass through one or more
types of
biological membranes.
[000313 ] For example, during the early stages of this research, the inventor
set out to
isolate and identify polypeptide sequences that could be used to efficiently
carry genetic
vectors and other payloads from nasal cavity into the blood circulation. To
accomplish
that, phage display libraries were nasally administered to mice. At various
times after
intranasal administration, animals were anesthetised, and blood was sampled.
The results
indicated that phages appeared within the blood circulation within 15 minutes,
and were
rapidly cleared from the blood.
[000314 ] Phages in the blood were reproduced in E. coli, and the resulting
phage
populations were tested by additional rounds of nasal-to-blood in vivo
selection. Increasing
numbers of phages were recovered with each selection round, during several
rounds of
testing. When fully-diverse scFv phage were used, initial nasal-to-blood
screening tests
yielded highly variable results. When similar tests were performed using scFv
phage that
previously had been enriched by sciatic nerve screening (as mentioned above,
and
described in more detail in WO 2003/091387), larger and more consistent
numbers of
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phages were obtained.
[000315 ] Accordingly, the phages selected by those types of screening tests
were
demonstrated to pass through endothelial membranes (i.e., the class of
membranes that
form capillaries and other blood vessels); and, the polypeptide sequences
carried by those
phages were shown to efficiently drive the transport of particles through
endothelial
membranes, into circulating blood.
[000316 ] However, even after multiple rounds of in vivo selection and
enrichment, the
number of scFv phage that could be recovered from the blood was only about 10
to 20-fold
higher than observed with a control phage population. This indicated that a
saturation
phenomenon was occurring, which presumably involves a limited number of
portals (such
as M cells) through which phage were passing from the nasal cavity, into
circulating blood.
Despite repeated rounds of in vivo selection, the maximum load of phage
particles that
could be delivered into the blood was estimated to be less than 0.0001 % of
the
administered dose. This posed an inherent limit on the usefulness of this
route, for
systemic delivery of drugs or vaccines, and it helped motivate the Inventor
herein to
redirect his efforts into developing a different type of nasal in vivo
screening strategy,
aimed not at isolating phages that could deliver payloads through M cells into
circulating
blood, but instead, at isolating phage-ligands that would be partially
retained within the
NALT, in a manner that could enable delivery of the phages to antigen-
presenting cells of
a mucosal immune system.
[000317 ] Since intravenous injection (and in some cases skin patches or other
modes of
administration) can introduce any pharmacological agent directly and
efficiently into
circulating blood, and since various approaches are known for directly
controlling the
sustained and prolonged release of drugs, it is not clear how that discovery
would have
substantial commercial or therapeutic value; furthermore, the "apparent
saturation" factor
mentioned above must also be taken into account. Nevertheless, those early
tests are
mentioned herein, partly for the sake of completeness, and partly because
others may be
able to identify and develop practical uses for that discovery.
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[000318 ] The disclosures herein also indicate that screening tests, using
phage display
libraries as candidate starting populations, can be used to isolate and
identify polypeptide
sequences that specifically target GALT cells (i.e., gut-associated lymphoid
tissues).
Because of the importance of food-borne microbes throughout the course of
evolution, the
intestinal tract contains specialized clusters of "lymphoid follicles",
including structures
called "Peyer's patches", located mainly in the small intestines (as an
indicator of their
importance, it has been estimated that 70% of a mammalian immune system, by
volume,
resides in the digestive tract). As with NALT tissues in the nose and throat,
GALT tissues
in the gut are covered by "M cells" having specialized microfolds, which
create expanded
surface areas that enable the M cells to "sample" molecules passing through
the intestines,
and to transfer those molecules or particles that appear unusual (such as
molecules with
"pathogen-associated molecular patterns", or PAMPs, described in the
Background
section) to immune cells that reside or travel beneath the outer layer of M
cells.
[000319 ] In general, unless and until experimental data indicates otherwise,
it is presumed
herein that NALT-targeting vaccines are like to be more efficient than
comparable orally-
ingested GALT-targeting vaccines. GALT-targeting vaccines will need to be
specially
formulated to survive stomach acidity, presumably by placing them in carriers
that will
release the vaccine particles only after the vaccine reaches the small
intestines (one such
carrier, developed by students working with Prof. Hai-Quan Mao at Johns
Hopkins
University, has been announced for a rotavirus vaccine being developed by
Aridis
Pharamaceuticals, www.aridispharma.com). However, even in such cases, the
vaccine
particles will be mixed with relatively large quantities of food that is being
digested and
converted into feces, in the intestines, and that natural process will
inevitably reduce the
contact and uptake of the vaccine, compared to a nasally administered vaccine
that can be:
(i) emplaced directly on NALT surfaces, in the nasal sinuses and (ii) held in
position for
sustained times by a mucoadherent compound, as described in the Background
section.
[000320 ] Nevertheless, several major research efforts are underway to try to
develop
genetically engineered plants (or microbial feed additives) that will create
proteins that,
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when eaten by poultry, livestock, and possibly humans, will effectively serve
as vaccines
against certain diseases. Accordingly, NALT-targeting and/or GALT-targeting
transport
polypeptide sequences can be merged and combined with such efforts, to utilize
the potent
and specialized transport activities of such polypeptide sequences.
[0003211 Because of the close similarities between, and the essentially
identical functions
of, NALT tissues in the nasal and throat regions, and GALT tissues in the
intestines, it is
likely that the same polypeptide sequences disclosed herein which drive NALT
intake
followed by APC intake and processing, will also drive GALT intake followed by
APC
intake and processing. Alternately or additionally, analogs (described below)
of any
NALT-targeting polypeptide sequence, having limited amino acid substitutions,
can be
evaluated for potent GALT uptake activity.
[0003221 If other polypeptide sequences that will potently drive both GALT
uptake and
APC phagocytosis are desired, such sequences can be isolated and identified by
using
screening tests that are directly comparable to the NALT screening tests
described herein,
modified in ways that will involve direct contact with GALT cells, rather than
NALT cells.
In general, instead of administering a phage library (with millions or
billions of candidate
phages) via nasal spray, a phage library can be fed to lab animals, using a
suitable carrier
system (such as the carrier mentioned above, for a rotavirus vaccine that will
be taken
orally). After a suitable time, the animals will be sacrificed, and Peyer's
patch tissues (or
similar intestinal tissues, or possibly certain types of "downstream" tissues
or cells, such as
macrophages in lymph nodes that serve the digestive tract) will be harvested.
The cell
membranes will be lysed, using a buffer that does not damage the coat proteins
of the
phages (as described for the NALT screening tests), to harvest enriched
populations of
phages that were preferentially taken into the Peyer's patch or other GALT
cells. If
desired, a phage library can be labeled, using FITC or other labeling agents,
to make the
tracking and harvesting steps easier or more efficient.
[000323 1 That type of screening process can be repeated as many times as
desired, and the
resulting enriched phage populations can be screened again, using macrophage
association
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and/or phagosomal entry screening tests, as described herein.
[000324 ] Accordingly, if desired (for example, if a NALT-targeting
polypeptide sequence
that functions in a certain class of animals does not also function optimally
for GALT
intake, in that same class of animals), a sequential combination of screening
tests, as
described above, can be used to identify one or more phages, from a phage
display library,
that will potently drive both steps of a two-step process: (i) intake into
GALT cells in the
intestines, followed by (ii) phagocytic intake, by antigen-presenting cells.
[000325 1 If desired, in vitro screening tests can also be used, in one or
more assays
designed to identify enrich any GALT-targeting polypeptide sequences. For
example,
O'Mahony et al 2004 describes the use of a tissue culture method for screening
a phage
display library, using "CACO" cells from a transformed cell line that was
initially derived
from a colon cancer tumor. CACO cells (which are anchorage-dependent) can be
grown
into a cohesive layer which will have an "apical" side (which normally would
be exposed
to semi-digested food passing through the intestines), and a "basal" side.
O'Mahony et al
contacted the apical sides of their CACO cell layers with phage populations,
and selected
phages which passed through those cell layers.
[000326 ] Those tests did not involve vaccine testing or development, and no
one should
rely on the presumption that transformed (cancerous) cell lines will behave
identically to
healthy cells or non-tumorous tissues. Nevertheless, that report can be
consulted for
information on how in vitro tests have been performed using cell lines derived
from
intestinal tissues.
POLYPEPTIDE ANALOGS FOR TARGETING-AND-DELIVERY USE
[000327 1 Any DNA or polypeptide sequence disclosed herein, and any other DNA
or
polypeptide sequence hereafter discovered to have potent targeting-and-
delivery activity
for vaccine use, can be used as a starting sequence (which can also be called
a baseline,
initial, or reference sequence, or similar terms), in efforts to find analogs
having
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comparable or even higher potency.
[000328 ] In conventional chemistry and pharmacology, an analog is a molecule
that
resembles a certain designated molecule (which can be called a baseline,
starting, initial,
reference, or referent molecule or compound, or similar terms), but which has
been
modified by substituting or altering one or more groups or substituents of the
starting
molecule. For example, if a molecule has a relatively small "moiety" (i.e., an
atom or
cluster of atoms, such as a hydrogen, sulfur, or halogen atom, or a hydroxyl,
amine,
methyl, or similar small group) at a specific location on the compound,
analogs can be
formed by replacing that moiety with various other atoms or small groups, or
by moving a
moiety to a different location on the molecule. Similarly, saturated bonds can
be replaced
by unsaturated bonds, and various other limited modifications can be made, to
create
molecules that are similar but not identical to a starting compound. Such
analogs can then
be screened, to determine whether any of them have a comparable (or improved)
level of a
desired activity, compared to the starting molecule. If a particular analog is
discovered to
offer a significant improvement, it can then be tested and studied more
closely, and it also
can be used as a new starting or baseline molecule, in subsequent efforts to
develop even
better analogs.
[000329 ] Most research labs which do this type of work use computerized
machines with
transport mechanisms that will sequentially deliver dozens or even hundreds of
small
containers (such as glass vials, wells in a multi-well plate, etc.) to one or
more
sophisticated detector devices. Each vial, well, or other container holds a
separate sample
(or "aliquot") of some test compound, usually created by an assay of some
sort, which
enables a product formed by some reaction or series of reactions to be tested.
The detector
device will analyze each aliquot, in turn. In some cases, this is done by
taking and
processing a sample of liquid from each container; in other cases, it is done
by
nondestructive means, such as by shining a light having a certain wavelength
(or range of
wavelengths) through each container, to measure one or more factors such as
turbidity or
color intensity (or to create a graph showing various peaks, as a function of
differing
wavelengths). The results of each test will be recorded in a computer, with
identifying
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numbers to correlate each analytical result with a specific container and
aliquot. The
computer software can even rank the output data, so that the best-performing
analogs can
be quickly identified and ranked.
[000330 ] These types of programs and machines, to create and test large
numbers of
analogs with minimal effort, expense, and delays, have become a standard part
of
biochemical research, and the types of automated machinery that enable "high-
throughput"
testing of large numbers of analog compounds are described in articles such as
Muller et
al, Catalysis Today 81: 337 (2003). In the specific field of polypeptide
chemistry, analogs
of a starting polypeptide are created, by altering one or more amino acid
residues in the
amino acid sequence of a starting (or initial, baseline, etc.) compound. Such
alterations can
take the form of: (i) substituting (or "swapping") one or more specific amino
acid residues,
without altering the number of residues in a sequence; (ii) inserting one or
more additional
amino acid residues into a known sequence, in a way that increases the number
of amino
acid residues in a sequence; or, (iii) deleting one or more amino acid
residues, to decrease
the number of residues in the sequence. These types of alterations can be
created by well-
known methods, such as by using automated machinery to create synthetic
segments of
DNA, which can be spliced into a known restriction site in a gene that encodes
a coat
protein, in a plasmid and/or phage. The synthetic segments of DNA can have
either: (i)
exact and known sequences, in "controlled mutagenesis"; or (ii) random and
assorted
sequences, in "random mutagenesis". The resulting modified genes can then be
expressed
into polypeptides (by using phage vectors, microbial fermentation, or other
methods), and
the resulting phages or polypeptides can be tested, using cell culture, small
animals, etc., to
determine which particular polypeptides happen to have the strongest levels of
activity,
using any screening test that is of interest.
[0003311 Accordingly, this invention anticipates that analogs can be prepared
and tested,
using any DNA or polypeptide sequence that is disclosed herein (or that is
hereafter
discovered to function potently as a targeting-and-delivery sequence, when
used as
disclosed herein), as a starting sequence. If some particular analog sequence
is found to be
more potent for the purposes disclosed herein than the starting sequence it
was derived
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from, then that analog may rise to the level of a patentable improvement;
nevertheless, if it
is created and tested as an analog of a known sequence, using processes such
as controlled
or random mutagenesis following by screening tests as disclosed herein, then
any such
analog that arises from the teachings herein is within the scope of this
invention.
[000332 ] The present invention is now described by the following non-limiting
Examples.
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EXAMPLE 1: SUPPLIES AND METHODS
[000333 ] Adult male BALBc mice were used for in vivo selection studies.
[000334 ] A phage display library, believed to contain 1.3x1010 individual
recombinants,
each containing a single chain variable fragment (scFv) gene sequence derived
from
human B-cells, and a CANTAB6 control phage lacking an scFv insert, were
obtained from
Cambridge Antibody Technology (United Kingdom). These phages carry an
ampicillin
resistance gene, as well as a plasmid origin of replication.
[000335 ] In vivo selection of the scFv phage library, to select and isolate
phages that were
taken into and retrogradely transported by nerve fibers in the sciatic nerve
bundle in rats, is
described in PCT application WO 2003/091387. In the discussion herein,
preselected
populations of phages that were used during specific steps are described by
phrases such
as, for example, "sciatic (18hr)Idiverse scfv", which refers to a line of
phages that were
isolated from rat sciatic nerve tissue which was harvested 18 hours after
administration of
a scFv phage library to the sciatic nerve at a different location.
[000336 ] A second type of "peptide display" phage library was obtained from
George
Smith (University of Missouri, USA). It is believed to contain approximately
108 different
clonal phages, and is derived from a filamentous phage designated as fd-tet,
which carries
a tetracycline resistance gene (as a selectable marker), and an origin of
replication that
allows the phage genome to be manipulated and reproduced in double-stranded
DNA form,
as a plasmid. These phages also are known as "type 88" phages (Smith 1993),
since their
genome (9273 bases) carries two different genes that encode coat protein VIII.
One of the
two protein VIII genes carried by "type 88" phages is a wild-type (unmodified)
gene, while
the other coat protein VIII gene carries a foreign gene sequence that encodes
a "15-mer"
polypeptide (i.e., an inserted polypeptide sequence containing 15 amino acid
residues,
spliced into the normal amino acid sequence of the phage's coat protein VIII).
This vector
is also referred to as a f88-15mer phage library (GenBank accession number
AF246448).
The random 15-mer polypeptide sequences typically are expressed at up to about
300
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copies per phage.
[000337 ] When appropriate, f88-15mer phages that were selected by some
particular
screening round as described below were reproduced ("amplified") in the K91Kan
strain of
E.coli, a lamba-derivative of a strain known as K-38. Unless transformed by a
phage or
plasmid carrying a resistance gene, K91 cells are susceptible to the
antibiotics kanamycin
(used at 50 micrograms/milliliter, ug/ml) or tetracycline (20 ug/ml). In
addition, an
inducible promoter was placed in front of the gene that encodes coat protein
VIII; this
allows a compound known as IPTG (1 mM) to be used, when desired, to increase
expression levels of recombinant coat proteins containing the various 15-mer
foreign
inserts that have been inserted into the coat protein VIII coding sequence.
[000338 ] The 15-mer phage library, or the scFv phage library, were cultured
and
reproduced, respectively, by methods described by George Smith (e.g., Smith
1993), by
Cambridge Antibody Technology (described in their guides to users), and by
references
such as Bonnycastle et al 2001.
[000339 ] For example, when a round of in vivo phage selection using the scFv
phage
library was completed by means of the sciatic nerve method, the harvested
phages (which
can be released from the neurons by mechanical or ultrasonic homogenization
and/or
dissolution of the nerve cell membranes using a detergent) were incubated for
1 hour with
a TG-1 strain of E. coli, in their log growth phase, in 2TY cell culture
medium. The phage-
infected E. coli cells were grown overnight in shaker flasks, in 400 ml of 2TY
medium
containing 100 ug/ml ampicillin. Infected E. coli (carrying phages with
ampicillin
resistance genes) were pelleted by centrifugation at 3000 rpm for 20 min, then
taken up in
10 ml of 2TY medium, and a 10-fold MOI of helper phage M13KO7 carrying a
kanamycin
resistance gene was added. After 60 min, doubly-infected E. coli were added to
800 ml
2TY medium containing 100 ug/ml ampicillin and 50 ug/ml kanamycin, and grown
for 24
to 48 hours at 37 C in a shaker flask at 300 rpm to allow secretion of phage
into the
medium. The E. coli were then removed by centrifugation at 10,000 rpm for 30
min. 20%
v/v of 16.7% polyethylene glycol (PEG) with 3.3 M NaCl (Bonnycastle et al
2001) was
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added to the supernatant, to precipitate the phage overnight at 4 C. The PEG-
precipitated
phage were pelleted at 10,000 rpm for 30 min, then dissolved in 40 ml of
Dulbecco's
phosphate buffered saline (PBS), pH 7.4, and centrifuged at 4000 rpm for 20
min to clear
the phage solution of any undissolved phage or other particulate matter. PEG
precipitation
was repeated at least two more times, prior to aliquoting the phages into
sterile screw-cap
vials. Phage were stored as PEG precipitates at 4 C until subsequent use.
Phages from
f88-15mer were produced using similar methods, using LB medium with 12.5 ug/ml
tetracycline in place of ampicillin, and without using helper phage. Phage-
infected host
cells in PBS or 2TY medium were stored at -80 C after mixing 1:1 with cell
freezing
medium, which contained 88 g/L glycerol, 12.6 g/L K2HPO4, 3.6 g/L KH2PO4, 1.8
g/L
(NH4)2SO4, 0.9 g/L Na3citrate, and 0.18 g/L MgSO4-7H20).
[000340 ] When a need arose to produce still larger quantities of phage, a
culture of TG1 E.
coli cells in 2TY medium was grown to logarithmic growth phase, then infected
with scFv
phages (either from the complete diverse library, or from a previously
selected population).
The phage-exposed cell population was inoculated into 800 ml of 2TY containing
ampicillin, and grown overnight in a shaker flask. The next morning, phage-
infected E.
coli were pelleted by centrifugation at 3000 rpm for 20 min, and the cell
pellet was taken
up in 10 ml of 2TY medium. M13KO7 helper phages were added, and allowed to
infect
the cells for 1 hour. To produce phage, infected host cells were grown in a 2L
shaker flask
containing ampicillin and kanamycin supplementing 800 ml of medium with a
recipe
based on Liu et al 2000; in addition to 40 g/L D-sorbitol as a carbon source,
this medium
contained 5 g/L yeast extract, 5 g/L tryptone, 7g/L NaH2PO4-12H20, 4 g/L
KH2PO4, 4 g/L
K2HPO4, 1.2 g/L (NH4)2SO4, 0.2 g/L NH4C1, 2.4 g/L MgSO4-7H20, and 0.02 g/L
CaCl2.
EXAMPLE 2: PURIFICATION OF PHA GE USING CERAMIC HYDROXYAPATITE
[0003411 Affinity column purification of phages was carried out using
particulate
hydroxyapatite (HA), a ceramic material containing calcium and phosphate,
purchased
from BioRad. Initial studies, using buffer recipes described in Smith and
Gingrich 2005 for
use with other types of hydroxyapatite affinity media, failed to achieve
desired purification
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when particulate HA was used with the phages, so a trial-and-error series of
tests were
performed to adapt the buffers and methods of Smith and Gingrich 2005 for use
with the
HA-phage combination of the inventor. Briefly, a 4.5 ml HA column was prepared
using
2.5 gm HA. 5 ml of a phage preparation, at a density of about 3 mg/ml in 20 mM
maleic
acid (pH 5.5) containing 2 mM CaCl2, was loaded onto the column under gravity
percolation. Subsequent elution buffers were prepared using 400 mM
NaH2PO4.2H20,
adjusted to pH 7.0 using NaOH.
[000342 ] A series of elution buffers was then passed through the loaded
column, using 1
ml aliquots of elution buffer, and collecting 1 ml liquid fractions that
emerged from the
column for analysis, using elution sequences such as listed below, which
provided good
results:
Fractions 1-10: 20 mM maleic acid, pH 5.5, plus 2 mM CaCl2
Fractions 11-20: 20 mM MOPS, pH 6.5
Fractions 21-30: 100 mM NaH2PO4, adjusted to pH 7.0
Fractions 31-40: 100 mM NaH2PO4, pH 7.0 plus 2.55 M NaCl
Fractions 41-50: 100 mM NaH2PO4, pH 7.0
Fractions 51-70: 250 mM NaH2PO4, pH 7.0
Fractions 71-80: 1M NaH2PO4 + 150 mM NaCl
[000343 ] Protein content of the eluted fractions were optically monitored at
a light
wavelength of 280 nanometers (nm), in part because maleic acid buffer absorbs
light
strongly, at wavelengths below 280 nm. The elution profiles were generally
similar to
those reported in Smith and Gingrich 2005, and highly purified phages eluted
in fractions
60 through 70, as illustrated in FIG. 10.
EXAMPLE 3: USE OF ANTI-PHA GE ANTIBODIES
[000344 ] To prepare antibodies that would bind to the phages, two sheep were
immunized
with phage. After a suitable delay, blood was sampled, serum was isolated, and
the serum
(containing IgG antibodies) was precipitated with 50% ammonium sulfate. The
antibodies
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were resuspended and passed through an affinity column containing immobilized
phages.
[000345 ] To immobilize phages in the affinity column, ethylene dichloride
(EDC) was
used for crosslinking. 10 mg EDC was added to 1 ml 40 mM NaH2PO4 (adjusted to
pH 7.0
by NaOH) containing 16 mg purified CANTAB6 phages. The mixture was allowed to
react for 4 hours at room temperature, then another 10 mg of EDC was added,
and the
mixture was incubated (with mixing) for another 4 hours. The EDC-crosslinked
phage
were precipitated overnight using polyethylene glycol, then dissolved in 5 ml
of coupling
buffer (0.1 M NaHCO3, pH 8.3, plus 0.5 M NaC1). In parallel, 1 gm of CNBr
Sepharose
4B was washed with 1 mM HC1, then added to the coupling buffer containing
EDC-crosslinked phages. The mixtures was incubated for 4 hours, then quenched
with
0.1M Tris (pH 7.6) and incubated overnight. Unbound phage and coat protein
components
were removed, using 4M MgC12 + 50 mM acetate, pH 5Ø
[000346 ] When the blood serum fraction (enriched in IgG by ammonium sulfate
precipitation, and processed to remove red and white blood cells from the
phage-injected
sheep) was passed through the affinity column, antibodies with phage affinity
remained
bound to the immobilized phages in the column, while other molecules were
washed out of
the column. A strong elution buffer (4M MgC12 plus 50 mM acetate, ph 5.0) was
then
passed through the column, to release and remove the phage-bound antibodies.
[000347 ] To prepare ELISA (enzyme-linked immuno-sorbent assay) plates, 20 ug
of
purified antibodies were mixed with an ELISA plate-coating buffer (0.5 M
carbonate
buffer, pH 9.6). The resulting mixture was coated onto Costar vinyl ELISA
plates.
[000348 ] ELISA assays, to determine the concentration of phages in a liquid
being
analyzed, involved: (i) a first incubation step, using a body fluid that was
being measured
to determine its phage concentration, followed by, (ii) a second incubation
step, using
wild-type phages crosslinked to a peroxidase enzyme. Unbound enzyme-linked
phages
were rinsed away and removed after the second incubation, and a liquid with a
color-
forming reagent was added to the plate. The color-forming reagent was
converted into a
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colored compound by the peroxidase enzyme, and its intensity was measured by a
spectrophotometer. A weak color change indicated that most of the anti-phage
antibodies
had become bound and occupied, by a high concentration of phage particles
(with no
peroxidase enzyme) in the body fluid, before the second incubation was carried
out using
phages carrying peroxidase enzymes. By contrast, a strong color change
indicated that
fewer anti-phage antibodies had been occupied by phage particles from the body
fluid,
thereby indicating a lower concentration of such phage particles.
EXAMPLE 4: IN VIVO SCREENING METHODS
[000349 ] To administer phage preparations to the nasal cavities of mice,
aliquots of PEG-
precipitated phage libraries were dissolved in sterile saline, and the phage
density of each
such solution was determined using spectrophotometry (Bonnycastle et al 2001).
When
necessary, a solution was adjusted by dilution with PBS, so that each mouse
would receive
about 2 x 10(11) phage particles. Each mouse was briefly anaesthetized with
halothane
vapor, and a pipette was used to blow a 2 uL aliquot of phage solution, via
each nostril,
into the nasal cavity. The animal was kept warm until it awoke from
anesthesia, then
returned to its cage. Any animals with nose bleeds (which were rare) were
euthanized and
removed from the study.
[000350 ]After a pre-determined time (which varied, depending on the test and
the type of
tissue being analyzed), a treated mouse was deeply anesthetized using
halothane,
euthanized, and its chest cavity was opened. If blood was to be analyzed, a
tuberculin
syringe containing heparin was used to remove 200 to 300 ul of blood from the
heart, for
transfer to pre-weighed microfuge tubes on ice containing 200 uL of lysis
buffer
containing 1 % Triton X-100, 10 mM Tris at pH 8.0, and 2 mM EDTA, which lysed
the
blood cells and released the phages. Tubes were weighed to determine the
volume of the
sample, and aliquots of the blood-bore phages were added to TG-1 E. coli cells
in log
growth phase (optical density approximately 0.2, at 600 nm). After 1 hour of
incubation,
the preparation (or a dilution thereof) was spread evenly across the surface
of agar plates
containing 2% glucose and 100 ug/ml ampicillin (200 ml of agar, in 234 mm x
234 mm
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Nunc tissue culture plates). The plates were sealed with parafilm and
incubated overnight
at 30 C. By 18 hours, typical colonies of E. coli cells infected by phages
carrying the
ampicillin resistance gene had grown to 0.5 to 2 mm diameters, without
evidence of
secondary colonies. Plates were then refrigerated, if necessary, at 4 C, and
the number of
colonies per plate were counted within 24 hours. The number of phage in each
blood
sample was determined from at least two titering tests, to calculate the mean
number of
phage recoverable per animal, when blood samples were tested.
[0003511 In some cases, colonies of phage-infected E. coli also were scraped
from the agar
plates, transferred to 2TY culture medium, and mixed with an equal volume of
glycerol-
based cell freezing medium, as listed above. Aliquots of the phage-infected E.
coli were
stored at -80 C. Glycerol scrapes from 30, 60 and 120 minute time points also
were
pooled, and used to generate phages for subsequent rounds of nasal-to-blood in
vivo
selection.
[000352 ] If tissue samples were to be analyzed, PBS was injected (perfused)
into the aorta,
using a syringe, to displace blood and rinse out blood-borne phages before the
tissue
samples were removed and phages were isolated.
EXAMPLE 5: IN VIVO SELECTION OF PHA GES THAT TARGET NALT CELLS
[000353 ] Sixty minutes after intranasal administration of phages, 10 mice
were
anesthetized, euthanized, and perfused with saline, and their olfactory bulbs
(OB) were
removed. Similarly, 45 minutes after phage administration, NALT tissue
flanking the
windpipe (Asanuma et al 1997) was removed from 9 mice. The OB or NALT tissue
was
dissected into 100 ul of cell lysing buffer and triturated, pooled, and given
two sonication
pulses, 1 second each. An equal volume of 2X stock of LB culture medium was
added,
then a sufficient quantity of LB culture medium carrying K91 E. coli cells in
logarithmic
growth phase was added to make 10 mL. After allowing 60 minutes for phage to
infect the
cells, cell broth was plated onto 22 x 22 cm LB agar with tetracycline, and
incubated
overnight. Glycerol scrapes of clonal colonies were prepared for production of
phage for
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additional tests, and for storage at -80 C.
EXAMPLE 6: PHA GE SELECTION USING PBMC WHITE BLOOD CELLS
[000354 ] A class of white blood cells from humans, known as peripheral blood
mononuclear cells (PBMC's), were separated by density-gradient centrifugation
of
heparinized blood, using Lymphoprep (Nycomed, Oslo, Norway). 10 ml of healthy
human
venous blood was collected into a heparinized syringe, and was diluted with 18
ml of
Dulbecco's PBS in 2 x 25 ml sterile flat-bottom specimen jars. 10 ml of
Lymphoprep
(Nycomed, Oslo, Norway) was gently delivered beneath the diluted blood layer,
using a 10
ml syringe fitted with 22 gauge catheter needle (Optiva Code 5060). Tubes were
centrifuged at 800 rpm for 25 min. The "buffy coat" layer (approx 3.5 ml) was
transferred
to a centrifuge tube, diluted to 15 ml with Dulbecco's PBS, and centrifuged at
1,600 rpm
for 15 min, to pelletized blood cells. Supernatant was removed by aspiration,
and the pellet
was resuspended in 15 ml of Dulbecco's PBS and recentrifuged. The washed
pellet was
resuspended in RPMI 1640, supplemented with 2 mmol/L glutamine, 100 ug/mL
penicillin, 100 ug/mL streptomycin, and 10% fetal calf serum (FCS), from GIBCO-
BRL
(Gaithersburg, MD) at 37 C. The mixture was diluted to 2x 10(6) cells/ml.
Viability of the
cells was >95% as determined by trypan blue exclusion.
[000355 J In separate microtubes, 20 ug of a 1 ug/ul suspension containing
either of two
types of phages were added to 500 ul batches of PBMC cell suspensions. One
batch, which
served as a control, contained fd-tet phages which had been fluorescently
labeled using
fluorescein isothiocyanate (abbreviated as FITC, from Sigma Chemical Company,
St.
Louis, Missouri). The other batch contained a subset of the 15-mer phage
library that had
been screened once for olfactory bulb (OB) targeting, and then screened twice
for NALT
targeting, by removing and isolating them from OB tissue and then NALT tissue
in mice,
after nasal administration, as described above; those active test phages were
also
fluorescently labeled, using FITC. The different test and control mixtures
were prepared in
3 0 microfuge tubes, mixed, and incubated for 30 min at room temp. Each
cell/phage mixture
was then diluted with 10 ml Dulbecco's PBS, centrifuged at 5 minutes at 2000
rpm, and
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resuspended in 2 ml Dulbecco's PBS. 5 uL aliquots were then removed and placed
on 4%
gelatin-coated slides, which were cover-slipped, and examined under a
fluorescent
microscope. Visual inspection indicated that labeling of about 5 to 20% of the
PBMC cells
clearly exceeded background levels.
[000356 ] To isolate a subpopulation of FITC-labeled phages from the OB/NALT-
selected
test populations that were most avidly taken up by PBMC cells, FITC-phage-
labeled
PBMC pellets were resuspended in 2 ml Dulbecco's PBS, and processed through a
BD-FACSAria Flow Cytometer (Becton-Dickinson AG, Basel, Switzerland) equipped
with 405 nm, 488 nm, and 633 nm lasers. Cells containing varying levels of
fluorescent
labeling, from adhering or ingested FITC-labeled phages, were identified at
488 nm, and
the control settings of the machine were adjusted to separate and capture
cells that ranked
in the top 3% of fluorescent intensity levels.
[000357 ] That population of strongly-labeled PBMC cells (approximately 5x
10(4) cells)
was suspended in 1.5 ml of culture medium. 1.5 ml of lysis buffer was added
and
incubated, to digest the cells without damaging the phages. The resulting
selected phages
were then used to infect K91 E. coli cells in logarithmic growth phase. After
allowing 1
hour for infection, cells were pelleted (10 min at 3,500 rpm), resuspended in
LB medium,
and plated on agar containing tetracycline, to select for cells infected by
phages carrying
the tetracycline resistance gene. After overnight incubation, numerous
colonies were
observed. Those colonies of phage-infected E. coli were expanded, and used to
produce
phage populations which were then screened for phagosome selection, as
described below.
[000358 ] Since those selected phage from the 15-mer phage library had passed
through
one round of selection in olfactory bulb tissue, two rounds of selection in
NALT tissues,
and a fluorescent-activated cell sorting (FACS) screening round, they were
designated as
FACS NNALT INALTIOB 115mer phages.
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EXAMPLE 7: PHA GE SELECTION USING MACROPHAGES AND PHA GOSOMES
[000359 ] As mentioned in the Background section, an important class of white
blood cells
passes through series of stages. They are called "monocytes" when circulating
in the blood,
in relatively compact form. They have special surface molecules that cause
them to grip
and permeate through capillary walls, causing them to leave the blood and
enter the lymph,
which slowly moves through soft tissues. In the lymph, they swell to a larger
size, and are
called macrophages, phagocytes, or phagocytic cells. If they encounter a
foreign microbe,
they will extend out projections, often called fingers, "pseudopods",
dendrites, etc., which
will surround the microbe. The cell will partially digest the microbe, using
internal
organelles called phagosomes. It will then position fragments of the digested
microbe on
the surface of the dendrite cell, in ways that "present" the foreign protein
to other cells of
the immune system. From this simplified description, it can be seen that a
cell which goes
through those stages can be called a monocyte, a macrophage or phagocyte, a
dendrite or
dendritic cell, and an antigen-presenting cell (APC).
[000360 ] Clearly, this class of cells is very important to the immune system.
Therefore, a
round of screening was used to identify particular 15-mer phages which showed
high
levels of activity in initiating and driving those processes, in human
macrophages. This
round of screening began with FACSINALTINALTIOBI15mer phage populations which
already had been identified and selected by four rounds of screening tests, as
described
above.
[0003611 The methods used were derived in part from starting information
described in
Luhrmann and Haas 2001, and Ramachandra et al 1998. Washed PBMC from 20 ml
blood
were prepared as above, and were taken up in 3 ml of Dulbecco's modified
Eagle's
medium (DME), supplemented by a standard cell culture mixture called Ham's
F12, and
by 10% fetal calf serum (FCS). The cell suspensions were divided between two
T25 flasks,
and were allowed to settle and attach to the bottom surfaces of the flasks,
for 30 minutes at
37 C.
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[000362 ] That attachment process is important, since it arises from the same
surface
molecules that enable certain monocytes to grip the interior walls of
capillaries, and then
pass through the capillaries, which is a crucial step in the conversion of
some monocytes
into macrophages. After 30 minutes of incubation, the culture medium was
removed, and
the cell layer that adhered to the flask surface was washed 5 times, using 2
ml volumes of
Dulbecco's PBS at 37 C, to remove and discard any monocytes or other white
blood cells
that had not adhered to a plate surface.
[000363 ] Suspensions of FACSINALTINALTIOB115mer phages, selected as described
above, were added to each culture plate (20 ug/ml of phage in 2 ml of
DME/F12/10% FCS,
estimated to contain 7.3x10(10) virions in each batch). After 30 minutes
incubation at
37 C, the liquid medium was removed, the cell layer was washed twice, using 2
ml
volumes of PBS at 37 C. Fresh DME/F12/10%FCS was added, and the cell/phage
mixture
was returned to the incubator. After 30 minutes, the liquid medium was
removed, and the
cell layers were washed twice with 2 ml Dulbecco's PBS at 37 C.
1000364 1 To release the cells from the surfaces of the flasks, a solution of
trypsin and
EDTA (catalog 15400-054, GIBCO) was added and spread across the flasks. After
4
minutes, 5 ml of DME/F12/10% FCS was added. The cells were gently scraped off
with a
rubber spatula, and poured into 15 ml centrifuge tubes. The flasks were rinsed
with 5 ml of
DME/F12/10% FCS, which was added to the centrifuge tubes. The cells were
centrifuged
at 1,600 rpm for 10 min at 4 C. Supernatant was discarded, and the pellet was
resuspended
in 10 ml DME/F12/10% FCS and centrifuged again. Supernatant was discarded, and
cells
taken up in 2 ml of 0.25M sucrose buffer with 10 mM HEPES, pH 7.2.
[000365 ] The cells were transferred to a 1 ml conical glass-glass homogenizer
(Wheaton-USA), and broken apart by 10 strokes of the homogenizer. The
homogenate was
then transferred to a centrifuge tube; the homogenizer also was rinsed twice,
using 2 ml of
sucrose solution each time, and the washings were added to the centrifuge
tube, making a
3 0 volume of 6 ml. The homogenate and washings were centrifuged at 900 rpm
for 10 min at
4 C, to pellet any undisrupted cells and cell nuclei, which were discarded.
The supernatant
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was transferred to another tube and centrifuged at 3,500 rpm for 10 min at 4
C, to pellet
the phagosomes. The supernatant was discarded, and the pellet was suspended in
500 ul of
a buffered lysis solution, which digested the phagosome membranes without
damaging the
phages. After trituration, 500 ul of 2x LB stock was added, and the resulting
suspension of
phages was added to 5 ml of K91 E. coli cells in logarithmic growth phase.
After allowing
1 hour for infection, E. coli cells were pelleted by centrifugation at 3,500
rpm for 10 min,
and the supernatant was discarded. The cells were suspended in 250 ul of LB
medium,
plated onto agar with tetracycline, and incubated overnight.
[000366 ] This process selected phages that, in addition to triggering and
driving NALT
uptake, also could efficiently trigger and drive uptake and processing by
macrophages and
antigen-presenting cells.
EXAMPLE 8: MHC-1 ANALYSIS
[000367 1 PBMC were prepared as above. Human autologous serum was prepared by
collecting 20 ml of donor blood in two serum clot separator tubes, then
centrifuging at
4,500 rpm for 10 min at room temperature (autologous serum was used to
minimize any
risk of activation of PBMC cells by foreign proteins, such as in fetal calf
serum). In a 50
ml Falcon tube was added 5 ml of autologous serum and 45 ml of
RPMI/NaHCO3/glutamine. Washed PBMC from 20 ml blood were taken up in 3 ml of
the
RPMI mixture with 10% serum, and 1 ml of each was placed in T25 flasks. Wild-
type
fdTet phages (used as controls), or phages selected by the phagosome isolation
process
described above, were added to PBMC cells that had adhered to the flask
surfaces, at viral
loads estimated at 20 ug/ml (7.3x10(10) virions per ml), and incubated for 30
min. The
phage-containing medium was then removed, and adhering cells were washed twice
with 2
ml of RPMI/10% serum, then incubated overnight in fresh serum. At 18 hours,
the cells
were gently scraped off and placed in a 15 ml centrifuge tube. The flask
surface was rinsed
with RPMI/10% serum, which also was transferred to the centrifuge tube, which
was
centrifuged at 1,600 rpm for 5 min at room temperature. The supernatant was
discarded,
and the cells were resuspended in 250 ul of fresh medium.
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[000368 ] 10 ul of FITC-labeled antibodies that bind to the MHC-1 protein were
added,
and incubated for 30 minutes. The cells were then diluted to 5 ml with
RPMI/serum, and
centrifuged. The supernatant was discarded, and cells were taken up in 500 ul
of
RPMI/10% serum on ice. They were then subjected to fluorescence-activated cell
sorting
(FACS), using a 530 nm laser beam.
[000369 ]After FACS analysis, an equal volume of 4% paraformaldehyde in 0.1 M
NaPO4
(pH 7.3) was added to the remaining cell suspension. After overnight fixation
at 4 C, 5 ul
aliquots of the cell suspension were applied to 4% gelatin-coated slides, and
were
examined under a fluorescent light source. The immunohistochemistry showed
clustering
of MHC-I on adherent PBMC cells treated with phagosome-targeting phage, and to
a
lesser extent, on cells treated by control phages. No clear MHC-I clustering
was seen on
adherent PBMC that were treated as controls, without phages.
[000370 ] It should be noted that clustering of MHC-I on phage-treated
adherent PBMC
cells suggested stimulation of a Th-1 response. Macrophages and "professional"
APCs
(i.e., APC cells that are presenting semi-digested foreign antigens to other
cells in the
immune system) can present antigens from certain types of viruses and other
pathogens in
a special way. To generate antigen-specific cytotoxic T lymphocytes (often
called "killer
cells" that can eliminate infected cells, thereby eliminating those cells as
"factories" for
making more of the pathogens), an APC cell can present exogenous virus
proteins, in ways
that involve MHC-1 proteins, by means of a recently-described mechanism called
"cross-presentation" (Houde et al 2003). In such macrophages, phagocytosis
proceeds by
means of endoplasmic reticulum (ER) recruitment at the cell surface. This
triggers a
process called ER-mediated phagocytosis (Gagnon et al 2002), in which a
transient fusing
of an endoplasmic reticulum with a phagosome brings an antigen into contact
with MHC-1
molecules and TLR9 toll-like receptors. This leads to "cross-presentation" of
the antigen
on MHC-1 as well as on MHC-2 molecules, and to maturation of "professional"
APC
cells, which present the antigens on their surfaces. Parallel processes
involving other cells
also leads to expression of co-stimulatory molecules (such as various
cytokines), and to
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robust stimulation of CD8+ and CD4+ receptors on T-cells, as described in
Desjardins et al
2005.
[0003711 The cross-presentation process also accounts for the observation that
the
immunostimulatory effects of CpG-ODN motifs, which is manifested by segments
of free
DNA, can be greatly amplified (such as 50 to 100 times higher) when a DNA
segment
having the CpG-ODN motif is conjugated to a proteinous antigen, rather than
simply
mixed with the antigen in a vaccine formulation, as reviewed in Wagner et al
2004.
[000372 ] In view of that observation, combined with the fact that CpG-motif
DNA strands
can directly activate macrophages to secrete ThI-like cytokines such as TNF-
alpha and
interleukins 6 and 12, and can also upregulate the expression of MHC and
costimulatory
molecules (as reviewed in Krieg 2002), it is believed and anticipated that, in
at least some
cases, by coupling a DNA strand having a CpG-ODN motif (see Klinman et al
2004) to a
NALT-targeting phage carrier (amination or other cationic modification also
can be used
to impart a positive charge to the phage carrier surface, to hold the DNA
segment in closer
proximity to the phage carrier), a robust MHC-I (Thl) and/or MHC-II (Th2)
immune
response may be generated.
EXAMPLE 9: IN VIVO TRACER STUDIES USING FITC LABELING
[000373 ] To evaluate where in vivo selected phage would travel, within
various tissues,
the FITC labeling reagent was coupled to phages that had been selected by a
screening
round which isolated them from olfactory bulb tissue. This was done by buffer-
exchanging PEG-precipitated phages into PBS (pH 7.4), using a 10 ml Sephadex G-
25
column. 1.8 mg of FITC dissolved in 180 ul of DMSO was added to the phage
solution,
the reaction proceeded for 2 hours at room temperature, or overnight at 4 C,
with mixing.
Phage were PEG-precipitated at least twice, to remove unreacted FITC, and the
FITC-labeled phages were nasally administered to mice, as described above.
[000374 ] At 30 hours after nasal administration, the animals were sacrificed,
and
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pre-perfused with 20 ml of Dulbecco's PBS. They were then perfusion fixed with
ice cold
0.1 M sodium phosphate buffer pH 7.4 containing 2% paraformaldehyde plus 0.2%
parabenzoquinone (40 ml over 15 minutes), followed by partial dissection and
immersion
in the same fixative for 2 hours (Conner, 1997). Tissues were cryoprotected in
PBS
containing 30% sucrose and embedded in a wax-type OCT cutting compound.
Sections 50
microns thick were made of the brain tissue, using a cryostat. Sections were
mounted in
buffered glycerol, and examined using a fluorescent microscope. Typical
results are
presented in FIGS. 5 and 6, which also have been posted (in downloadable
versions that
provide better resolution) at www.tetraheed.net/ferguson. Other organs (such
as heart,
lung, liver, etc.) also were analyzed using similar methods, and were found to
have lower
yet significant levels of fluorescently-labeled phages present.
EXAMPLE 10: TRANSPORT OF PROTEINS INTO BLOOD, BY PHA GES
[000375 ] In another studies, selected phage populations that already had
passed an in vivo
screening test were studied, to determine whether they could transport a
protein into
circulating blood. That type of transport activity poses a potentially
important challenge for
candidate vaccines, since much of the immune system relies heavily on cells
that circulate
in blood.
[000376 ] To enable convenient analysis, the horseradish peroxidase (HRP)
protein was
used, since it will generate a color change that can be easily measured, when
certain types
of substrate molecules (such as tetramethyl benzidine, TMB) are added to a
liquid being
analyzed.
[000377 ] Crosslinked HIP-avidin complexes were used, since avidin will bind
very
tightly to a compound called biotin. To crosslink biotin to a phage
population, PEG
precipitated phages (either fd-tet phages as controls, or phages selected by
in vivo
screening) were dissolved in PBS, twice centrifuged at 14,000 rpm to pelletize
the phage,
and resuspended and diluted to 0.75 mg/ml. To 1 ml of phage was added 25 ul of
1.94 mg
Biotin dissolved in 194 ul dimethyl sulfoxide (DMSO). The reaction proceeded
overnight
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with gentle mixing at 4 C. The biotinylated phage were PEG-precipitated at
least twice, to
remove unreacted biotin.
[000378 ] One mg of biotinylated phage were dissolved in 550 ul PBS, and 50 ul
of 1
mg/ml HRP-avidin was added. The mixture was incubated overnight at 4 C with
mixing,
before PEG precipitation, twice, to remove any unbound HRP. Biotinylation and
HRP
coupling was monitoring by applying 1 ul samples from reaction mixtures to
nitrocellulose
membrane, blocking with 10% FCS in 0.1M Tris (pH 7.3), and probing with HRP-
avidin
and visualizing, after reaction with diaminobenzadine.
[000379 ] After nasal administration of phage-biotin-avidin-HRP complexes,
concentrations of the HRP component of the phage-HRP complexes, in blood
samples,
was determined by ELISA assay, using TMB as a chromogenic substrate. The
results
confirmed that the phages could and did transport the HRP protein into
circulating blood.
EXAMPLE 11: BINDING OF DNA STRANDS TO PHA GE PARTICLES
[000380 ] For two reasons, the binding of double-stranded DNA, to the outside
of phage
particles, also was evaluated. One reason involves the potential use of dsDNA
segments,
having controlled nucleotide sequences which will emulate certain known
"pathogen
associated molecular patterns" (such as CpG motifs), as vaccine components
that can
activate certain types of toll-like receptors, which will help ensure a strong
immune
response rather than an allergic or tolerance response, when a vaccine is
administered. The
second reason is that various types of DNA vaccines are showing good promise,
and may
provide practical alternatives (or possibly additions and/or enhancements) to
immunization
using foreign protein antigens. In a DNA vaccine, the DNA (usually in plasmid
form)
encodes the foreign protein antigen; accordingly, when a DNA plasmid is
delivered into a
cell and then expressed, the resulting foreign protein stimulates an immune
response.
[0003811 To test whether DNA plasmids can bind to phage particles in solution,
the
change in percentage light transmission at 370 nm (Tang et al 1997) was used
to monitor
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for DNA plasmid binding to phage particles. Phages were dissolved in 1 ml of
3.3 M NaCl
with 50 mM Tris (pH 9.05), with or without 100 ng/ml of dsDNA from a standard
laboratory plasmid known as pCMVLacZ. Aliquots of 20% v/v of 16.7%
polyethylene
glycol (PEG) plus 3.3M NaCl in 50 mM Tris pH 9.05 were added, and any changes
in
opacity were monitored by a spectrophotometer, at 370 rim. It was found that
DNA
plasmids did not affect the amount of PEG that was needed to effect a state
transition; this
indicated that DNA plasmid would not normally bind to filamentous phages, in
solution,
unless the phages were first treated to create positive charges on their
surfaces.
EXAMPLE 12: COUPLING OF CATIONS TO PHA GE SURFACES
[000382 ] It can be desirable to create positive surface charges, on viral
particles being
used as vaccines. This was indicated by the results of Example 11, above,
showing that
dsDNA will not bind to bacteriophages unless the phage surfaces are modified.
[000383 ] In addition, as mentioned in the Background section, most mammalian
cells have
negatively-charged cell surfaces, while many pathogens have positively-charged
surfaces.
That helps pathogens rapidly bind to and infect cells, but it also helps
macrophages
recognize such pathogens as foreign, which triggers the process of
phagocytosis by the
macrophages. Therefore, giving positive surface charges to phage particles in
vaccines
may increase the likelihood that an immune system will respond rapidly and
efficiently to
the vaccine.
[000384 ] In addition, as mentioned above, surface-exposed DNA strands having
certain
types of "pathogen associated molecular patterns" (such as CpG motifs) can
help activate
certain toll-like receptors, which will help ensure that an immune system
generates a
desired antibody response, rather than an undesired allergic or tolerance
reaction.
[000385 ] Finally, vaccines made of dsDNA segments (which will be taken into
cells, and
then expressed into antigenic foreign proteins) are being developed, and
appear to offer
good promise in a number of situations. Accordingly, efficient creation of
positive charges
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on phage surfaces can provide another set of options, for use with such DNA
vaccines.
[000386 ] For all of those reasons, it can be desirable to impart positive
charges to a virus
particle being used in a vaccine. This was done by treating phages with
ethylenediamine,
which will bond amine groups (-NHX) to carboxyl groups, which are present in
the side
chains of several types of amino acids that are incorporated into the viral
coat proteins. A
nitrogen atom in an amine group will bring at least one hydrogen proton with
it, and it will
also attract another hydrogen proton (from an aqueous solution) to an unshared
electron
pair on the surface of the nitrogen. Those protons will impart a local
positive charge to a
protein.
[000387 ] Accordingly, carboxyl groups on phage were aminated with
ethylenediamine
(EDA), using a method derived from Futami et a! 2001. 2M ethylenediamine (pH
5.0) was
prepared by HCl neutralization of EDA. A preparation of phages from the scFv
phage
display library (from Cambridge Antibody Technology) that had been selected
for sciatic
nerve uptake and transport in rats (as described in PCT application WO
2003/091387) was
suspended in 1 ml of 2M EDA and 20 mg of EDC (Sigma E-6383). The mixture was
reacted, with mixing, at room temperature for 2 hours, then another 20 mg EDC
was
added, and incubation with mixing continued overnight. Cationised phage was
separated
from unreacted reagents by passage through a Sephadex G25 gel (Pharmacia PD
10) into
Dulbecco's PBS. Compared to uncationised phage controls, elution of cationised
phage
from the column was significantly retarded. Emergent fractions that contained
purified
cationised phage (indicated by spectroscopic monitoring at 280 nm) were
collected,
pooled, and used. While unmodified phage could be precipitated by adding 2%
PEG,
precipitation of 1.5 ml of cationised phage required 600 ul of PEG stock.
[000388 ] To couple dsDNA strands to the treated phages, 10 ul of cationised
phage (final
concentration: 0 to 500 ng/50 ul), 10 ul of 200 ng dsDNA from the pCMV1acZ
plasmid, 1
ug/ul TE buffer (pH8.0, Clonetech), and 10 ul of tracking dye and glycerol
were mixed in a
microfuge tubes. Samples were allowed to complex for 1 hour, then loaded into
0.9%
agarose gel/20mM Tris-borate/1mM EDTA (pH 8.0) with 0.5 ug/ml ethyl bromide,
and
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subjected to electrophoresis at 150 volts for 30 minutes. Gel shift assays
indicated that 200
ng of DNA plasmid was neutralised by 100 to 200 ng cationised phage. Further
evidence
that the cationised phage would complex with DNA plasmids was seen when it was
observed that a mixture of 10 ug of DNA added to10 ug in 0.5 ul of cationised
phage led to
gradual precipitation from solution of a DNA/plasmid matrix or gel.
EXAMPLE 13: DNA AND AMINO ACID SEQUENCING
[000389 ] Several clonal colonies of phages, originally from the 15-mer
diverse phage
display library (Smith 1993), and progressively selected by means of a series
of screening
and selection tests (including two rounds of isolation from olfactory bulb
tissue after nasal
administration, two rounds of isolation from NALT tissue after nasal
administration,
selection for binding to PBMC white blood cells using fluorescent cell
sorting, and
isolation from the phagosomes of macrophages) were sequenced, to analyze the
15-mer
inserts that were present in the coat protein VIII subunits of those
particular phages. The
entire DNA sequence of the genomes of these phages is already known and
published, via
Genbank accession number AF246448. The only variable sequences, in the phages
contained in the display library, is in a specific 45 base sequence containing
15 codons,
with each variable codon specifying a single amino acid residue that will
appear in the
15-mer variable portion of the coat protein VIII polypeptide.
[000390 ] Accordingly, preparations of selected phages which had passed all
screening
tests listed above were used to infect E. coli, which were plated at low
concentration on LB
agar-tetracycline plates, to allow individual monoclonal phages to be isolated
after 24
hours growth at 30 C. Individual phage clones were picked from the agar plates
and
transferred to 4 ml of high cell density culture medium containing
tetracycline and were
grown for 48 hours. Phage were PEG-precipitated from culture medium and ssDNA
prepared by phenol-chloroform extraction and ethanol precipitation. Foreign
peptide
inserts in the pVIII coat proteins of isolated phages were sequenced, using 5'-
TCG-GCA-
3 0 AGC-TCT-TTT-AGG-3' as a primer strand (Villard et a! 2003). Sequencing
reactions
were carried out according to the dideoxy chain termination method. The
nucleotide and
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amino acid sequences for peptides displayed on 42 clones isolated from within
PBMC
phagosomes were determined. Of the 42 clones, 39 clones (92.9%) contained the
sequence
HSPLPPPLFHLLQSMet (SEQIDNO4), 2 clones (4.8%) contained
PAYIKQVPDFCNVLL (SEQIDNO5), and 1 clone (2.4%) contained
HAGIGGLTCNLPILP (SEQIDNO6).
[000391 ] A search of genetic databases showed that a 7-residue sequence,
PPLFHLL,
within the first phage peptide sequence listed above was also present in two
apparent
proteins from a fungal pathogen, Cryptococcus neoformas (UniProtKB/TrEMBL
accession
nos Q5KLZ8 and Q55XZ2). The complete pathogen, often found in bird droppings,
has
several interesting properties (e.g., Del Poeta 2004) and can cause
cryptococcosis, which
also can manifest in a form of meningoencephalitis, as described in articles
such as
(Buchanan and Murphy, 1998). Since it is an airborne pathogen, it may be
speculated there
may have been selection pressure for certain M cells receptors and/or
phagosome receptors
to evolve that will recognize this motif as a possible PAMP. However, it must
be
emphasized that the overlap between the short sequence of the phage insert,
and a short
segment of a pathogenic microbe, does not imply that the phage insert was
derived in any
way from any toxin, and it should not be referred to as a toxin or a toxin
adjuvant.
[000392 ] Also, none of the phagosome-selected peptide sequences showed
significant
homology with any human or animal sequences on any publicly available genetic
databases. Unlike the first phage peptide sequence, no good matches were found
between
the second two phage peptides sequences of 6 or more amino acids length and
sequences
on any publicly available genetic databases.
[000393 ] Thus, there has been shown and described a new and useful means for
creating
specialized vaccine cassettes and vaccines, using phage particles that have
been selected
and modified to provide a number of advantages for inducing desired responses
by
mammalian immune systems. Although this invention has been exemplified for
purposes
of illustration and description by reference to certain specific embodiments,
it will be
apparent to those skilled in the art that various modifications, alterations,
and equivalents
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of the illustrated examples are possible. Any such changes which derive
directly from the
teachings herein, and which do not depart from the spirit and scope of the
invention, are
deemed to be covered by this invention and the claims below.
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