Note: Descriptions are shown in the official language in which they were submitted.
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INNATE IMMUNE SYSTEM-DIRECTED VACCINES
FIELD OF THE INVENTION
The present invention relates to novel vaccines, the production of such
vaccines and methods of using such vaccines. More specifically, this invention
provides unique vaccine molecules comprising an isolated Pathogen Associated
Molecular Pattern (DAMP) and an antigen. Even more specifically, this
invention
provides novel fusion proteins comprising an isolated PAMP and an antigen such
that
vaccination with these fusion proteins provides the two signals required for
native T-
cell activation. The novel vaccines of the present invention provide an
efficient way
of making and using a single molecule to induce a robust T-cell immune
response that
activates other aspects of the adaptive immune responses. The methods and
compositions of the present invention provide a powerful way of designing,
producing
and using vaccines targeted to specific antigens, including antigens
associated with
selected pathogens, tumors, allergens and other disease-related molecules.
BACKGROUND OF THE INVENTION
All articles, patents and other materials referred to below are specifically
incorporated herein by reference.
1. Immunity
Multicellular organisms have developed two general systems of immunity to
infectious agents. The two systems are innate or natural immunity (also known
as
"innate immunity") and adaptive (acquired) or specific immunity. The major
difference between the two systems is the mechanism by which they recognize
infectious agents.
1.
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The innate immune system uses a set of germline-encoded receptors for the
recognition of conserved molecular patterns present in microorganisms. These
molecular patterns occur in certain constituents of microorganisms including:
lipopolysaccharides, peptidoglycans, lipoteichoic acids, phosphatidyl
cholines,
bacteria-specific proteins, including lipoproteins, bacterial DNAs, viral
single and
double-stranded RNAs, unmethylated CpG-DNAs, mannans and a variety of other
bacterial and fungal cell wall components. Such molecular patterns can also
occur in
other molecules such as plant alkaloids. These targets of innate immune
recognition
are called Pathogen Associated Molecular Patterns (PAMPs) since they are
produced
by microorganisms and not by the infected host organism. (Janeway et al.
(1989)
Cold Spying Ha~b. Syrup. Quant. Biol. 54: 1-13; Medzhitov et al. (1997) Curr.
Opin.
Immunol. 94: 4-9).
The receptors of the innate immune system that recognize PAMPs are called
Pattern Recognition Receptors (PRRs). (Janeway et al. (1989) Cold Spning Ha~b.
Synap. Quant. Biol. 54: 1-13; Medzhitov et al. (1997) Cur. Opin. Immunol. 94:
4-9).
These receptors vary in structure and belong to several different protein
families.
Some of these receptors recognize PAMPs directly (e.g., CD 14, DEC205,
collectins),
while others (e.g., complement receptors) recognize the products generated by
PAMP
recognition. Members of these receptor families can, generally, be divided
into three
types: 1) humoral receptors circulating in the plasma; 2) endocytic receptors
expressed on immune-cell surfaces, and 3) signaling receptors that can be
expressed
either on the cell surface or intracellularly. (Medzhitov et al. (1997) Cunn.
Opin.
Immunol. 94: 4-9; Fearon et al. (1996) Science 272: 50-3).
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Cellular PRRs are expressed on effector cells of the innate immune system,
including cells that function as professional antigen-presenting cells (APC)
in
adaptive immunity. Such effector cells include, but are not limited to,
macrophages,
dendritic cells, B lymphocytes and surface epithelia. This expression profile
allows
PRRs to directly induce innate effector mechanisms, and also to alert the host
organism to the presence of infectious agents by inducing the expression of a
set of
endogenous signals, such as inflammatory cytokines and chemokines, as
discussed
below. This latter function allows efficient mobilization of effector forces
to combat
the invaders.
In contrast, the adaptive irmnune system, which is found only in vertebrates,
uses two types of antigen receptors that are generated by somatic mechanisms
during
the development of each individual organism. The two types of antigen
receptors are
the T-cell receptor (TCR) and the immunoglobulin receptor (IgR), which are
expressed on two specialized cell types, T-lymphocytes and B-lymphocytes,
respectively. The specificities of these antigen receptors are generated at
random
during the maturation of lymphocytes by the processes of somatic gene
rearrangement, random pairing of receptor subunits, and by a template-
independent
addition of nucleotides to the coding regions during the rearrangement.
Recent studies have demonstrated that the innate immune system plays a
crucial role in the control of initiation of the adaptive immune response and
in the
induction of appropriate cell effector responses. (Fearon et al. (1996)
Scieyace 272:
50-3; Medzhitov et al. (1997) Cell 91: 295-8). Indeed, it is now well
established that
the activation of naive T-lymphocytes requires two distinct signals: one is a
specific
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antigenic peptide recognized by the TCR, and the other is the so called co-
stimulatory
signal, B7, which is expressed on APCs and recognized by the CD28 molecule
expressed on T-cells. (Lenschow et al. (1996) Annu. Rev. Imfyauhol. 14: 233-
58).
Activation of naive CD4+ T-lymphocytes requires that both signals, the
specific
antigen and the B7 molecule, are expressed on the same APC. If a naive CD4 T-
cell
recognizes the antigen in the absence of the B7 signal, the T-cell will die by
apoptosis. Expression of B7 molecules on APCs, therefore, controls whether or
not
the naive CD4 T-lymphocytes will be activated. Since CD4 T-cells control the
activation of CD8 T-cells for cytotoxic functions, and the activation of B-
cells for
antibody production, the expression of B7 molecules determines whether or not
an
adaptive immune response will be activated.
Recent studies have also demonstrated that the iimate immune system plays a
crucial role in the control of B7 expression. (Fearon et al. (1996) Science
272: 50-3;
Medzhitov et al. (1997) Cell 91: 295-8). As mentioned earlier, innate immune
recognition is mediated by PRRs that recognize PAMPs. Recognition of PAMPs by
PRRs results in the activation of signaling pathways that control the
expression of a
variety of inducible immune response genes, including the genes that encode
signals
necessary for the activation of lymphocytes, such as B7, cytokines and
chernokines.
(Medzhitov et al. (1997) Cell 91: 295-8; Medzhitov et al. (1997) Nature 388:
394-
397). Induction of B7 expression by PRR upon recognition of PAMPs thus
accounts
for self/nonself discrimination and ensures that only T-cells specific for
microorganism-derived antigens are normally activated. This mechanism normally
prevents activation of autoreactive lymphocytes specific for self antigens.
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Receptors of the innate immune system that control the expression of B7
molecules and cytokines have recently been identified. (Medzhitov et al.
(1997)
Nature 388: 394-397; Rock et al. (1998) P~°oc. Natl. Acad. Sci. USA,
9S: S88-93).
These receptors belong to the family of Toll-like receptors (TLRs), so called
because
S they are homologous to the Drosophila Toll protein which is involved both in
dorsoventral patterning in Drosophila embryos and in the immune response in
adult
flies. (Lemaitre et al. (1996) Cell 86: 973-83). In mammalian organisms, such
TLRs
have been shown to recognize PAMPs such as the bacterial products LPS,
peptidoglycan, and lipoprotein. (Schwandner et al. (1999) J. Biol. Chem. 274:
17406-
9; Yoshimura et al. (1999) J. ImmufZOl. 163: 1-S; Aliprantis et al. (1999)
Science 285:
736-9).
2. Vaccine Development
Vaccines have traditionally been used as a means to protect against disease
caused by infectious agents. However, with the advancement of vaccine
technology,
1S vaccines have been used in additional applications that include, but are
not limited to,
control of mammalian fertility, modulation of hormone action, and prevention
or
treatment of tumors.
The primary purpose of vaccines used to protect against a disease is to induce
immunological memory to a particular microorganism. More generally, vaccines
are
needed to induce an immune response to specific antigens, whether they belong
to a
microorganism or are expressed by tumor cells or other diseased or abnormal
cells.
Division and differentiation of B- and T-lymphocytes that have surface
receptors
specific for the antigen generate both specificity and memory.
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Tn order for a vaccine to induce a protective immune response, it must fulfill
the following requirements: 1) it must include the specific antigens) or
fragments)
thereof that will be the target of protective immunity following vaccination;
2) it
must present such antigens in a form that can be recognized by the immune
system,
e.g., a form resistant to degradation prior to immune recognition; and 3) it
must
activate APCs to present the antigen to CD4+ T-cells, which in turn induce B-
cell
differentiation and other immune effector functions.
Conventional vaccines contain suspensions of attenuated or killed
microorganisms, such as viruses or bacteria, incapable of inducing severe
infection by
themselves, but capable of counteracting the unmodified (or virulent) species
when
inoculated into a host. Usage of the term has now been extended to include
essentially any preparation intended for active immunologic prophylaxis (e.g.,
preparations of killed microbes of virulent strains or living microbes of
attenuated
(variant or mutant) strains; microbial, fungal, plant, protozoan, or metazoan
derivatives or products; synthetic vaccines). Examples of vaccines include,
but are
not limited to, cowpox virus for inoculating against smallpox, tetanus toxoid
to
prevent tetanus, whole-inactivated bacteria to prevent whooping cough
(pertussis),
polysaccharide subunits to prevent streptococcal pneumonia, and recombinant
proteins to prevent hepatitis B.
Although attenuated vaccines are usually immunogenic, their use has been
limited because their efficacy generally requires specific, detailed knowledge
of the
molecular determinants of virulence. Moreover, the use of attenuated pathogens
in
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vaccines is associated with a variety of risk factors that in most cases
prevent their
safe use in humans.
The problem with synthetic vaccines, on the other hand, is that they are often
non-immunogenic or non-protective. The use of available adjuvants to increase
the
immunogenicity of synthetic vaccines is often not an option because of
unacceptable
side effects induced by the adjuvants themselves.
An adjuvant is defined as any substance that increases the immunogenicity of
admixed antigens. Although chemicals such as alum are often considered to be
adjuvants, they are in effect akin to carriers and are likely to act by
stabilizing
antigens and/or promoting their interaction with antigen-presenting cells. The
best
adjuvants are those that mimic the ability of microorganisms to activate the
innate
immune system. Pure antigens do not induce an immune response because they
fail
to induce the costimulatory signal (e.g., B7.1 or B7.2) necessary for
activation of
lymphocytes. Thus, a key mechanism of adjuvant activity has been attributed to
the
induction of costimulatory signals by microbial, or microbial-like,
constituents
carrying PAMPs that are routine constituents of adjuvants. (Janeway et al.
(I989)
Cold Sp~ir2g Harb. Symp. Quaht. Biol., 54: 1-13). As discussed above, the
recognition
of these PAMPs by PRRs induces the signals necessary for lymphocyte activation
(such as B7) and differentiation (effector cytokines).
Because adjuvants are often used in molar excess of antigens and thus trigger
an innate immune response in many cells that do not come in contact with the
target
antigen, this non-specific induction of the innate immune system to produce
the
signals that are required for activation of an adaptive immune response
produces an
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excessive inflammatory response that renders marry of the most potent
adjuvants
clinically unsuitable. Alum is currently approved for use as a clinical
adjuvant, even
though it has relatively limited efficacy, because it is not an innate immune
stimulant
and thus does not cause excessive inflammation. However, a vaccine that
included
the use of an innate immune stimulant in such a way as not to elicit excess
inflammation could be far more effective than vaccines comprising an antigen
together with an adjuvant such as alum. Fusion of an antigen with a PAMP, such
as
bacterial lipoprotein (BLP), optimizes the stoichiometry of the two signals
and
coordinates their effect on the same APC, thus minimizing the unwanted
excessive
inflammatory responses that occur when antigens are mixed with adjuvants
comprising innate immune stimulants to increase their immunogenicity. In
addition,
the chimeric constructs of the present invention will prevent activation of
APCs that
do not take up the antigen. Activation of such APCs in the absence of uptake
and
presentation of the target antigen can lead to the induction of autoimmune
responses,
which, again, is one of the problems with commonly used innate immunity-
stimulating adjuvants that prevents or limits their use in humans. Notably,
the
chimeric constructs of the present invention exhibit the essential
immunological
characteristics or properties expected of a conventional vaccine supplemented
with an
adjuvant, but the chimeric constructs do not induce an excessive inflammatory
reaction as is often induced by an adjuvant. Thus, the vaccine approach
described in
the present invention minimizes or eliminates undesired side effects (e.g.,
excessive
inflammatory reaction, autoimmunity) yet induces a very potent and specific
immune
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response, and provides a favorable alternative to vaccines comprising mixtures
of
antigens and adjuvants.
3. Alternative Vaccine Strategies
Immune Stimulating Complexes for Use as Vaccines. Immune stimulating
complexes (ISCOMS) are cage-like structures comprising Quil-A, cholesterol,
adjuvant active saponin and phospholipids that induce a wide range of systemic
immune responses. (Mowat et al. (1999) Immuhol. Lett. 65: 133-140; Smith et
al.,
(1999) J. Immuraol. 162(9): 5536-5546). ISCOMS are suitable for repeated
administration of different antigens to an individual because these complexes
allow
the entry of antigen into both MHC I and II processing pathways. (Mowat et al.
(1991) Immunol. 72: 317-322).
ISCOMS have been used with conjugates of modified soluble proteins. (Reid
(1992) haccine IO(9): 597-602). These complexes also produce a Thl type
response,
as would be expected for such a vaccine. (Morein et al. (1999) Methods 19: 94-
102).
However, in contrast to the molecules of the present invention, ISCOMS are
far more complex structures that present potential problems of reproducibility
and
dosing; nor do they contain conjugates between an antigen and a PAMP. Since
ISCOMS do not specifically target APCs their use can result in problems of
toxicity
and a lack of specificity.
Multiple Antigenic Recombinant Vaccines. Various U.S. patents disclose
chimeric proteins consisting of multiple antigenic peptides (MAPS) for use as
vaccines. For example, I~lein et al. were granted a family of patents (e.g.,
U.S. Patent
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No. 6,033,668; 6,017,539; 5,998,169; and 5,968,776) which describe genes
encoding
multimeric hybrids comprising an immunogenic region of a protein from a first
antigen linked to an immunogenic region from a second pathogen. While the
patents
are focused on human Parainfluenza/Respiratory syncytial virus protein
chimeras, the
first and second antigens may be more broadly selected from bacterial and
viral
pathogens. Although the vaccines contemplated by Klein et al. are fusion
proteins, all
the component peptides are all selected by virtue of their being antigens
(i.e., being
recognized by a TCR or IgR) rather than a pairing of antigens with PAMPs, and
thus
the vaccines are not designed to stimulate the innate immune system.
Sinugalia (U.S. Patent No. 5,114,713) discloses vaccines consisting of
peptides from the circumsporozoite protein of Plasmodium falciparum (P.
falciparum) as universal T-cell epitopes that can be coupled to B-cell
epitopes, such
as surface proteins derived from pathogenic agents (e.g., bacteria, viruses,
fungi or
parasites). These combined peptides can be prepared by recombinant means.
These
universal T-cell epitopes are not known to be PAMPs, and they act via the
adaptive
immune system rather than the innate immune system.
Russell-Jones et al. (IJ.S. Patent No. 5,928,644) disclose T-cell epitopes
derived from the TraT protein of E. coli that is used to produce hybrid
molecules to
raise immune responses against various targets to include parasites, soluble
factors
(e.g., LSH) and viruses. Thus, these constructs provide strategies for
increasing the
complexity of the antigenic nature of the vaccines, thereby promoting
strengthened or
multiple adaptive immune responses. However, the epitopes are not known to be
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PAMPs, and they act via the adaptive immune system rather than the innate
immune
system.
Thus, the aforementioned inventions are very different in intent, concept,
strategy and mode of action from the present invention.
4. Overview of the Novel Vaccines of the Present Invention
The novel vaccines of the present invention comprise one or more isolated
PAMPs in combination with one or more antigens. The antigens used in the
vaccines
of the present invention can be any type of antigen (e.g., including but not
limited to
pathogen-related antigens, tumor-related antigens, allergy-related antigens,
neural
defect-related antigens, cardiovascular disease antigens, rheumatoid arthritis-
related
antigens, other disease-related antigens, hormones, pregnancy-related
antigens,
embryonic antigens and/or fetal antigens and the like). Examples of various
types of
vaccines, which can be produced by the present invention, are provided in
Figure 1.
In one preferred embodiment, the vaccines are recombinant proteins, or
recombinant lipoproteins, or recombinant glycoproteins, which contain a PAMP
(e.g.,
BLP or Flagellin) and one or more antigens. The basic concept for preparing a
fusion
protein of the present invention is provided in Figure 1.
Upon administration into human or animal subjects, the vaccines of the
present invention will interact with APCs, such as dendritic cells and
macrophages.
This interaction will have two consequences: First, the PAMP portion of the
vaccine
will interact with a PRR such as a TLR and stimulate a signaling pathway, such
as the
NF-xB, JNK andlor p3~ pathways. Second, due to the PAMP's interaction with
TLRs and/or other pattern-recognition receptors, the recombinant vaccine will
be
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taken up into dendritic cells and macrophages by phagocytosis, endocytosis, or
macropinocytosis, depending on the cell type, the size of the recombinant
vaccine,
and the identity of the PAMP.
Activation of TLR-induced signaling pathways will lead to the induction of
the expression of cytokines, chemokines, adhesion molecules, and co-
stimulatory
molecules by dendritic cells and macrophages and, in some cases, B-cells.
Uptake of
the vaccines will lead to the processing of the antigens) fused to the PAMP
and their
presentation by the MHC class-I and MHC class-II molecules. This will generate
the
two signals required for the activation of naive T-cells - a specific antigen
signal and
the co-stimulatory signal. In addition, chemokines induced by the vaccine (due
to
PAMP interaction with TLR) will recruit naive T-cells to the APC and
cytokines, like
IL-12, which will induce T-cell differentiation into Th-1 effector cells. As a
result, a
robust T-cell immune response will be induced, which will in turn activate
other
aspects of the adaptive immune responses, such as activation of antigen-
specific B-
cells and macrophages.
Thus, the novel vaccines of the present invention provide an efficient way to
produce an immune response to one or more specific antigens without the
adverse
side effects normally associated with conventional vaccines.
SUMMARY OF THE INVENTION
The present invention relates generally to vaccines, methods of making
vaccines and methods of using vaccines.
More specifically, the present invention provides vaccines comprising an
isolated PAMP, immunostimulatory portion or immunostimulatory derivative
thereof
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and an antigen or an immunogenic portion or immunogenic derivative thereof. An
example of a preferred vaccine of the present invention is a fusion protein
comprising
a PAMP, immunostimulatory portion or immunostimulatory derivative thereof and
an
antigen or an immunogenic portion or immunogenic derivative thereof.
The vaccines of the present invention can comprise any PAMP peptide or
protein, including, but not limited to, the following PAMPs: peptidoglycans,
lipoproteins and lipopeptides, Flagellins, outer membrane proteins (OMPs),
outer
surface proteins (OSPs), other protein components of the bacterial cell walls,
and
other PRR ligands.
One preferred PAMP of the present invention is BLP, including the BLP
encoded by the polypeptide of SEQ m NO: 2, set forth in Figure 15. In addition
to
protein PAMPs, also useful in the vaccines of the present invention are
peptide
mimetics of any non-protein PAMP.
Antigens useful in the present invention include, but are not limited to,
those
that are microorganism-related, and other disease-related antigens, including
but not
limited to those that are allergen-related and cancer-related. The antigen
component
of the vaccine can be derived from sources that include, but are not limited
to,
bacteria, viruses, fungi, yeast, protozoa, metazoa, tumors, malignant cells,
plants;
animals, humans, allergens, hormones and amyloid-(3 peptide. The antigens,
immunogenic portions or immunogenic derivatives thereof can be composed of
peptides, polypeptides, lipoproteins, glycoproteins, mucoproteins and the
like.
The various vaccines of the present invention include, but are not limited to:
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1) one or more PAMPs, immunostimulatory portions or immunostimulatory
derivatives thereof, conjugated to one or more antigens, immunogenic portions
or
immunogenic derivatives thereof;
2) a PAMP/antigen fusion protein comprising one or more PAMPs,
immunostimulatory portions or immunostimulatory derivatives thereof, and one
or
more antigens, immunogenic portions or immunogenic derivatives thereof; and
3) a modified antigen, irmnunogenic portion or immunogenic derivative
thereof, that comprises a leader sequence fused to a lipidation or
glycosylation
consensus sequence that is further fused to the antigen, or an immunogenic
portion or
immunogenic derivative thereof.
The present invention also encompasses such vaccines further comprising a
pharmaceutically acceptable carrier, including, but not limited to, alum.
More specifically, the present invention provides fusion proteins comprising
one or more PAMPs, immunostimulatory portions or immunostimulatory derivatives
thereof, and one or more antigens, immunogenic portions or immunogenic
derivatives
thereof. The PAMP domains of the fusion proteins of the present invention can
be
composed of amino acids, amino acid polymers, peptidoglycans, glycoproteins,
and
lipoproteins or any other suitable component. One preferred PAMP to use in the
fusion proteins of the present invention is BLP, including the BLP encoded by
the
polypeptide of SEQ m NO: 2. Flagellin is another P~ to use in the fusion
proteins of the present invention, and is provided by (but not limited to)
accession
numbers P04949 (E. Coli Flagellin) and A24262 (Salmonella Flagellin). Useful
antigen domains) in the fusion proteins of the present invention include, but
are not
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limited to, Ea (a peptide antigen derived from mouse MHC class-II I-E),
Iisteriolysin,
PSMA, HIV gp120, RaSG and TRP-2. In one preferred embodiment, the fusion
proteins of the present invention include a construct comprising the following
components: a leader peptide that signals lipidation or glycosylation of the
consensus
sequence, a lipidation and/or glycosylation consensus sequence, and antigen.
More
specifically, the fusion proteins of the present invention include a construct
comprising a leader sequence-CXXN-antigen, wherein the leader peptide is a
signal for lipidation of the consensus sequence and wherein X is any amino
acid,
preferably serine. Examples of leader peptides useful in the present invention
include,
but are not limited to, those selected from the peptides of SEQ ID NO: 3
(shown in
Figure 15), SEQ ID NO: 4 (shown in Figure 16), SEQ ID NO: 5 (shown in Figure
17),
SEQ ID NO: 6 (shown in Figure 18) and SEQ ID NO: 7 (shown in Figure 19).
In another embodiment, the present invention provides also provides a fusion
protein comprising an isolated PAMP and an antigen, wherein the antigen is a
self
antigen.
The present invention further provides methods of recombinantly producing
the fusion proteins of the present invention. Thus, the present invention
provides
recombinant expression vectors comprising a nucleotide sequence encoding the
chimeric constructs of the present invention as well as host cells transformed
with
such recombinant expression vectors. Any cell that is capable of expressing
the
fusion proteins of the present invention is suitable for use as a host cell.
Such host
cells include, but are not limited to, the cells of bacteria, yeast, insects,
plants and
animals. More preferably for certain PAMPs such as BLP, the host cell is a
bacterial
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cell. Even more preferably, the host cell is a bacterial cell that can
appropriately
modify (e.g., lipidation, glycosylation) the PAMP domain of the fusion protein
when
such modification is necessary or desirable.
The present invention also provides methods of immunizing an animal with
the vaccines of the present invention, where such methods include, but are not
limited
to, administering a vaccine parenterally, intravenously, orally, using
suppositories, or
via the mucosal surfaces. In one preferred embodiment the animal being
vaccinated is
a human.
The immune response can be measured using any suitable method including,
but not limited to, direct measurement of peripheral blood lymphocytes,
natural killer
cell cytotoxicity assays, cell proliferation assays, immunoassays of immune
cells and
subsets, and skin tests for cell-mediated immunity.
The present invention also provides methods of treating a patient susceptible
to an allergic response to an allergen by administering a vaccine of the
present
invention and thereby stimulating the TLR-mediated signaling pathway.
The present invention also provides methods of treating a patient susceptible
to or suffering from Alzheimer's disease by administering a vaccine of the
present
invention in which the target antigen is a peptide or protein associated with
Alzheimer's disease, including but not limited to amyloid- peptide.
The present invention further provides a method of stimulating an innate
immune response in an animal and thereby enhancing the adaptive immune
response
to a foreign or self antigen which comprises co-administering a PAMP with the
foreign or self antigen.
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The present invention also provides a vaccine which comprises a PAMP
conjugated with a foreign or self antigen that stimulates an innate inunune
response in
an animal and thereby enhances the adaptive immune response to a foreign or
self
antigen but does not lead to undesirable levels of inflammation.
Additionally, the present' invention provides a vaccine which comprises a
PAMP conjugated with a foreign or self antigen which, when administered at a
therapeutically active dose, stimulates an innate immune response in an animal
and
thereby enhances the adaptive immune response to a foreign or self antigen but
does
not lead to undesirable levels of inflammation.
The present invention also provides a method of treatment comprising the
steps of administering to an individual a vaccine which comprises a PAMP
conjugated with a foreign or self antigen which stimulates an innate immune
response
in an animal and thereby enhances the adaptive immune response to a foreign or
self
antigen but does not lead to undesirable levels of inflammation.
Additional embodiments of the present invention will be obvious to those
skilled in the art of vaccine preparation and vaccine administration. Such
obvious
variations of the present invention are also contemplated by the present
inventor.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows examples of alternative fusion proteins according to the
present invention. Permutations and combinations of these fusion proteins can
also be
prepared according to the methods of the present invention.
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Figure 2 shows a basic outline for generating different recombinant protein
vaccines containing different antigens and a signal to trigger the innate
immune
response (PAMP). Each antigen is represented by a different shape of the
central
portion of the vaccine.
Figure 3 shows the BLP/Ea construct.
Figure 4 shows that BLP/Ea activates NF-xB in dose-dependent manner.
Figure 5 shows IL-6 production by dendritic cells stimulated with BLP/Ea.
Figure 6 shows the induction of dendritic cell activation and vaccine antigen
processing and presentation by the MHC class-II pathway.
Figure 7 shows the immunostimulatory effect of the chimeric construct
BLP/Ea on specific T-cells in vitro.
Figure 8 shows the effect of the chimeric construct, BLP/Ecc, on specific T-
cell proliferation iyz vivo.
Figure 9 shows that CpG-induced B-cell activation is dependent upon
MyD88. MyD88 -~- , MyD88-deficient cells; ICE -~- , caspase-1-deficient cells;
B10/ScCr, TLR4-deficient cells derived from C57BL/lOScCr mice; TLR2 -~- , TLR2-
deficient cells.
Figure 10 shows that IL-6 production by macrophages during CpG
stimulation and CpG-DNA-induced IkBa degradation is mediated by a signaling
pathway dependent on MyD88.
Figure 11 shows that wild-type and B10/ScCr dendritic cells, but not dendritic
cells from MyD88-~- animals produce IL-12 when stimulated with CpG
oligonucleotides.
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Figure 12 shows activation of NF-~cB by Flagellin fusions.
Figure 13 shows induction of NF-~cB in macrophages by Flagellin fusions.
Figure 14 shows NF-xB activity in RAW xB cells.
Figure 15 shows SEQ ID NO: 3.
3 Figure 16 shows SEQ ID NO: 4.
Figure 17 shows SEQ ID NO: 5.
Figure 18 shows SEQ ID NO: 6.
Figure 19 shows SEQ ID NO: 7.
Figure 20 shows SEQ ID NO: 10.
Figure 21 shows SEQ ID NO: 11.
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DETAILED DESCRIPTION OF THE INVENTION
1. General Description
The present invention discloses a novel strategy of vaccine design based on
the inventor's recent findings in the field of innate immunity. This approach
is not
limited to any particular antigen or immunogenic portions or derivatives
thereof (e.g.,
microorganism-related, allergen-related or tumor-related, and the like) nor is
it limited
to any particular PAMP or immunostimulatory portions or immunostimulatory
derivatives thereof. The term "vaccine", therefore, is used herein in a
general sense to
refer to any therapeutic or immunogenic or immunostimulatory composition that
includes the features of the present invention. A more detailed definition of
vaccine is
disclosed elsewhere herein.
The activation of an adaptive immune response requires both the specific
antigen or derivative thereof, and a signal (e.g. PAMP) that can induce the
expression
of B7 on the APCs. The present invention combines, in a single chimeric
construct,
both signals required for the induction of the adaptive immune responses - a
signal
recognized by the innate immune system (DAMP), and a signal recognized by an
antigen receptor (antigen).
According to the present invention, neither the PAMP nor the antigen need
consist of a polypeptide. However, either the PAMP or the antigen, or both,
may be a
peptide or polypeptide. In one embodiment of the present invention,
recombinant
DNA technology may be utilized in the production of chimeric constructs, for
use in
vaccines, when both the PAMP, or an immunogenic portion or derivative thereof,
and
the antigen, or an immunostimulatory portion or derivative thereof, are
polypeptides.
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Alternatively, recombinant techniques may also be utilized to produce a
protein
chimeric construct when a peptide mimetic is used in lieu of a non-protein
antigen,
such as a polysaccharide or a nucleic acid and the like, and/or a non-protein
PAMP,
such as a lipopolysaccharide, CpG-DNA, bacterial DNA, single or double-
stranded
viral RNA, phosphatidyl choline, lipoteichoic acids and the like, for example.
The
present invention contemplates in one embodiment the use of BLP, the bacterial
outer
membrane proteins (OMP), the outer surface proteins A (OspA) of bacteria,
Flagellins
and other DNA-encoded PAMPs in the recombinant production of chimeric
constructs. These PAMPs are known to induce activation of the innate immune
response and therefore would be particularly suitable for use in vaccine
formulations.
(Henderson et al. (1996) Microbiol. Rev. 60: 316-41). Furthermore, BLP has
been
shown to be recognized by TLRs. (Aliprantis et al. (1999) Science 285: 736-9).
The
details of the approach are described using BLP as the PAMP domain of a
PAMP/antigen fusion protein; however any inducers of the innate immune system
are
equally applicable for such purpose in the present invention.
In another embodiment, one or more PAMP mimetics is substituted in place of
a PAMP in a fusion protein.
This invention further provides methods for producing chimeric constructs
where either the PAMP or an immunostimulatory portion or derivative thereof,
or the
antigen or an immunogenic portion or derivative thereof, or both the PAMP and
the
antigen are non-protein. Generally, these methods utilize chemical means to
conjugate a PAMP to an antigen thereby producing a non-protein chimeric
construct.
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This invention further provides ways to exploit recombinant DNA technology
in the synthesis of the peptide vaccines. Many of the surface antigens present
on the
pathogens of interest would not be amenable to encoding by nucleic acids as
they are
not proteins (e.g., lipopolysaccharides) or possess low protein content (e.g.,
peptidoglycans).
The present invention contemplates the use of peptide mimetics for these
surface antigens or an immunogenic protein or derivative thereof, and the use
of
peptide mimetics in vaccines.
As discussed in greater detail herein, the present invention contemplates
vaccines comprising chimeric constructs that comprise at least one antigen, or
an
immunogenic portion or derivative thereof, and at least one PAMP, or an
immunogenic portion or derivative thereof. Thus, the present invention
encompasses
vaccines comprising fusion proteins where one or more protein antigens are
linked to
one or more protein PAMPs or a peptide mimetic of a PAMP. Preferably, the
fusion
protein has maximal immunogenicity and induces only a modest inflammatory
response.
In instances in which a target antigen, or a domain of a target antigen, has a
relatively low molecular weight and is not adequately immunogenic because of
its
small size, that antigen or antigen domain can act as a hapten and can be
combined
with a larger carrier molecule such that the molecular weight of the combined
molecule will be high enough to evoke a strong immune response against the
antigen.
In one embodiment of this invention, the antigen itself serves as the carrier
molecule.
In another embodiment of this invention, the PAMP serves as the carrier
molecule. In
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yet another embodiment, a hapten is combined, by either fusion or conjugation,
with
the PAMP or the antigen domain of the vaccine to elicit an antibody response
to the
hapten. In yet another embodiment, which would, without limitation, be
preferable
when the molecular weight of both antigen and PAMP are low, the PAMP and the
antigen are combined with a third molecule that serves as the Garner molecule.
Such
carrier molecule can be keyhole limpet hemocyanin or any of a number of
carrier
molecules for haptens that are known to the artisan. In yet another
embodiment, a
fusion protein contains an antigen or antigen domain, a PAMP or a portion of a
PAMP or a PAMP mimetic, and a carrier protein or carrier peptide. Once again,
such
carrier protein can be keyhole limpet hemocyanin or any of a number of carrier
proteins or carrier peptides for haptens that are known to the artisan.
Increasing the
number of antigens or antigen epitopes, by using multiple antigen proteins
and/or
multiple domains of the same antigen protein or of different antigen proteins
and/or
some combination of the foregoing, are contemplated in this invention. Also
contemplated are fusion proteins in which the number of PAMPs or PAMP
derivatives or PAMP mimetics is increased. It is within the skill of the
artisan to
determine the optimal ratio of PAMP to antigen domains to maximize
immunogenicity and minimize inflammatory response.
2. Definitions
"Adaptive immunity" refers to the adaptive immune system, which involves
two types of receptors generated by somatic mechanisms during the development
of
each individual organism. As used herein, the "adaptive immune system" refers
to
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both cellular and humoral immunity. hnmune recognition by the adaptive immune
system is mediated by antigen receptors.
"Adaptive immune response" refers to a response involving the characteristics
of the "adaptive immune system" described above.
"Adapter molecule" refers to a molecule that can be transiently associated
with some TLRs, mediates immunostimulation by molecules of the innate immune
system, and mediates cytokine-induced signaling. "Adapter molecule" includes,
but
is not limited to, myeloid differentiation marker 88 (MyD88).
"Allergen" refers to an antigen, or a portion or derivative of an antigen,
that
induces an allergic or hypersensitive response.
"Amino acid polymer" refers to proteins, or peptides, and other polymers
comprising at least two amino acids linked by a peptide bond(s), wherein such
polymers contain either no non-peptide bonds or one or more non-peptide bonds.
As
used herein, "proteins" include polypeptides and oligopeptides.
"Antigen" refers to a substance that is specifically recognized by the antigen
receptors of the adaptive immune system. Thus, as used herein, the term
"antigen"
includes antigens, derivatives or portions of antigens that are immunogenic
and
immunogenic molecules derived from antigens. Preferably, the antigens used in
the
present invention are isolated antigens. Antigens that are particularly useful
in the
present invention include, but are not limited to, those that are pathogen-
related,
allergen-related, or disease-related.
"Antigenic determinant" refers to a region on an antigen at which a given
antigen receptor binds.
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"Antigen-presenting cell" or "APC" or "professional antigen-presenting cell"
or "professional APC" is a cell of the immune system that functions in
triggering an
adaptive immune response by taking up, processing and expressing antigens on
its
surface. Such effector cells include, but are not limited to, macrophages,
dendritic
cells and B cells.
"Antigen receptors" refers to the two types of antigen receptors of the
adaptive
immune system: the T-cell receptor (TCR) and the irnmunoglobulin receptor
(IgR),
which are expressed on two specialized cell types, T-lymphocytes and B-
lymphocytes, respectively. The secreted form of the immunoglobulin receptor is
referred to as antibody. The specificities of the antigen receptors are
generated at
random during the maturation of the lymphocytes by the processes of somatic
gene
rearrangement, random pairing of receptor subunits, and by a template-
independent
addition of nucleotides to the coding regions during the rearrangement.
"Chimeric construct" refers to a construct comprising an antigen and a PAMP,
or PAMP mimetic, wherein the antigen and the PAMP are comprised of molecules
such as amino acids, amino acid polymers, nucleotides, nucleotide polymers,
carbohydrates, carbohydrate polymers, lipids, lipid polymers or other
synthetic or
naturally occurring chemicals or chemical polymers. As used herein, a
"chimeric
construct" refers to constructs wherein the antigen is comprised of one type
of
molecule in association with a PAMP or PAMP mimetic, which is comprised of
either
the same type of molecule or a different type of molecule.
"CpG" refers to a dinucleotide in which an unmethylated cytosine (C) residue
occurs immediately 5' to a guanosine (G) residue. As used herein, "CpG-DNA"
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refers to a synthetic CpG repeat, intact bacterial DNA containing CpG motifs,
or a
CpG-containing derivative thereof. The immunostimulatory effect of CpG-DNA on
B-cells is mediated through a TLR and is dependent upon a "protein adapter
molecule".
"Derivative" refers to any molecule or compound that is structurally related
to
the molecule or compound from which it is derived. As used herein,
"derivative"
includes peptide mimetics (e.g., PAMP mimetics).
"Domain" refers to a portion of a protein with its own function. The
combination of domains in a single protein determines its overall function. An
"antigen domain" comprises an antigen or an immunogenic portion or derivative
of an
antigen. A "PAMP domain" comprises a PAMP or a PAMP mimetic or an
immunostimulatory portion or derivative of a PAMP or a PAMP mimetic.
"Fusion protein" and "chimeric protein" both refer to any protein fusion
comprising two or more domains selected from the following group consisting of
proteins, peptides, lipoproteins, lipopeptides, glycoproteins, glycopeptides,
mucoproteins, mucopeptides, such that at least two of the domains are either
from
different species or encoded by different genes or such that one of the two
domains is
found in nature and the second domain is not known to be found in nature or
such that
one of the two domains resembles a molecule found in nature and the other does
not
resemble that same molecule. The term "fusion protein" also refers to an
antigen or
an immunogenic portion or derivative thereof which has been modified to
contain an
amino acid sequence that results in post-translational modification of that
amino acid
sequence or a portion of that sequence, wherein the post-translationally
modified
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sequence is a ligand for a PRR. As yet another definition of a fusion protein,
in the
foregoing sentence, the amino acid sequence that results in post-translational
modification to form a ligand for a PRR can comprise a consensus sequence, or
that
amino acid sequence can contain a leader sequence and a consensus sequence.
"Hapten" refers to a small molecule that is not by itself immunogenic but can
bind antigen receptors and can combine with a larger carrier molecule to
become
immunogenic.
"In association with" refers to a reversible union between two chemical
entities, whether alike or different, to form a more complex substance.
"In combination with" refers to either a reversible or irreversible (e.g.
covalent) union between two chemical entities, whether alike or different, to
form a
more complex substance.
"Immunostimulatory" refers to the ability of a molecule to activate either the
adaptive immune system or the innate immune system. As used herein, "antigens"
are examples of molecules that are capable of stimulating the adaptive immune
system, whereas PAMPs or PAMP mimetics are examples of molecules that are
capable of stimulating the innate immune system. As used herein, "activation"
of
either immure system includes the production of constituents of humoral and/or
cellular immune responses that are reactive against the immunostimulatory
molecule.
"Innate immunity" refers to the innate immune system, which, unlike the
"adaptive immune system", uses a set of germline-encoded receptors for the
recognition of conserved molecular patterns present in microorganisms.
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"Innate immune response" refers to a response involving the characteristics of
the "innate immune system" described above.
"Isolated" refers to a substance, cell, tissue, or subcellular component that
is
separated from or substantially purified with respect to a mixture or
naturally
occurnng material.
"Linker" refers to any chemical entity that links one chemical moiety to
another chemical moiety. Thus, something that chemically or physically
connects a
PAMP and an antigen is a linker. Examples of linkers include, but are not
limited to,
complex or simple hydrocarbons, nucleosides, nucleotides, nucleotide
phosphates,
oligonucleotides, polynucleotides, nucleic acids, amino acids, small peptides,
polypeptides, carbohydrates (e.g., monosaccharides, disaccharides,
trisaccharides),
and lipids. Additional examples of linkers are provided in the Detailed
Description
Selection included herein. Without limitation, the present invention also
contemplates
using a peptide bond or an amino acid or a peptide linker to link a
polypeptide PAMP
and a polypeptide antigen. The present invention further contemplates
preparing such
a linked molecule by recombinant DNA procedures. A linker can also function as
a
spacer.
"Malignant" refers to an invasive, spreading tumor.
"Microorganism" refers to a living organism too small to be seen with the
naked eye. Microorganisms include, but are not limited to bacteria, fungi,
protozoans,
microscopic algae, and also viruses.
"Mimetic" refers to a molecule that closely resembles a second molecule and
has a similar effect or function as that of the second molecule.
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"Moiety" refers to one of the component parts of a molecule. While there are
normally two moieties in a single molecule, there may be more than two
moieties in a
single molecule.
"Molecular pattern" refers to a chemical structure or motifthat is typically a
component of microorganisms, or certain other organisms, but which is not
typically
produced by normal human cells or by other normal animal cells. Molecular
patterns
are found in, or composed of, the following types of molecules:
lipopolysaccharides,
peptidoglycans, lipoteichoic acids, phosphatidyl cholines, lipoproteins,
bacterial
DNAs, viral single and double-stranded RNAs, certain viral glycoproteins,
unmethylated CpG-DNAs, mannans, and a variety of other bacterial, fungal and
yeast
cell wall components and the like.
"Non-protein chimeric construct" or "non-protein chimera" refers to a
"chimeric construct" wherein either the antigen or the PAMP or the PAMP
mimetic,
or two or more of them, is not an amino acid polymer.
"Pathogen-Associated Molecular Pattern" or "PAMP" refers to a molecular
pattern found in a microorganism but not in humans, which, when it binds a
PRR, can
trigger an innate immune response. Thus, as used herein, the term "PAMP"
includes
any such microbial molecular pattern and is not limited to those associated
with
pathogenic microorganisms or microbes. As used herein, the term "PAMP"
includes
a PAMP, derivative or portion of a PAMP that is immunostimulatory, and any
immunostimulatory molecule derived from any PAMP. These structures, or
derivatives thereof, are potential initiators of innate immune responses, and
therefore,
ligands for PRRs, including Toll receptors and TLRs. "PAMPs" are
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immunostimulatory structures that are found in, or composed of molecules
including,
but not limited to, lipopolysaccharides; phosphatidyl choline; glycans,
including
peptidoglycans; teichoic acids, including lipoteichoic acids; proteins,
including
lipoproteins and lipopeptides; outer membrane proteins (OMPs), outer surface
proteins (OSPs) and other protein components of the bacterial cell walls and
Flagellins; bacterial DNAs; single and double-stranded viral RNAs;
unmethylated
CpG-DNAs; mannans; mycobacterial membranes; porins; and a variety of other
bacterial and fungal cell wall components, including those found in yeast.
Additional
examples of PAMPs are provided in the Detailed Description section included
herein.
"PAMP/antigen conjugate" refers to an antigen and a PAMP or PAMP mimetic that
are covalently or noncovalently linked. A conjugate may be comprised of a
protein
PAMP or antigen and a non-protein PAMP or antigen.
"PAMP/antigen fusion" or "PAMP/antigen chimera" refers to any protein
fusion formed between a PAMP or PAMP mimetic and an antigen.
"Passive immunization" refers to the administration of antibodies or primed
lymphocytes to an individual in order to confer immunity.
"PAMP mimetic" refers to a molecule that, although it does not occur in
microorganisms, is analogous to a PAMP in that it can bind to a PRR and such
binding can trigger an innate immune response. A PAMP mimetic can be a
naturally-
occurring molecule or a partially or totally synthetic molecule. As an
example, and
not by way of limitation, certain plant alkaloids, such as taxol, are PRR
ligands, have
an immunostimulatory effect on the innate immune system, and thus behave as
PAMP
mimetics. (Kawasaki et al. (2000) J. Biol. Che~ra. 275(4): 2251-2254).
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"Pattern Recognition Receptor" or "PRR" refers to a member of a family of
receptors of the innate immune system that, upon binding a PAMP, an
immunostimulatory portion or derivative thereof, can initiate an innate immune
response. Members of this receptor family are structurally different and
belong to
several different protein families. Some of these receptors recognize PAMPs
directly
(e.g., CD14, DEC205, collectins), while others (e.g., complement receptors)
recognize
the products generated by PAMP recognition. Members of these receptor families
can, generally, be divided into three types: 1) humoral receptors circulating
in the
plasma; 2) endocytic receptors expressed on immune-cell surfaces, and 3)
signaling
receptors that can be expressed either on the cell surface or intracellularly.
Cellular
PRRs may be expressed on effector cells of the innate immune system, including
cells
that function as professional APCs in adaptive immunity, and also on cells
such as
surface epithelia that are the first to encounter pathogens during infection.
PRRs may
also induce the expression of a set of endogenous signals, such as
inflammatory
cytokines and chemokines. Examples of PRRs useful for the present invention
include, but are not limited to, the following: C-type lectins (e.g., humoral,
such as
collectins (MBL), and cellular, such as macrophage C-type lectins, macrophage
mannose receptors, DEC205); proteins containing leucine-rich repeats (e.g.,
Toll
receptor and TLRs, CD14, RP105); scavenger receptors (e.g., macrophage
scavenger
receptors, MARCO, WC1); and pentraxins (e.g., C-reactive proteins, serum,
amyloid
P, LBP, BPIF, CDllb,C and CD18.
"Peptide mimetic" refers to a protein or peptide that closely resembles a non-
protein molecule and has a similar effect or function to the non-protein
molecule.
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Alternatively, a peptide mimetic can be a non-protein molecule or non-peptide
molecule that closely resembles a peptide or protein and has a similar effect
or
function to the peptide or protein.
"Pharmaceutically acceptable carrier" refers to a carrier that can be
tolerated
by a recipient animal, typically a mammal.
"Protein chimeric construct" refers to a chimeric construct wherein both the
antigen and the PAMP or PAMP mimetic are amino acid polymers.
"Recombinant" refers to genetic material that is produced by splicing genes,
gene derivatives or other genetic material. As used herein, "recombinant" also
refers
to the products produced from recombinant genes (e.g. recombinant protein).
"Spacer" refers to any chemical entity placed between two chemical moieties
that serves to physically separate the latter two moieties. Thus, a chemical
entity
placed between a PAMP or PAMP mimetic and an antigen is a spacer. Examples of
spacers include, but are not limited to, nucleic acids (e.g. untranscribed DNA
between
two stretches of transcribed DNA), amino acids, carbohydrates (e.g.,
monosaccharides, disaccharides, trisaccharides), and lipids.
"Strong immune response" refers to an immune response, induced by the
chimeric construct, that has about the same intensity or greater than the
response
induced by an antigen mixed with Complete Freund's Adjuvant (CFA).
"Therapeutically effective amount" refers to an amount of an agent (e.g., a
vaccine) that can produce a measurable positive effect in a patient.
"Toll-like receptor" (TLR) refers to any of a family of receptor proteins that
are homologous to the Drosophila melahogaster Toll protein. TLRs also refer to
type
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I transmembrane signaling receptor proteins that are characterized by an
extracellular
leucine-rich repeat domain and an intracellular domain homologous to that of
the
interleukin 1 receptor. The TLR family includes, but is not limited to, mouse
TLR2
and TLR4 and their homologues, particularly in other species including humans.
This
invention also defines Toll receptor proteins and TLRs wherein the nucleic
acids
encoding such proteins have at least about 70% sequence identity, more
preferably, at
least about 80% sequence identity, even more preferably, at least about 85%
sequence
identity, yet more preferably at least about 90% sequence identity, and most
preferably at least about 95% sequence identity to the nucleic acid sequence
encoding
the Toll protein and the TLR proteins TLR2, TLR4 and other members of the TLR
family. In addition, this invention also contemplates Toll receptors and TLRs
wherein
the amino acid sequences of such Toll receptors and TLRs have at least about
70%
sequence identity, more preferably, at least about 80% sequence identity, even
more
preferably, at least about 85% sequence identity, yet more preferably at least
about
90% sequence identity, and most preferably at least about 95% sequence
identity to
the amino acid sequences of the Toll protein and the TLRs, TLR2, TLR4 and
their
homologues.
"Tumor" refers to a mass of proliferating cells lacking, to varying degrees,
normal growth control. As used herein, "tumors'.' include, at one extreme,
slowly
proliferating "benign" tumors, to, at the other extreme, rapidly proliferating
"malignant" tumors that aggressively invade neighboring tissues.
"Vaccine" refers to a composition comprising an antigen, and optionally other
ancillary molecules, the purpose of which is to administer such compositions
to a
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subject to stimulate an immune response specifically against the antigen and
preferably to engender immunological memory that leads to mounting of an
immune
response should the subject encounter that antigen at some future time.
Examples of
other ancillary molecules are adjuvants, which are non-specific
immunostimulatory
molecules, and other molecules that improve the pharniacokinetic and/or
pharmacodynamic properties of the antigen. Conventionally, a vaccine usually
consists of the organism that causes a disease (suitably attenuated or killed)
or some
part of the pathogenic organism as the antigen. Attenuated organisms, such as
attenuated viruses or attenuated bacteria, are manipulated so that they lose
some or all
of their ability to grow in their natural host. There are now a range of
biotechnological approaches used to producing vaccines. (See, e.g., W. Bains
(1998)
Biotechnology From A to Z, Second Edition, Oxford University Press). The
various
methods include, but are not limited to, the following:
1) Viral vaccines consisting of genetically altered viruses. The viruses
can be engineered so that they are harmless but can still replicate (albeit
inefficiently, sometimes) in cultured animal cells. Another approach is to
clone the gene for a protein from a pathogenic virus into another, harmless
virus, so that that resulting, engineered virus has certain immunologic
properties of the pathogenic virus but does not cause any disease. Examples
of the latter method include, but are not limited to, altered vaccinia and
adenoviruses used as rabies vaccines for distribution with meat bait, and a
vaccinia virus engineered to produce haemagglutinin and fusion proteins of
rindepest virus of cattle;
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2) Enhanced bacterial vaccines consisting of bacteria genetically
engineered to enhance their value as vaccines when the bacteria are dead
(e.g.,
E. coli vaccine for pigs, bacterial vaccine for furunculosis in salmon).
Recombinant DNA techniques can be used to delete pathogenesis-causing
genes in the bacteria or to engineer the protective epitope from a pathogen
into
a safe bacterium;
3) Biopharmaceutical vaccines consist of proteins, or portions of
proteins, that are the same as the proteins in a viral, fungal or bacterial
coat or
wall, which can be made by recombinant DNA methods;
4) Multiple antigen peptides (MAPS) are peptide vaccines that are
chemically attached (usually on a polylysine backbone), enabling several
vaccines to be delivered at the same time;
5) Polyprotein vaccines consist of a single protein made by genetic
engineering so that the different peptides from the organisms of interest form
part of a continuous polypeptide chain; and
6) Vaccines produced in transgenic plants that can be used as food to
provide oral vaccines (e.g., vaccine delivery by eating bananas).
3. Specific Embodiments
A. Fusion Proteins
The present invention is based in part on the unexpected discovery that
vaccines comprising chimeric constructs of a PAMP and an antigen (e.g., the
fusion
protein BLP/Ea) exhibit the essential immunological characteristics or
properties
expected of a conventional vaccine supplemented with an adjuvant.
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In one aspect, the present invention is based on the finding that BLPBa
induces activation of NF-xB and production of IL-6 in macrophages and
dendritic
cells, respectively, demonstrating that the vaccine is capable of activating
the innate
immune system. The activity of BLP/Ea is comparable to that of LPS, and is not
due
to endotoxin contamination, as demonstrated by the lack of inhibition by
polymyxin
B.
In another aspect, the present invention is based on the fording that the
BLP/Ea fusion protein induces maturation of dendritic cells, as demonstrated
by the
induction of the cell surface expression of the co-stimulatory molecule, B7.2.
Additionally, BLP/Ea is appropriately targeted to the antigen processing and
presentation pathway, and a functional Ea peptide/MHC class-II complex is
generated. This result is demonstrated by FAGS analysis using an antibody
specific
for the Ea peptide complexed with MHC class-II.
Moreover, the present invention is based on the surprising discovery that a
recombinant vaccine comprising a BLP/Ea chimeric construct activates antigen-
specific T-cell responses ih vitro by stimulating dendritic cell activation
and
generating a specific Iigand (Ea/MHC-II) for the T-cell receptor. Furthermore,
the
results of immunization of mice with BLP/Ea and the resultant antigen-specific
T-cell
responses demonstrate that the recombinant vaccine activates antigen-specific
T-cell
responses ih vivo. For comparison, mice were immunized with Ea peptide mixed
with Complete Fremld's Adjuvant (CFA). The recombinant vaccine of the present
invention induced an immune response in the mice that is stronger than that
produced
by Ea peptide mixed with CFA.
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The present invention is also based on the surprising discovery that
immunization with the recombinant vaccines that comprise the chimeric
constructs of
the present invention induce a minimal inflammatory reaction when compared to
that
induced by an adjuvant. However, as noted above, in spite of a reduced
inflammatory
response, the vaccine unexpectedly induced a strong immune response. Thus, the
vaccine approach described in the present invention minimizes an undesired
side
effect (e.g., an excessive inflammatory reaction) yet induces a very potent
and
specific immune response. The present invention also provides fusion proteins
comprising at least one antigen molecule or antigen domain and at least one
PAMP or
PAMP mimetic for use as vaccines. Preferably, the fusion protein has maximal
immunogenicity and induces only a modest inflammatory response. Increasing the
number of antigens or antigen epitopes, by using multiple antigen proteins
and/or
multiple domains of the same antigen protein or of different antigen proteins,
andlor
some combination of the foregoing, are contemplated in this invention. It is
within
the skill of the artisan to determine the optimal ratio of PAMP to antigen
molecules to
maximize immunogenicity and minimize or control the inflammatory response.
There are several advantages of using a fusion system for the production of
recombinant polypeptides. First, heterologous proteins and peptides are often
degraded by host proteases; this may be avoided, especially for small
peptides, by
using a gene fusion expression system. Second, general and efficient
purification
schemes are established for several fusion partners. The use of a fusion
partner as an
affinity handle allows rapid isolation of the recombinant peptide. Third, by
using
different fusion partners, the recombinant product may be localized to
different
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compartments or it might be secreted; such strategy could lead to facilitation
of
purification of the fusion partner and/or directed compartmentalization of the
fusion
protein.
Additionally, various methods are available for chemical or enzymatic
cleavage of the fusion protein that provides efficient strategies to obtain
the desired
cleavage product in large quantities. Frequently employed fusion systems are
the
Staphylococcal protein A fusion system and the synthetic ZZ variant which have
IgG
affinity and have been used for the generation of antibodies against short
peptides; the
glutathione S-transferase fusion system (Smith et al. (1988) Gehe 60); the (3-
galactosidase fusion system; and the trpE fusion system (Yansura (1990)
Methods
Enzym. 185: 61). Some of these systems are commercially available as kits,
including
vectors, purification components and detailed instructions.
The present invention also contemplates modified fusion proteins having
affinity for metal (metal ion) affinity matrices, whereby one or more specific
metal-
binding or metal-chelating amino acid residues are introduced, by addition,
deletion,
or substitution, into the fusion protein sequence as a tag. Optimally, the
fusion
partner, e.g., the antigen or PAMP sequence, is modified to contain the metal-
chelating amino acid tag; however the antigen or PAMP could also be altered to
provide a metal-binding site if such modifications could be achieved without
adversely effecting a ligand-binding site, an active site, or other functional
sites,
and/or destroying important tertiary structural relationships in the protein.
These
metal-binding or metal-chelating residues may be identical or different, and
can be
selected from the group consisting of cysteine, histidine, aspartate,
tyrosine,
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tryptophan, lysine, and glutamate, and are located so to permit binding or
chelation of
the expressed fusion protein to a metal. Histidine is the preferred metal-
binding
residue. The metal-binding/chelating residues are situated with reference to
the
overall tertiary structure of the fusion protein to maximize binding/chelation
to the
metal and to minimize interference with the expression of the fusion protein
or with
the protein's biological activity.
A fusion sequence of an antigen, PAMP and a tag may optionally contain a
linker peptide. The linker peptide might separate a tag from the antigen
sequence or
the PAMP sequence. If the linker peptide so used encodes a sequence that is
selectively cleavable or digestible by conventional chemical or enzymatic
methods,
then the tag can be separated from the rest of the fusion protein after
purification. For
example, the selected cleavage site within the tag may be an enzymatic
cleavage site.
Examples of suitable enzymatic cleavage sites include sites for cleavage by a
proteolytic enzyme, such as enterokinase, Factor Xa, trypsin, collagenase, and
thrombin. Alternatively, the cleavage site in the linker may be a site capable
of
cleavage upon exposure to a selected chemical (e.g., cyanogen bromide,
hydroxylamine, or low pH).
Cleavage at the selected cleavage site enables separation of the tag from the
antigen/PAMP fusion protein. The antigen/PAMP fusion protein may then be
obtained in purified form, free from any peptide fragment to which it was
previously
linked for ease of expression or purification. The cleavage site, if inserted
into a
linker useful in the fusion sequences of this invention, does not limit this
invention.
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Any desired cleavage site, of which many are known in the art, may be used for
this
purpose.
The optional linker peptide in a fusion protein of the present invention might
serve a purpose other than the provision of a cleavage site. As an example,
and not by
limitation, the linker peptide might be inserted between the PAMP and the
antigen to
prevent or alleviate steric hindrance between the two domains. In addition,
the linker
sequence might provide for post-translational modification including, but not
limited
to, e.g., phosphorylation sites, biotinylation sites, sulfation sites,
carboxylation sites,
lipidation sites, glycosylation sites and the like.
In one embodiment, the fusion protein of this invention contains an antigen
sequence fused directly at its amino or carboxyl terminal end to the sequence
of a
PAMP. In another embodiment, the fusion protein of this invention, comprising
an
antigen and a PAMP sequence, is fused directly at its amino or carboxyl
terminal end
to the sequence of a tag. The resulting fusion protein is a soluble
cytoplasmic fusion
protein. In another embodiment, the fusion sequence further comprises a linker
sequence interposed between the antigen, sequence and a PAMP sequence or
sequence
of a tag. This fusion protein is also produced as a soluble cytoplasmic
protein.
B. Anti_~ens
As used herein, an "antigen" is any substance that induces a state of
sensitivity
and/or immune responsiveness after any latent period (normally, days to weeks
in
humans) and that reacts in a demonstrable way with antibodies and/or immune
cells
of the sensitized subject if2 vivo or in vitro. Examples of antigens include,
but are not
limited to, (1) microbial-related antigens, especially antigens of pathogens
such as
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viruses, fungi or bacteria, or immunogenic molecules derived from them; (2)
"self
antigens, collectively comprising cellular antigens including cells containing
normal
transplantation antigens and/or tumor-related antigens, RR-Rh antigens and
antigens
characteristic of, or specific to particular cells or tissues or body fluids;
(3) allergen-
related antigens such as those associated with environmental allergens (e.g.,
grasses,
pollens, molds, dust, insects and dander), occupational allergens (e.g.,
latex, dander,
urethanes, epoxy resins), food (e.g., shellfish, peanuts, eggs, milk
products), drugs
(e.g., antibiotics, anesthetics) and (4) vaccines (e.g., flu vaccine).
Antigen processing and recognition of displayed peptides by T-lymphocytes
depends in large part on the amino acid sequence of the antigen rather than
the three-
dimensional structure of the antigen. Thus, the antigen portion used in the
vaccines of
the present invention can contain epitopes or specific domains of interest
rather than
the entire sequence. In fact, the antigenic portions of the vaccines of the
present
invention can comprise one or more immunogenic portions or derivatives of the
antigen rather than the entire antigen. Additionally, the vaccine of the
present
invention can contain an entire antigen with intact three-dimensional
structure or a
portion of the antigen that maintains a three-dimensional structure of an
antigenic
determinant, in order to produce an antibody response by B-lymphocytes against
a
spatial epitope of the antigen.
1. Pathogen-Related Antigens. Specific examples of pathogen-related
antigens include, but are not limited to, antigens selected from the group
consisting of
vaccinia, avipox virus, turkey influenza virus, bovine leukemia virus, feline
leukemia
virus, avian influenza, chicken pneumovirosis virus, canine parvovirus, equine
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influenza, FHV, Newcastle Disease Virus (NDV), Chicken/Pennsylvaniall/83
influenza virus, infectious bronchitis virus; Dengue virus, measles virus,
Rubella
virus, pseudorabies, Epstein-Barr Virus, HIV, SIV, EHV, BHV, HCMV, Hantaan, C.
tetani, mumps, Morbillivirus, Herpes Simplex Virus type 1, Herpes Simplex
Virus
type 2, Human cytomegalovirus, Hepatitis A Virus, Hepatitis B Virus, Hepatitis
C
Virus, Hepatitis E Virus, Respiratory Syncytial Virus, Human Papilloma Virus,
Tnfluenza Virus, Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella, and
Plasmodium and Toxoplasma, Cryptococcus, Streptococcus, Staphylococcus,
Haenaoplailus, Diptheria, Tetanus, Pertussis, Escherichia, Gandida,
Aspergillus,
Entamoeba, Giardia, and Trypanasonaa.
2. Cancer-Related Antigens. The methods and compositions of the present
invention can also be used to produce vaccines directed against tumor-
associated
protein antigens such as melanoma-associated antigens, mammary cancer-
associated
antigens, colorectal cancer-associated antigens, prostate cancer-associated
antigens
and the like.
Specific examples of tumor-related or tissue-specific protein antigens useful
in
such vaccines include, but are not limited to, antigens selected from the
group in the
following table.
Cancer a Anti ens
Prostate prostate-specific antigen (PSA), prostate-specific
membrane anti en PSMA , Her-2neu, SPAS-1
Melanoma TRP-2, tyrosinase, Melan A/Mart-1, gp100,
BAGS,
GAGE, GM2 an lioside
Breast Her2-neu, kinesin 2, TATA element modulatory
factor 1,
tumor protein D52, MAGE D, ING2, HIP-55,
TGF -1
anti-a: o totic factor, HOM-Mel-40/SSX2
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Testis MAGE-1, HOM-Mel-401SSX2, NY-ESO-1
Colorectal EGFR, CEA
Lun MAGE D, EGFR
Ovarian Her-2neu
Several cancers NY-ESO-1, glycoprotein MLJC1 and MUC10
mucins,
53 es eciall mutated versions , EGFR
Miscellaneous CDC27 (including the mutated form of
tumor the protein),
anti ens triose hos hate isomerase
In order for tumors to give rise to proliferating and malignant cells, they
must
become vascularized. Strategies that prevent tumor vascularization have the
potential
for being therapeutic. The methods and compositions of the present invention
can also
be used to produce vaccines directed against tumor vascularization. Examples
of
target antigens for such vaccines are vascular endothelial growth factors,
vascular
endothelial growth factor receptors, fibroblast growth factors and fibroblast
growth
factor receptors and the like.
3. Allergen-Related Antigens. The methods and compositions of the present
invention can be used to prevent or treat allergies and asthma. According to
the
present invention, one or more protein allergens can be linked to one or more
PAMPs,
producing a PAMP/allergen chimeric construct, and admiW stered to subj ects
that are
allergic to that antigen. Thus, the methods and compositions of the present
invention
can also be used to construct vaccines that may suppress allergic reactions.
In this
case, the allergen is associated with or combined with a PAMP, including but
not
limited to BLP or Flagellin, that can initiate a Thl response upon binding to
a TLR.
Initiation of innate immunity via a TLR, fox example, tends to be
characterized by
production and secretion of cytokines, such as IL-12, that elicit a so-called
Thl
response in a subject, rather than the typical Th2 response that triggers B-
cells to
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produce immunoglobulin E, the initiator of typical allergic and/or
hypersensitive
responses. IL-12 produced by dendritic cells and macrophages upon PAMP binding
to their TLRs will direct T-cell differentiation into Thl effector cells.
Cytokines
produced by Thl cells, such as Interferon-gamma, will block the
differentiation of IL-
4 producing Th2 cells and would thus prevent production of antibodies of the
IgE
isotype, which are responsible for allergic responses.
Specific examples of allergen-related protein antigens useful in the methods
and compositions of the present invention include, but are not limited to:
allergens
derived from pollen, such as those derived from trees such as Japanese cedar
(CYyptonaeria, CYyptofraeria japonica), grasses (Gramineae), such as orchard-
grass
(Dactylic, Dactylic glomerata), weeds such as ragweed (Ambrosia, Ambrosia
aYtemisiifolia); specific examples of pollen allergens including the Japanese
cedar
pollen allergens Cry j 1 (J. AlleYgy Clin. Immunol. (1983)71: 77-86) and Cry j
2
(FEBS Letters (1988) 239: 329-332), and the ragweed allergens Amb a L 1, Amba
L2,
Amb a L3, Amb a L4, Amb a II etc.; allergens derived from fungi (Aspergillus,
Candida, Alternaria, etc.); allergens derived from mites (allergens from
Dey~matophagoides pteronyssinus, Dermatophagoides farinae etc.; specific
examples
of mite allergens including Der p I, Der p II, Der p III, Der p VII, Der f I,
Der f II, Der
f III, Der f VII etc.); house dust; allergens derived from animal skin debris,
feces and
hair (for example, the feline allergen Fel d I); allergens derived from
insects (such as
scaly hair or scale of moths, butterflies, Chi~~onomidae etc., poisons of the
hespidae,
such as Vespa mandarinia); food allergens (eggs, milk, meat, seafood, beans,
cereals,
fruits, nuts and vegetables etc.); allergens derived from parasites (such as
roundworm
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and nematodes, for example, Anisakis); and protein or peptide based drugs
(such as
insulin). Many of these allergens are commercially available.
In another embodiment, prophylactic treatment of chronic allergies can be
accomplished by the administration of a protein PAMP. In a preferred
embodiment,
the PAMP of the prophylactic vaccine is an OMP, more preferably OspA, and most
preferably BLP. Alternatively, Flagellin can be used as the PAMP.
4. Other Disease Anti ens. Also contemplated in this invention are vaccines
directed against antigens that are associated with diseases other than cancer,
allergy
and asthma. As one example of many, and not by limitation, an extracellular
accumulation of a protein cleavage product of (3-amyloid precursor protein,
called
"amyloid-(3 peptide", is associated with the pathogenesis of Alzheimer's
disease.
(Janus et al., Nature (2000) 408: 979-982; Morgan et al., Nature (2000) 408:
982-
985). Thus, the chimeric construct used in the vaccines of the present
invention can
include amyloid-[3 peptide, or antigenic domains of amyloid-(3 peptide, as the
antigenic portion of the construct, and a PAMP or PAMP mimetic. Examples of
other
diseases in which vaccines might be generated against self proteins or self
peptides
are shown in the following table.
Disease Anti ens
Autoimmune diseases disease-linked HLA-alleles
(e.g., HLA-
DRB l, HLA-DRl, HLA-DR6 B 1
proteins or fragments thereof,
chain genes); TCR chain sub-groups;
CDlla (leukocyte function-associated
antigen 1; LFA-1); IFNy; IL-lO;TCR
analogs; IgR analogs; 21-hydoxylase
(for
Addison's disease); calcium
sensing
rece for for ac wired
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hypoparathyroidism); tyrosinase
(for
vitiligo)
Cardiovascular disease LDL rece for
Diabetes glutamic acid decarboxylase
(GAD);insulin B chain; PC-1;
IA-2, IA-
2b; GLIMA-3 8
E ile s NMDA
C. PAMPs
PAMPs are discrete molecular structures that are shared by a large group of
microorganisms. They are conserved products of microbial metabolism, which are
not subj ect to antigenic variability and are distinct from self antigens.
(Medzlutov et
al. (1997) CurYent Opinion in Immunology 9: 4).
PAMPs can be composed of or found in, but are not limited to, the following
types of molecules: Flagellins, lipopolysaccharides (LPS), porins, lipid A-
associated
proteins (LAP), lipopolysaccharides, fimbrial proteins, unmethylated CpG
motifs,
bacterial DNAs, double-stranded viral RNAs, mannans, cell wall-associated
proteins,
heat shock proteins, glycoproteins, lipids, cell surface polysaccharides,
glycans (e.g.,
peptidoglycans), phosphatidyl cholines, teichoic acids (e.g., lipoteichoic
acids),
mycobacterial cell wall components/membranes, bacterial lipoproteins (BLP),
outer
membrane proteins (OMP), and outer surface protein A (Osp A). (Henderson et
al.
(1996) Microbiol. Review 60: 316; Medzhitov et al. (1997) Cu~~rent Opinion in
Immunology 9: 4-9).
The preferred PAMPs of the present invention include those that contain a
DNA-encoded protein component, such as BLP, Neisseria porin, OMP, Flagellin
and
OspA, as these can be used as fusion partners to prepare the preferred
embodiment of
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the invention, i.e., fusion proteins comprising a PAMP and an antigen,
preferably a
self antigen. One preferable PAMP for use in the present invention is BLP
because
BLP is known to induce activation of the innate immune response (Henderson et
al.
(1996) MicYObiol. Review 60: 316) and has been shown to be recognized by TLRs
(Aliprantis et al. (1999) Science 285: 763). Flagellin has similarly been
demonstrated
to induce features of innate immunity (Eaves-Pyles et al., (2001) J. Immunol.
166:1248; Gewirtz et al., (2001) .l Clin Invest. 107: 99); Aderem,
Py~esehtation at
Keystorze Symposium, Keystone, CO, 2001 ).
Additionally, the present invention contemplates derivatives, portions, parts,
or peptides of PAMPs that are recognized by the innate immune system for
generating
vaccines. As used herein, the terms "fragments of PAMPs", "portions of PAMPs",
"parts of PAMPs" and "peptides of PAMPs", all refer to an immunostimulatory
part
of an entire PAMP molecule. Thus, the PAMPs used in the vaccines of the
present
invention can comprise an immunostimulatory portion or derivative of the PAMP
rather than the entire PAMP, for example E. Coli murein lipoprotein amino
acids 1 to
24.
In another embodiment, the present invention contemplates peptide mimetics
of non-protein PAMPs. Peptide mimetics of polysaccharides and peptidoglycans
are
examples of peptide mimetics which can be used in the present invention. The
present invention contemplates using phage selection methods to identify
peptide
mimetics of these non-protein PAMPs. For example, an antibody raised against a
non-protein PAMP can be used to screen a phage library containing randomized
short-peptide sequences. Selected sequences are isolated and assayed to
determine
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their usefulness as a protein derivative of a non-protein PAMP in the chimeric
constructs of the present invention. Such peptide mimetics are useful to
produce the
recombinant vaccines disclosed herein.
In yet another embodiment, the present invention contemplates further
examples of PAMP mimics or PAMP mimetics in which analogs of amino acids
and/or peptides are substituted for the amino acid and/or peptide residues,
respectively, of a peptide-containing PAMP or a protein PAMP.
In another embodiment, the chimeric construct is a construct comprising CpG
or CpG-DNA, and an antigen. The CpG or CpG-DNA can be conjugated to a protein
or non-protein antigen. In addition, peptide mimetics of CpG or CpG-DNA, that
mimic the structural, functional, antigenic or immunogenic properties of CpG,
can be
produced and used to generate an antigen-PAMP (where PAMP is a CpG peptide
mimetic) protein chimeric construct. This chimeric construct can be produced
by
recombinant DNA techniques and the expressed fusion protein can be used in the
compositions and methods of the present invention.
D. Peptide Mimetics
This invention also includes a mimetic of the three-dimensional structure of a
PAMP or antigen. In particular, this invention also includes peptides that
closely
resemble the three-dimensional structure of non-peptide PAMPs and antigens.
Such
peptides provide alternatives to non-polypeptide PAMPs or antigens,
respectively, by
providing the advantages of, for example: more economical production, greater
chemical stability, enhanced pharmacological properties (half life,
absorption,
4S
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potency, efficacy, and/or altered specificity (e.g., a broad-spectrum of
biological
activities), and other advantages.
Conversely, analogs of PAMP and/or antigen proteins can be synthesized such
that one or both consists partially or entirely of amino acid and /or peptide
analogs.
Such analogs can contain non-naturally-occurring amino acids, or naturally-
occurring
amino acids that do not commonly occur in proteins, including but not limited
to, D-
amino acids or amino acids such as (3-alanine, ornithine or canavanine, and
the like,
many of which are known in the art. Alternatively, analogs of PAMPs and/or
antigens can be synthesized such that one or both consists partially or
entirely of
peptide analogs containing non-peptide bonds, many examples of which are known
in
the art. Such analogs may provide greater chemical stability, enhanced
pharmacological properties (half life, absorption, potency, efficacy, etc.)
and/or
altered specificity (e.g., a broad-spectrum of biological activities) when
compared
with the naturally-occurring PAMP and/or antigen as well as other advantages.
In one form, the contemplated molecular structures are peptide-containing
molecules that mimic elements of protein secondary structure. (see, for
example,
Johnson et al. (1993) Peptide Turn Mimetics, in Biotechnology and Pharmacy,
Pezzuto et al., (editors) Chapman and Hall). Such molecules are expected to
permit
molecular interactions similar to the natural molecule.
In another form, analogs of peptides are commonly used in the pharmaceutical
industry as non-peptide drugs with properties analogous to those of a subject
peptide.
These types of non-peptide compounds are also referred to as "peptide
mimetics" or
"peptidomimetics" (Fauchere (1986) Adv. Drug Res.15, 29-69; Veber et al.
(1985)
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Trends Neuy~osci. 8: 392-396; Evans et al. (1987) J. Med. Claem. 30: 1229-
1239) and
are usually developed with the aid of computerized molecular modeling.
Peptide mimetics that are structurally similar to therapeutically useful
peptides
may be used to produce an equivalent therapeutic or prophylactic effect.
Generally,
peptide mimetics are structurally similar to a paradigm polypeptide (e.g., a
polypeptide that has a biochemical property or pharmacological activity), but
have
one or more peptide linkages optionally replaced by a linkage selected from
the group
consisting of: -CHzNH-, -CHZS-, -CHa-CH2-, -CH=CH- (cis and trans), -COCH2-, -
CH(OH)CHZ-, -CHZSO- and the like. (Money (1980) TYends Pharmacol. Sci. 1: 463-
468 (general review); Hudson et al. (1979) hat. J. Pept. Protein Res. l4: 177-
185 (-
CH2NH-, CHzCH2-); Spatola et al. (1986) Life Sci. 38: 1243-1249 (-CHz-S); Hann
(1982) J. Chem. Soc. Perkin Trans. 1: 307-314 (-CH-CH-, cis and trans);
Alinquist et
al. (1980) J. Med. Chem. 23: 1392-1398 (-COCHz-); Jennings-White et al. (1982)
Tetrahedron Lett. 23: 2533 (-COCHZ-); Holladay et al. (1983) TetrahedYOn Lett.
24:
4401-4404 (-C(OH)CHa-); and Hruby (1982) Life Sci. 31: 189-199 (-CHzS-); each
of
which is incorporated herein by reference.).
Labeling of peptide mimetics usually involves covalent attachment of one or
more labels, directly or through a spacer (e.g., an amide group), to non-
interfering
positions) on the peptide mimetic that are predicted by quantitative structure-
activity
data and molecular modeling. Such non-interfering positions generally are
positions
that do not form direct contacts with the macromolecules) (e.g., in the
present
example they are not contact points in PAMP-PRR complexes) to which the
peptide
mimetic binds to produce the therapeutic effect. Derivitization (e.g.,
labeling) of
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peptide mimetics should not substantially interfere with the desired
biological or
pharmacological activity of the peptide mimetic.
PAMP peptide mimetics can be constructed by structure-based drug design
through replacement of amino acids by organic moieties. (Hughes (1980) Philos.
Trans. R. Soc. Lond. 290: 387-394; Hodgson (1991) BiotechfZOl. 9: 19-21;
Suckling
(1991) Sci. Prog. 75: 323-359).
The design of peptide mimetics can be aided by identifying amino acid
mutations that increase or decrease binding of PAMP to its PRR. Approaches
that
can be used include the yeast two-hybrid method (Chien et al. (1991) P~oc.
Natl.
Acad. Sci. USA 88: 9578-9582) and using the phage display method. The two-
hybrid
method detects protein-protein interactions in yeast. (Fields et al. (1989)
Nature 340:
245-246). The phage display method detects the interaction between an
immobilized
protein and a protein that is expressed on the surface of phages such as
lambda and
M13. (Amberg et al. (1993) Strategies 6: 2-4; Hogrefe et al. (1993) GefZe 128:
119-
126). These methods allow positive and negative selection for protein-protein
interactions and the identification of the sequences that determine these
interactions.
Conventional methods of peptide synthesis are known in the art. (Jones
(1992) Amino Acid and Peptide Synthesis, Oxford University Press; Jung (1997)
Combinatorial Peptide and Nonpeptide Libraries: A Handbook, John Wiley;
Bodanszky et al. (1993) Peptide Chemistry - A Practical Textbook, Springer
Verlag).
E. Fla~ellin PAMPs
Bacterial flagella are made up of the structural protein Flagellin, which
induces expression of chemokine IL-8 and activation of NF-~cB in human and
mouse
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cells. Additionally Flagellin activates mammalian cells via a Toll-Like
Receptor,
TLRS. These findings, as well as the fact that Flagellin proteins are
extremely
conserved in bacteria, indicate that Flagellin is a pathogen-associated
molecular
pattern (PAMP) that would be recognized by the innate immune system.
Because Flagellin is a protein and a PAMP, it is also be suitable for the
generation of recombinant fusion vaccines. As described in the Examples
section
below, a series of fusion constructs were tested for their ability to activate
the
mammalian innate immune system. Activation of NF-~cB was used as a read-out in
the experiments because it is a critical pathway indicative of the triggering
of the Toll-
Like Receptors, and has been demonstrated to be a property of the recombinant
fusion
vaccines.
F. Conservative Variants of PAMPs
The present invention also contemplates conservative variants of naturally-
occurring protein PAMPs, peptides of PAMPs, and peptide mimetics of PAMPs that
recognize the corresponding PRRs. Such variants are examples of PAMP mimetics.
The conservative variations include mutations that substitute one amino acid
for
another within one of the following groups:
1. Small aliphatic, nonpolar or slightly polar residues: AIa, Ser, Thr, Pro
and
Gly;
2. Polar, negatively charged residues and their amides: Asp, Asn, Glu and Gln;
3. Polar, positively charged residues: His, Arg and Lys;
4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and
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5. Aromatic residues: Phe, Tyr and Trp.
The types of substitutions selected may be based on the analysis of the
frequencies of
amino acid substitutions among the PAMPs of different species (Schulz et al.
Principles of Protein Structure, Springer-Verlag, 1978, pp. 14-16) on the
analyses of
structure-forming potentials developed by Chou and Fasman (Chow et al. (1974)
Biochemistry 13: 211; Schulz et al. (1978) Principles in Protein Structure,
Springer-
Verlag, pp. 108-130), and on the analysis of hydrophobicity patterns in
proteins
developed by Kyte and Doolittle (Kyte et al. (1982) J. Mol. Biol. 157: 105-
132).
The present invention also contemplates conservative variants that do not
affect the ability of the PAMP to bind to its PRR. The present invention
includes
PAMPs with altered overall charge, structure, hydrophobicity/hydrophilicity
properties produced by amino acid substitution, insertion, or deletion that
retain
and/or improve the ability to bind to their receptor. Preferably, the mutated
PAMP
has at least about 70% sequence identity, more preferably at least about 80%
sequence
identity, even more preferably, at least about 85% sequence identity, yet more
preferably at least about 90% sequence identity, and most preferably at least
about
95% sequence identity to its corresponding wild-type PAMP.
Numerous methods for determining percent homology are known in the art.
Version 6.0 of the GAP computer program is available from the University of
Wisconsin Genetics Computer Group and utilizes the alignment method of
Needleman and Wunsch, as revised by Smith and Waterman. (Needleman et al.
(1970) J. Mol. Biol. 48: 443; Smith et al. (1981) Adv. Appl. Math. 2: 482).
Numerous methods for determining percent identity are also known in the art,
and a
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preferred method is to use the FASTA computer program, which is also available
from the University of Wisconsin Genetics Computer Group.
G. Combination Treatments
The present invention provides methods of treating subj ects comprising
passively immunizing an individual by administering antibodies or activated
immune
cells to a subject to confer immunity, and administering a vaccine comprising
a fusion
protein of the present invention, preferably wherein the administered antibody
or
activated immune cells are directed against the same antigen of the fusion
protein of
the vaccine. Such treatments can be sequential, in either order or
simultaneous. This
combination therapy contemplates the use of either monoclonal or polyclonal
antibodies that are directed against the antigen of the PAMP/antigen fusion.
The present invention provides methods of treating subjects comprising
passively immunizing an individual by administering antibodies or activated
immune
cells to a subj ect to confer immunity, and administering a vaccine comprising
a
chimeric construct of the present invention, wherein the administered antibody
or
activated immune cells are preferably directed against the same antigen of the
chimeric construct. Such treatments can be sequential, in either order, or
simultaneous. This combination therapy contemplates the use of either
monoclonal or
polyclonal antibodies that are directed against the antigen of the
PAMP/antigen
chimeric construct.
The present invention also contemplates the use of a vaccine comprising a.
chimeric construct of the present invention in combination with a second
treatment
where such second treatment is not an immune-directed therapy. A non-limiting
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example of such combination therapy is the combination of a vaccine comprising
a
fusion protein of the present invention in combination with a chemotherapeutic
agent,
such as an anti-cancer chemotherapeutic agent. A further non-limiting example
of
such combination therapy is the combination of a vaccine comprising a fusion
protein
construct of the present invention in combination with an anti-angiogenic
agent. A
further non-limiting example of such combination therapy is the combination of
a
vaccine comprising a fusion protein of the present invention in combination
with
radiation therapy, such as an anti-cancer radiation therapy. Yet a further non-
limiting
example of combination therapy is the combination of a vaccine comprising a
fusion
protein of the present invention in combination with surgery, such as surgery
to
remove or reduce vascular blockage.
Also contemplated in this invention is a combination of more than one other
therapeutic with a vaccine contemplated in this invention. A non-limiting
example is
a combination of a vaccine contemplated in this invention in combination with
passive immunotherapy treatment and chemotherapy treatment.
In such combination treatments as can be contemplated herein, treatments can
be
sequential or simultaneous.
The PAMP domain can comprise the entire PAMP or an immunostimulatory
portion of the PAMP. Preferably, the fusion protein has maximal immunogenicity
and induces minimal inflammatory response. Such desirable properties might be
achieved, for example, by using two or more different antigens, and/or
portions of
different antigens, and/or by using more than one copy of the same antigen or
portions
of the same antigen, and/or by a combination of both. Alternatively, two or
more
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different PAMPs, or portions of different PAMPs, and/or two or more copies of
the
same PAMP, or portions of the same PAMP, and/or a combination of both can be
used. A further embodiment contemplates fusion proteins containing multiple
antigens, andlor portions of antigens, together with multiple PAMPs, and/or
portions
of PAMPs. It is within the skill of the artisan to determine the desirable
ratio of
PAMP to antigen domains to maximize immunogenicity and minimize inflammatory
response.
There are several advantages of using a fusion system for the production of
recombinant polypeptides. First, heterologous proteins and peptides are often
degraded by host proteases; this may be avoided, especially for small
peptides, by
using a gene fusion expression system. Second, general and efficient
purification
schemes are established for several fusion partners. The use of a fusion
partner as an
affinity handle allows rapid isolation and purification of the recombinant
peptide.
Third, by using different fusion partners, the recombinant product may be
localized to
different compartments or it might be secreted; such strategy could lead to
facilitation
of purification of the fusion partner and/or directed compartmentalization of
the
fusion protein.
Additionally, various methods are available for chemical or enzymatic
cleavage of the fusion protein that provides efficient strategies to obtain
the desired
peptide in large quantities. Frequently employed fusion systems include: the
Staphylococcal protein A fusion system and the synthetic ZZ variant, both of
which
have IgG affinity and have been used for the generation of antibodies against
short
peptides; the glutathione S-transferase fusion system (Smith et al. (1988)
Gene 60);
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the (3-galactosidase fusion system; and the trpE fusion system (Yansura (1990)
Methods Efzzy~ra. 1 ~5: 61). Some of these systems are commercially available
as kits,
including vectors, purification components and detailed instructions.
The present invention also contemplates modified fusion proteins having
affinity for metal ion affinity matrices, whereby one or more specific metal-
binding or
metal-chelating amino acid residues are introduced, by addition, deletion, or
substitution, into the fusion protein sequence as a tag. Optimally, a fusion
parhier,
either an antigen or a PAMP domain, is modified to contain an added metal-
chelating
amino acid tag. The sequence of an antigen or PAMP domain, however, could also
be
altered to provide a metal-binding site if such modifications could be
achieved
without adversely affecting a ligand-binding site, an active site, or other
functional
sites, and/or destroying important tertiary structural relationships in the
protein.
These metal-binding or metal-chelating residues may be identical or different,
and can
be selected from the group consisting of cysteine, histidine, aspartate,
tyrosine,
tryptophan, lysine, and glutamate, and are located so to permit binding or
chelation of
the expressed fusion protein to a metal. Histidine is the preferred metal-
binding
residue. The metal-binding/chelating residues are situated with reference to
the
overall tertiary structure of the fusion protein to maximize binding/chelation
to the
metal and to minimize interference with the expression of the fusion protein
its
biological activity.
A fusion sequence of an antigen, PAMP and a tag, may optionally contain a
linker peptide. The linker peptide might separate a tag from the antigen
sequence or
the PAMP sequence. If the linker peptide so used encodes a sequence that is
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selectively cleavable or digestible by conventional chemical or enzymatic
methods,
then the tag can be separated from the rest of the fusion protein after
purification. For
example, the selected cleavage site within the tag may be an enzymatic
cleavage site.
Examples of suitable enzymatic cleavage sites include sites for cleavage by a
proteolytic enzyme, such as enterokinase, Factor Xa, trypsin, collagenase,
thrombin
and the like. Alternatively; the cleavage site in the linker may be a site
capable of
cleavage upon exposure to a selected chemical or condition, e.g., cyanogen
bromide,
hydroxylamine, or low pH, or other chemicals or conditions known in the art.
Cleavage at the selected cleavage site enables separation of the tag from the
antigen/PAMP fusion protein. The antigen/PAMP fusion protein may then be
obtained in purified form, free from any peptide derivative to which it was
previously
linked for ease of expression or purification. The cleavage site, if inserted
into a
linker useful in the fusion sequences of this invention, does not limit this
invention.
Any desired cleavage site, of which many are known in the art, may be used for
this
purpose.
Another use of linker peptides might be to direct cleavage of the antigen in
intracellular processing so as to facilitate peptide presentation on the
surface of the
APC. Appropriate cleavage sites might be inserted via linkers such that the
fusion
protein is not cleaved until it is internalized by the APC. Under such
circumstances,
such a peptide cleavage site can be introduced via a linker between the PAMP
and the
antigen to generate intracellular antigen free of PAMP. Such directed cleavage
could
also be used particularly to facilitate production within the APC of specific
peptides
that have been identified as interacting with particular HLA haplotypes.
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Alternatively, different domains from a single antigen or from more than one
antigen
might be separated by linkers containing cleavage sites so that these epitopes
could be
appropriately processed for presentation on the surface of the APC.
The optional linker peptide in a fusion protein of the present invention might
serve a purpose other than the provision of a cleavage site. As an example,
and not by
limitation, the linker peptide might be inserted between a PAMP domain and an
antigen domain to prevent or alleviate steric hindrance between the two
domains. In
addition, the linker sequence might provide for post-translational
modification
including, but not limited to, e.g., phosphorylation sites, biotinylation
sites, sulfation
sites, carboxylation sites, glycosylation sites, lipidation sites, and the
like.
In one embodiment, the fusion protein of this invention contains a domain of
an antigen or an immunogenic portion of an antigen fused directly at its amino
or
carboxyl terminal end to the domain of a PAMP or an immunostimulatory portion
of a
PAMP. In another embodiment, the fusion protein of this invention contains a
domain of a PAMP, or an immunostimulatory portion of a PAMP, or a sequence
that
can be post-translationally modified to produce a PAMP, inserted within the
domain
of an antigen, or an immunogenic portion of an antigen. In yet another
embodiment,
the fusion protein of this invention contains a domain of an antigen, or an
immunogenic portion of an antigen, inserted within the domain of a PAMP, or an
immunostimulatory portion of a PAMP, or a sequence that can be post-
translationally
modified to produce a PAMP. In another embodiment, the fusion protein of this
invention, comprising an antigen and a PAMP sequence, is fused directly at its
amino
or carboxyl terminal end to the sequence of a tag. The resulting fusion
protein is a
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soluble cytoplasmic fusion protein. In another embodiment, the fusion sequence
farther comprises a linker sequence interposed between the antigen sequence
and a
PAMP sequence or sequence of a tag. This fusion protein is also produced as a
soluble cytoplasmic protein.
H. Recombinant Technolo~y
Protein PAMPs, protein antigens, and derivatives thereof can be generated
using standard peptide synthesis technology. Alternatively, recombinant
methods can
be used to generate nucleic acid molecules that encode protein PAMPs, protein
antigens and derivatives thereof.
Nucleic acids encoding PAMP/antigen fusions (e.g., synthetic oligo- and
polynucleotides) can easily be synthesized by chemical techniques, for
example, the
phosphotriester method of Matteucci, et al. ((1981) J. Am. Chem. Soc. 103:
3185-
3191) or using automated synthesis methods. In addition, larger nucleic acids
can
readily be prepared by well known methods, such as synthesis of a group of
oligonucleotides that define various modular segments of the nucleic acid
encoding
the PAMP/antigen fusion, followed by ligation of oligonucleotides to build the
complete nucleic acid molecule.
The present invention further provides recombinant nucleic acid molecules
that encode PAMP/antigen fusion proteins. As used herein, a "recombinant
nucleic
acid molecule" refers to a nucleic acid molecule that has been subjected to
molecular
manipulation in vitro. Methods for generating recombinant nucleic acid
molecules
are well known in the art. (Sambrook et al. (1989) Molecular Cloning: A
Laboratory
Manual, Cold Spring Harbor Press). In the preferred recombinant nucleic acid
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molecules, a nucleotide sequence that encodes a PAMP/antigen fusion is
operably
linked to one or more expression control sequences and/or vector sequences.
The choice of vector and/or expression control sequences to which one of the
PAMP/antigen fusion encoding sequences of the present invention is operably
linked
depends directly, as is well known in the art, on the functional properties
desired (e.g.,
protein expression), and the host cell to be transformed. A vector
contemplated by the
present invention is at least capable of directing the replication or
insertion into the
host chromosome, and preferably also expression, of a nucleotide sequence
encoding
a PAMP/antigen fusion.
Expression control elements that are used for regulating the expression of an
operably linked protein encoding sequence are known in the art and include,
but are
not limited to, inducible promoters, constitutive promoters, secretion
signals,
enhancers, transcription terminators and other regulatory elements.
Preferably, an
inducible promoter that is readily controlled, such as being responsive to a
nutrient in
the medium, is used.
In one embodiment, the vector containing a nucleic acid molecule encoding a
PAMP/antigen fusion will include a prokaryotic replicon, e.g., a nucleotide
sequence
having the ability to direct autonomous replication and maintenance of the
recombinant nucleic acid molecule intrachromosomally in a prokaryotic host
cell,
such as a bacterial host cell, transformed therewith. Such replicons are well
known in
the art. In addition, vectors that include a prokaryotic replicon may also
include a
gene whose expression confers a detectable marker such as a drug resistance.
Typical
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bacterial drug resistance genes are those that confer resistance to ampicillin
(Amp) or
tetracycline (Tet).
Vectors that include a prokaryotic replicon can further include a prokaryotic
or
viral promoter capable of directing the expression (transcription and
translation) of
the PAMP/antigen fusion in a bacterial host cell, such as E. coli. A promoter
is an
expression control element formed by a nucleic acid sequence that permits
binding of
RNA polymerase and transcription to occur. Promoter sequences compatible with
bacterial hosts are typically provided in plasmid vectors containing
convenient
restriction sites for insertion of a nucleic acid segment of the present
invention.
Typical of such vector plasmids are pUCB, pUC9, pBR322 and pBR329 available
from Biorad Laboratories (Richmond, CA), pPL and pKK223 available from
Amersham Pharmacia Biotech, Piscataway, NJ.
Expression vectors compatible with eukaryotic cells, preferably those
compatible with vertebrate cells, can also be used to express nucleic acid
molecules
that contain a nucleotide sequence that encodes a PAMP/antigen fusion.
Eukaryotic
cell expression vectors are well known in the art and are available from
several
conunercial sources. Typically, such vectors provide convenient restriction
sites for
insertion of the desired DNA segment. Typical of such vectors are pSVL and
pKSV-
10 (Amersham Pharmacia Biotech), pBPV-1/pML2d (International Biotechnologies,
Inc.), pTDTl (ATCC, #31255), the vector pCDM8 described herein, and other like
eukaryotic expression vectors.
Eukaryotic cell expression vectors used to construct the recombinant
molecules of the present invention may further include a selectable marker
that is
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effective in a eukaryotic cell, preferably a drug resistance selection marker.
A
preferred drug resistance marker is the gene whose expression results in
neomycin
resistance, e.g., the neomycin phosphotransferase (neo) gene. (Southern et al.
(1982)
J. Mol. Afaal. Gehet. 1:327-341). Alternatively, the selectable marker can be
present
on a separate plasmid, and the two vectors are introduced by cotransfection of
the host
cell, and selected by culturing in the presence of the appropriate drug for
the
selectable marker.
The present invention further provides host cells transformed with a nucleic
acid molecule that encodes a PAMP/antigen fusion protein of the present
invention.
The host cell can be either prokaryotic or eukaryotic. Eukaryotic cells useful
for
expression of a PAMP/antigen fusion protein are not limited, so long as the
cell line is
compatible with cell culture methods and compatible with the propagation of
the
expression vector and expression of the fusion protein. Preferred eukaryotic
host cells
include, but are not limited to, yeast, insect and mammalian cells, preferably
vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic
cell
line.
Any prokaryotic host can be used to express a recombinant nucleic acid
molecule. The preferred prokaryotic host is E. coli. In embodiments where the
PAMP is a lipoprotein, expression of the PAMP/antigen fusion protein in a
bacterial
cell is preferred. Expression of the nucleic acid in a bacterial cell line is
desirable to
ensure proper post-translational modificatiomof the protein portion of the
lipoprotein.
Preferably, the host cells selected for expression of the PAMP/antigen fusion
(e.g.
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lipoprotein/antigen fusion) is the cell that natively produces the lipoprotein
of the
lipoprotein/antigen fusion.
Transformation of appropriate cell hosts with nucleic acid molecules encoding
a PAMP/antigen fusion of the present invention is accomplished by well known
methods that typically depend on the type of vector and host system employed.
With
regard to transformation of prokaryotic host cells, electroporation and salt
treatment
methods are typically employed. (See e.g., Cohen et al. (1972) PYO.C Natl.
Acad. Sci.
USA 69:2110; Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold
Spring
Harbor Laboratory, Cold Spring Harbor, NY (1982); Sambrook et al. (1989)).
With
regard to transformation of vertebrate cells with vectors containing rDNAs,
electroporation, cationic lipid or salt treatment methods are typically
employed. (See
e.g., Graham et al., Virology (1973) 52:456; Wigler et al. (1979) Proc. Natl.
Acad.
Sci. U.S.A. 76:1373-76).
Successfully transformed cells, e.g., cells that contain a nucleic acid
molecule
encoding the PAMPlantigen fusions of the present invention, can be identified
by well
known techniques. For example, cells resulting from the introduction of a
nucleic
acid molecule encoding the PAMP/antigen fusions of the present invention can
be
cloned to produce single colonies. Cells from those colonies can be harvested,
lysed
and their nucleic acids content examined for the presence of the recombinant
molecule using a method such as that described by Southern (1975) (J. Mol.
Biol. 98:
503), or Berent et al. (1985) (Biotech. 3: 208) or the proteins produced from
the cell
assayed via an immunological method.
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The present invention further provides methods for producing a
PAMP/antigen fusion protein that uses one of the nucleic acid molecules herein
described. In general terms, the production of a recombinant protein typically
involves the following steps.
First, a nucleic acid molecule is obtained that encodes a PAMP/antigen fusion
protein. Said nucleic acid molecule is then preferably placed in an operable
linkage
with suitable control sequences, as described above. The expression unit is
used to
transform a suitable host and the transformed host is cultured under
conditions that
allow the production of the PAMP/antigen fusion protein. Optionally, the
fusion
protein is isolated from the medium or from the cells; recovery and
purification of the
fusion protein may not be necessary in some instances where some impurities
may be
tolerated.
Each of the foregoing steps can be done in a variety of ways. For example, the
desired coding sequences may be obtained from genomic fragments and used
directly
in an appropriate host. The construction of expression vectors that are
operable in a
variety of hosts is accomplished using an appropriate combination of replicons
and
control sequences. The control sequences, expression vectors, and
transformation
methods are dependent on the type of host cell used to express the gene and
were
discussed in detail earlier. A skilled artisan can readily adapt any
host/expression
system known in the art for use with the nucleotide sequences described herein
to
produce a PAMP/antigen fusion protein.
Endonucleases are nucleases that are able to break internal phosphodiester
bonds within a nucleic acid molecule. Examples of nucleases include, but are
not
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limited to, S 1 endonuclease from the fungus Aspergillus oryzae,
deoxyribonuclease
(DNase I), and restriction endonucleases. The cutting and joining processes
that
underlie DNA manipulation are carned out by enzymes called restriction
endonucleases (for cutting) and ligases (for joining). Suitable restriction
endonuclease
cleavage sites can, if not normally available, be added to the ends of the
coding
sequence so as to provide an excisable nucleic acid sequence to insert into
these
vectors.
In addition, restriction endonuclease cleavage sites may also be inserted in
the
nucleic acid sequence encoding the PAMP/antigen fusion protein. Preferably,
these
cleavage sites are engineered between nucleotide sequences encoding identical
or
different PAMPs; between identical or different antigens, or between
nucleotide
sequences encoding PAMP and antigen. Appropriate cleavage sites well know to
those skilled in the art include, but are not limited to, the following:
EcoRI, BamHI,
BgllII, PvuI, PvuII, HindIII, Hihfl, Sau3A, AIuI, TaqI, HaeIII and NotI. (T.A.
Brown
(1996) Gene Cloning: An Introduction, Second Edition, Chapman & Hall, Chapter
4:49-83).
I. Coniu~ates
The present invention also includes "conjugates" which comprise two or more
molecules that are covalently linked, or noncovalently linked but in
association with
each other. Thus, vaccines of the present invention include PAMP/antigen
conjugates
such as, but not limited to, the following: protein/nucleic acid conjugates,
nucleic
acid/protein conjugates, nucleic acid/nucleic acid conjugates, peptide-
mimetic/nucleic
acid conjugates, nucleic acid/peptide mimetic conjugates, peptide
mimetic/peptide
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mimetic conjugates, lipopolysaccharide/protein conjugates, lipoprotein/protein
conjugates, RNA/protein conjugates, CpG-DNA/protein conjugates, nucleic acid
analog/protein conjugates, and mannan/protein conjugates. To the extent that
PAMPs
identified in the future are comprised of yet other chemical classes,
conjugates
containing such chemicals in combination with antigen domains can also be
contemplated.
Methods for the conjugation of polypeptides, carbohydrates, and lipids with
DNA are well known to the artisan. See e.g., U.S. Pat. Nos. 4,191,668,
4,650,625,
5,162,515, 5,700,922, 5,786,461, 6,06,0056; and J. ClifZ. Iyavest. (1988)
82:1901-1907.
Non-protein PAMPs such as CpG or CpG-DNA, and lipopolysaccharides may
be conjugated to protein or non-protein antigens by conventional techniques.
For
example, PAMP/antigen conjugates may be linked through polymers such as PEG,
poly-D-lysine, polyvinyl alcohol, polyvinylpyrollidone, irnmunoglobulins, and
copolymers of D-lysine and D-glutamic acid. Conjugation of the PAMP and
antigen
to the polymer linker may be achieved in any number of ways, typically
involving one
or more crosslinking agents and functional groups on the PAMP and antigen.
Polypeptide PAMPs and antigens will contain amino acid side chains such as
amino,
carbonyl, or sulfliydryl groups that will serve as sites for linking the PAMP
and
antigen to each other. Residues that have such functional groups may be added
to
either the PAMP or antigen. Such residues may be incorporated by solid phase
synthesis techniques or recombinant techniques, both of which are well known
in the
peptide synthesis arts.
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In the case of carbohydrate or Lipid analogs, functional amino and sulfhydryl
groups may be incorporated therein by conventional chemistry. For instance,
primary
amino groups may be incorporated by reaction with ethylenediamine in the
presence
of sodium cyanoborohydride and sulfhydryls may be introduced by reaction of
cysteamine dihydrochloride followed by reduction with a standard disulfide
reducing
agent. In a similar fashion the polymer linker may also be derivatized to
contain
functional groups if it does not already possess appropriate functional
groups.
Heterobifunctional crosslinkers, such as sulfosuccinimidyl(4-iodoacetyl)
aminobenzoate, wluch link the .epsilon. amino group on the D-lysine residues
of
copolymers of D-lysine and D-glutamate to a sulfhydryl side chain from an
amino
terminal cysteine residue on the peptide to be coupled, are also useful to
increase the
ratio PAMPs or antigens in the conjugate.
J. Vaccine Formulation and Delivery
The vaccines of the present invention contain one or more PAMPs,
immunostimulatory portions, or irmnunostimulatory derivatives thereof (e.g., a
domain recognized by the innate immune system), and one or more antigens,
immunogenic portions, or immunogenic derivatives thereof (e.g., a domain
recognized by the adaptive immune system). Since a PAMP mimetic, by
definition,
has the ability to bind PRRs and initiate an innate immune response, vaccine
formulations contemplated by this invention include PAMP mimetics in place of
PAMPs. Thus, the present invention contemplates vaccines comprising chimeric
constructs including at least one antigen domain and at least one PAMP domain.
In
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one specific embodiment, the vaccines of the present invention comprise a
BLP/Ea
fusion protein.
The vaccines, comprising the chimeric constructs of the present invention, can
be formulated according to known methods for preparing pharmaceutically useful
compositions, whereby the chimeric constructs are combined in a mixture with a
pharmaceutically acceptable carrier. A composition is said to be a
"pharmaceutically
acceptable carrier" if its administration can be tolerated by the recipient
and if that
composition renders the active ingredients) accessible at the site where the
action is
required. Sterile phosphate-buffered saline is one example of a
pharmaceutically
acceptable earner. Other suitable earners are well-known to those in the art.
(Ansel
et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th Edition
(Lea &
Febiger 1990); Gennaro (ed.), Remington's Pharmaceutical Sciences 18th Edition
(Mack Publishing Company 1990)).
Examples of several other excipients that can be contemplated may include,
water, dextrose, glycerol, ethanol, and combinations thereof. The vaccines of
the
present invention may further contain auxiliary substances, such as wetting or
emulsifying agents, pH buffering agents, stabilizers or other carriers that
include, but
are not limited to, agents such as aluminum hydroxide or phosphate (alum),
commonly used as a 0.05 to 0.1 percent solution in phosphate buffered saline,
to
enhance the effectiveness thereof.
The chimeric constructs of the present invention can be used as vaccines by
conjugating to soluble immunogenic carrier molecules. Suitable earner
molecules
include protein, including keyhole limpet hemocyanin, which is a preferred
carrier
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protein. The chimeric construct can be conjugated to the carrier
molecule.using
standard methods. (Hancock et al., "Synthesis of Peptides for Use as
hnmunogens,"
in Methods in Molecular Biology: Imrnunochemical Protocols, Manson (ed.),
pages
23-32 (Humane Press 1992)).
Furthernlore, the present invention contemplates a vaccine composition
comprising a pharmaceutically acceptable injectable vehicle. The vaccines of
the
present invention may be administered in conventional vehicles with or without
other
standard carriers, in the form of injectable solutions or suspensions. The
added
carriers might be selected from agents that elevate total immune response in
the
course of the immunization procedure.
Liposomes have been suggested as suitable carriers. The insoluble salts of
aluminum, that is aluminum phosphate or aluminum hydroxide, have been utilized
as
carriers in routine clinical applications in humans. Polynucleotides and
polyelectrolytes and water soluble carriers such as muramyl dipeptides have
been
used.
Preparation of injectable vaccines of the present invention, includes mixing
the chimeric construct with muramyl dipeptides or other carriers. The
resultant
mixture may be emulsified in a mannide monooleate/squalene or squalane
vehicle.
Four parts by volmne of squalene and/or squalane are used per part by volume
of
mannide monooleate. Methods of formulating vaccine compositions are well-known
to those of ordinary skill in the art. (Role, Immunizing Agents and Diagnostic
Skin
Antigens. In: Remington's Pharmaceutical Sciences,l8th Edition, Gennaro (ed.),
(Mack Publishing Company 1990) pages 1389-1404).
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Additional pharmaceutical Garners may be employed to control the duration of
action of a vaccine in a therapeutic application. Control release preparations
can be
prepared through the use of polymers to complex or adsorb chimeric construct.
For
example, biocompatible polymers include matrices of polyethylene-co-vinyl
acetate)
and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic
acid.
(Sherwood et al. (1992) BiolTechyaology 10: 1446). The rate of release of the
chimeric construct from such a matrix depends upon the molecular weight of the
construct, the amount of the construct within the matrix, and the size of
dispersed
particles. (Saltzman et al. (1989) Biophys. J. 55: 163; Sherwood et al.,
supra.; Ansel
et al. Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th Edition (Lea
&
Febiger 1990); and Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th
Edition (Mack Publishing Company 1990)). The chimeric construct can also be
conjugated to polyethylene glycol (PEG) to improve stability and extend
bioavailability times (e.g., Katre et al.; U.S. Patent 4,766,106).
The vaccines of this invention may be administered parenterally. The usual
modes of adminstration of the vaccine are intramuscular, sub-cutaneous, and
intra-
peritoneal injections. Moreover, the administration may be by continuous
infusion or
by single or multiple boluses.
The gene gun has also been used to successfully deliver plasmid DNA for
inducing immunity against an intracellular pathogen for which protection
primarily
depends on type 1 CDB + T-cells. (Kaufmann et al. (1999) J. Imnzuh.
163(8):
4510-4518).
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Gene transfer-mediated vaccination methods have become a rapidly expanding
field and the compositions of the present invention are applicable to the
treatment of
both noninfectious and infectious diseases and noninfectious diseases,
including but
not limited to genetic disorders, using such vaccination methods. (See e.g.,
Eck et al.
(1996) Gene-Based Therapy, In: Goodman & Gilman's The Pharmacological Basis of
Therapeutics, Ninth Edition, Chapter 5, McGraw Hill).
Alternatively, the vaccine of the present invention, particularly as regards
use
of Flagellin as a PAMP, may be formulated and delivered in a manner designed
to
evoke an immune response at a mucosal surface. Thus, the vaccine compositions
may
be administered to mucosal surfaces by, for example, nasal or oral
(intragastric)
routes. Other modes of administration include suppositories and oral
formulations.
For suppositories, binders and Garners may include polyalkalene glycols or
triglycerides. Oral formulations may include normally employed incipients such
as
pharmaceutical grades of saccharine, cellulose and magnesium carbonate. These
compositions can take the form of solutions, suspensions, tablets, pills,
capsules,
sustained release formulations or powders and contain about 1 to 95% of the
chimeric
construct. The vaccines are administered in a manner compatible with the
dosage
formulation, and in such amount as will be therapeutically effective,
protective and
immunogenic dosages.
The quantity of vaccine employed will of course vary depending upon the
patient's age, weight, height, sex, general medical condition, previous
medical history,
the condition being treated and its severity, and the capacity of the
individual's
immune system to synthesize antibodies, and produce a cell-mediated immune
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response. Typically, it is desirable to provide the recipient with a dosage of
the
chimeric construct which is in the range of from about 1 ~,g agent /kg body
weight of
patient to 100 mg agent/kg body weight of patient, although a lower or higher
dosage
may also be administered. Precise quantities of the active ingredient,
however,
depend on the judgment of the practitioner. Suitable dosage ranges are readily
determinable by one skilled in the art and may be on the order of nanograms of
the
chimeric construct to grams of the chimeric construct, depending on the
particular
construct. Preferably the dosage range of the active ingredient is nanograms
to
micrograms; more preferably nanograms to milligrams; and most preferably
micrograms to milligrams. Suitable regimes for initial administration and
booster
doses are also variable, but may include an initial administration followed by
subsequent administrations. The dosage may depend on the route of
administration
and will vary according to the size of the subject.
The present invention encompasses vaccines containing antigen and PAMPs
from a single organism, such as from a specific pathogen. The present
invention also
encompasses vaccines that contain antigenic material from several different
sources
and/or PAMP material isolated from several different sources. Such combined
vaccines contain, for example, antigen and PAMPs from various microorganisms
or
from various strains of the same microorganism, or from combinations of
various
microorganisms.
For purposes of therapy, the antigen/PAMP fusion proteins are administered to
a mammal in a therapeutically effective amount. A vaccine preparation is said
to be
administered in a "therapeutically effective amount" if the amount
administered is can
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produce a measurable positive effect in a recipient. In particular, a vaccine
preparation of the present invention produces a positive effect in a recipient
if it
invokes a measurable humoral and/or cellular immune response in the recipient.
In
particular, this invention contemplates a desirable therapeutically effective
amount as
one in which the vaccine invokes in the recipient a measurable humoral andlor
cellular immune response versus the target antigen but causes neither
excessive non-
specific inflammation nor an autoimmune response versus non-target antigen(s).
As used herein, the term "treatment" refers to both therapeutic treatment and
prophylactic or preventative treatment. In one embodiment, the present
invention
contemplates using the disclosed vaccines to treat patients in need thereof.
The
patients may be suffering from diseases such as, but not limited to, cancer,
allergy,
infectious disease, autoimmune disease, neurological disease, cardiovascular
disease,
or a disease associated with an allergic reaction. In another embodiment, the
present
invention contemplates administering the disclosed vaccines to passively
immunize
patients against diseases such as but not limited to, cancer, allergy,
infectious disease,
autoimmune disease, neurological disease, cardiovascular disease, or disease
associated with an allergic reaction. In yet another embodiment the present
invention
contemplates administering the disclosed vaccines to immunize patients against
diseases in addition to those cited in the previous sentence in which the
objective is to
rid the body of specific molecules or specific cells. A non-limiting example
might be
the removal or prevention of deposition of plaque in cardiovascular disease.
K. TreatmentlEnhancement of Immunity
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The vaccines of the present invention can be used to enhance the immunity of
animals, more specifically mammals, and even more specifically humans (e.g.,
patients) in need thereof. Enhancement of immunity is a desirable goal in the
treatment of patients diagnosed with, for example, cancer, immune deficiency
syndrome, certain topical and systemic infections, leprosy, tuberculosis,
shingles,
warts, herpes, malaria, gingivitis, and atherosclerosis.
The advantages of the vaccines of the present invention are that they induce a
strong immune response against the target antigen with minimal undesired
inflammatory reaction, as well as minimal instances of autoimmune disease.
Such a
reduced side effect profile has a distinct advantage over other vaccine
approaches,
particularly with respect to targeting of self antigens, because with many
other
vaccine strategies, in order to elicit a robust response against the self
antigen, strong
adjuvants are used and they result in excessive inflammation and can increase
the risk
of autoimmune disease.
As used herein, "immunoenhancement" refers to any increase in an organism's
capacity to respond to foreign antigens or other targeted antigens, such as
those
associated with cancer, which includes an increased number of immune cells,
increased activity and increased ability to detect and destroy such antigens,
in those
cells primed to attack such antigens.
The strength of an immune response can be measured by standard tests
including, but not limited to, the following: direct measurement of peripheral
blood
lymphocytes by means known to the art; natural killer cell cytotoxicity assays
(Provinciali et al. (1992) J. Immunol. Meth. 155: 19-24), cell proliferation
assays
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(Vollenweider et al. (1992) J. Immunol. Meth. 149: 133-135), immunoassays of
immune cells and subsets (Loeffler et al. (1992) Cytom. 13: 169-174; Rivoltini
et al.
(1992) Cafz. Immunol. Immunotlaef°. 34: 241-251); and skin tests for
cell- mediated
irmnunity (Chang et al. (1993) Cancer Res. 53: 1043-1050). For an excellent
text on
methods and analyses for measuring the strength of the immune system, see, for
example, Coligan et al. (Ed.) (2000) Current Protocals in Immunology, Vol. l,
Wiley
& Sons.
Any statistically significant increase in the strength of immune response, as
measured by the above tests, is considered "enhanced immune response" or
"immunoenhancement". An increase in T-cells in S-phase of greater than 5
percent
has been achieved by the methods of this invention. Enhanced immune response
is
also indicated by physical manifestations such as fever and inflammation,
although
one or both of these manifestations might not be observed with the recombinant
vaccines of the present invention. Enhanced immune response is also
characterized
by healing of systemic and local infections, and reduction of symptoms in
disease,
e.g. decrease in tumor size, alleviation of symptoms of leprosy, tuberculosis,
malaria,
naphthous ulcers, herpetic and papillomatous warts, gingivitis,
atherosclerosis, the
concomitants of AIDS such as I~aposi's sarcoma, bronchial infections, and the
like.
L. Vaccine Production
The procedures of the present invention can be used to generate a chimeric
construct comprising one or more antigens of interest and one or more PAMPs. A
small, non-immunogenic epitope tag (such as a His tag) can be added to
facilitate the
purification of fusion protein expressed in bacteria. The combination of
antigen with
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a PAMP such as BLP or Flagellin provides signals necessary for the activation
of the
antigen-specific adaptive and innate immune responses.
A large number of differing fusion proteins comprising different combinations
of antigens and PAMPs can be readily generated using recombinant DNA
technology
or conjugation chemistry that is well known in the art. Virtually any antigen
can be
used to generate a vaccine by this approach using the same technology. This
novel
approach, therefore, is very versatile.
Large amounts of recombinant vaccine product can be generated using a
bacterial expression system. The product can be purified from bacterial
cultures using
standard techniques. The approach is thus extremely economical and cost
efficient.
Alternatively, recombinant vaccine product can be produced and purified from
cultures of yeast or other eukaryotic cells including, without limitation,
insect cells or
mammalian cells. Conjugated non-protein vaccine product can also be produced
chemically in relatively large amounts. This is particularly the case if the
PAMP and
the antigen can both be obtained by relatively straightforward purification
procedures
and then conjugated together with relatively simple and efficient conjugation
chemistry.
Alternatively, a chimeric construct containing a protein component and a non-
protein component can be conveniently obtained by preparing the protein
component
by recombinant means and the non-protein component by chemical means and then
linking the two components with linker chemistry well known in the art, some
of
which is described herein. Additionally, since the antigens and PAMPs
contemplated
in this invention can be naturally occurring, they can be purified from their
natural
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sources and then linked together chemically. Both T-cell and B-cell antigens
can be
used to generate vaccines by this approach.
Fusion of an antigen with a PAMP such as BLP or Flagellin optimizes the
stoichiometry of the two signals thus minimizing the unwanted excessive
inflammatory responses (which occur, for example, when antigens are mixed with
adjuvants to increase their immunogenicity).
Fusion of an antigen with a PAMP such as BLP increases the likelihood that
APCs activated in response to the vaccine productively trigger the desired
adaptive
immune response. Activation of such APCs in the absence of uptake and
presentation
of the antigen can lead to the induction of autoimmune responses, which,
again, is one
of the problems with commonly used adjuvants that prevents or limits their use
in
humans.
In a preferred embodiment, the fusion proteins of the present invention
comprise an antigen or an immunogenic portion thereof which has been modified
to
contain an amino acid sequence comprising a leader sequence and a consensus
sequence, that results in the post-translational modification of the consensus
sequence
or a portion of that sequence, wherein the post-translationally modified
sequence is a
ligand for a PRR. The modified antigens include, but are not limited to,
antigens that
contain the bacterial lipidation consensus sequence CXXN (SEQ ID NO: 1),
wherein
X is any amino acid, but preferably serine. Numerous leader sequences are well
.
known in the art, but a preferred leader sequence is described by the first 20
amino
acids of SEQ ID NO: 2, wherein the first 20 amino acids of SEQ ID NO: 2 are
set
forth in set forth in SEQ ID NO: 3. Examples of additional suitable leader
sequences
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are described in the Sequence Listing as SEQ ID NO: 4-7. A preferred chimeric
construct comprises a leader sequence fused, in frame, to a sequence
comprising the
bacterial lipidation consensus sequence of SEQ ID NO: 1 further fused to an
antigen
(e.g. leader sequence-CXXN-antigen). Although this modification of the antigen
can be referred tb as a fusion, this modification can be achieved without
fusing DNA,
but rather by introducing, by mutagenesis, a leader sequence followed by the
CXX
sequence into DNA encoding any antigen of interest. Expression of a nucleic
acid
molecule encoding this chimeric construct, in a bacterial host cell, produces
a
substrate, first for bacterial proteases, that cleave the leader sequence from
the
I O modified antigen, and bacterial lipid transferases, which Iipidate the
sequence, or a
portion thereof, comprising the lipidation consensus sequence. The resultant
product
is a chimeric construct or fusion protein that is a ligand for a PRR and is
capable of
stimulating both the innate and adaptive immune systems. In an additional
embodiment, this chimeric construct or fusion protein comprises additional
polar or
15 charged amino acids to increase the hydrophilicity of the chimeric
construct or fusion
protein without altering the irnmunogenic or immunostimulatory properties of
the
construct.
Without further description, it is believed that one of ordinary skill in the
art
can, using the preceding description and the following illustrative examples,
practice
20 the methods of the present invention. The following working examples,
therefore,
specifically point out the preferred embodiments of the present invention, are
illustrative only, and are not to be construed as limiting in any way the
remainder of
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the disclosure. Other generic and specific configurations will be apparent to
those
persons skilled in the art.
EXAMPLES
Example 1. Model Vaccine Cassette with an Antigen Domain and a PAMP
Domain
In order to produce a model vaccine cassette of the present invention, we
fused
a pathogen-associated molecular pattern (DAMP) to the characterized mouse
antigen,
Ea. The PAMP we selected, BLP, is known to stimulate innate immune responses
through the receptor, Toll-like-receptor-2 (TLR-2).
The protein sequence of the bacterial lipoprotein (BLP) used in the vaccine
cassette for fusion with an antigen of interest is as follows:
MKATKLVLGAVILGSTLLAGCSSNAKIDQLSSDVQTLNAKVDQLSNDVNAM
RSDVQAAKDDAAR.ANQRLDNMATKYRK (SEQ ID NO: 2). The leader
sequence includes amino acid number 1 through amino acid number 20 of SEQ ID
NO: 2. The first cysteine (amino acid number 21 of SEQ ID NO: 2) is lipidated
in
bacteria. This lipidation, which can only occur in bacteria, is essential for
BLP
recognition by Toll and TLRs. The C-terminal lysine (amino acid number 78 of
SEQ
ID NO: 2) was mutated to increase the yield of a recombinant vaccine, because
this
lysine can form a covalent bond with the peptidoglycan.
To assist in identification and purification of the antigen, a hexa-histidine
tag
was engineered on the C-terminal of the protein. The final construct is shown
in
Figure 3.
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The fusion protein was expressed in bacteria and induced with IPTG. The
protein was purified by lysis and sonication in 8 M Urea, 20 mM Tris, 20 rnM
NaCl,
2% Triton-X-100, pH 8Ø The lysate was passed over a 100 ml Q-Sepharose ion
exchange column in the same buffer and washed with 5 column volumes of 8 M
Urea,
20 mM Tris, 20 mM NaCI, 0.2% Triton-X-100, pH 8Ø The protein was eluted by
salt gradient (20 mM NaCI to 800 mM NaCI). Positive fractions were identified
by
immunoblotting using an antibody to the Histidine tag. These fractions were
pooled
and passed over a 2 ml nickel-agarose column. The column was extensively
washed
with the same buffer (10 column volumes) and then washed with 5 column volumes
of phosphate buffer (20 mM) containing 200 mM NaCI, 0.2% Triton-X-100, 20 mM
imidazole, pH 8Ø The purified protein was eluted in 20 mM phosphate buffer,
200
mM NaCl, 0.1% Triton-X-100, 250 mM imidazole and fractions were again tested
for
protein by immunoblotting. Positive fractions were pooled and dialyzed
overnight
against phosphate buffered saline containing 0.1% Triton-X-100. The sample was
then decontaminated of any endotoxin by passage over a polymyxin B column, and
concentrated in an Amicon concentrator by centrifugation and tested by
immunoblotting and protein concentration for protein content.
Example 2. Stimulation of NF-KB by BLP/Ea model antigen in RAW cells
To test whether the model antigen could stimulate signal transduction
pathways necessary for an immune response, we assayed NF-xB activation in the
RAW mouse macrophage cell line i~a vitro. We developed a stable RAW cell line
that
harbors an NF-xB-dependent firefly luciferase gene. Stimulation of these cells
with
activators of NF-~cB leads to production of luciferase which is measured in
cell lysates
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by use of a luminometer. Cells were stimulated with the indicated amounts of
BLP/Eoc left 5 hours and harvested for luciferase measurement.
As a control, RAW cells were stimulated with LPS in the presence and
absence of polymyxin B (PmB). PmB inactivates endotoxin and as expected the
activation of NF-xB activity in the LPS+PmB sample is diminished by 98%.
BLP/Ea
also activates NF-KB in a dose-dependent manner as shown in Figure 4, however,
treatment with PmB does not inactivate the stimulus to a statistically
significant
degree. These results suggest that the activation of NF-xB seen with BLP/Ea is
not
due to contamination of the preparation with endotoxin.
Example 3. BLP/Ea Model Vaccine Induces the Production of IL-6 by
Dendritic Cells Ira VitYo
An effective vaccine must be able to stimulate dendritic cells (DC)to mature
and present antigen. To test whether BLP/Ea could induce DC function, we
tested
the ability of bone marrow-derived DC to produce IL-6 after stimulation in
vitro.
Bone marrow dendritic cells were isolated and grown for 5 days in culture in
the
presence of 1 % GM-CSF. After 5 days, cells were replated at 250,000
cells/well in a
96-well dish and treated with either Ea peptide (0.3~g/ml), LPS (100ng/ml) +
Ea
peptide (0.3~g/ml), or BLP/Ea. BLP/Ea was able to stimulate IL-6 production in
these cells as measured in a sandwich ELISA (Figure 5).
Example 4. BLP/Ea Stimulates Maturation of Immature Dendritic Cells
To determine whether BLP/Ea vaccine can be processed and presented by
dendritic cells, we stimulated dendritic cells with the vaccine and tested
them for the
surface expression of B7.2 and Ea peptide bound to MHC Class II. Cultured bone
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marrow-derived dendritic cells (5 days) were stimulated with Ea peptide or
BLP/Ea
and were stained with an antibody to the B7.2 costimulatory molecule and/or
with
Yae antibody which recognizes Ea peptide bound to MHC Class II. Analysis was
performed by FRCS (Figure 6).
Example 5. BLP/Ea Model Vaccine Stimulates Specific T-Cells In Vitro
We next assayed whether BLP/Ea that was processed and presented by DC
could stimulate the proliferation of antigen-specific T-cells in vitro. Bone
marrow
derived mouse DC were isolated and plated into mediwn containing 1 % GM-CSF at
750,000 cells/well. Cells were cultured for 6 days and then the DC were
collected,
washed, and counted then replated in 96-well dishes at 250,000 cells per well.
Cells
were stimulated with the above indicated antigens and left three days to
mature. After
3 days, the DC were resuspended and plated in a 96-well dish at either 5,000
or
10,000 cells/well. T-cells from lymph nodes from a 1H3.1 TCR transgenic mouse
(1H3.1 TCR is specific for the Eoc peptide)were plated on the DC at 100,000
cells/well. Cells were left for 3 days in culture then "pulsed" with
O.S~,Ci/well of 3H-
thymidine. The cells were harvested 24 hours later and incorporation of
thymidine
(T-cell proliferation) was measured in cpm (Figure 7).
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Example 6. BLPlEoc Activates Specific T-cells In Viyo
To assess the ability of the vaccine to generate a specific T-cell response in
vivo, we injected the fusion protein into a mouse. Three mice were injected as
follows:
Mouse # Sample injected # of lymph node cells
1 Ea peptide 30~g in 1.9x10"
PBS
2 Ea peptide 30~,g in 3.29x10'
CFA*
3 BLP/ Ea 100~,g 5.2x10"
*Complete Freund's Adjuvant
The injected footpad of mouse #2 was considerably swollen for the duration of
the experiment, but the footpads of mice #1 and #3 appeared normal. After 6
days,
the mice were euthanized and the associated draining lymph node was harvested
for a
T-cell proliferation assay. T-cells were plated in a 96-well plate at 400,000
cells/well
and were restimulated with either Ea peptide or with BLP/Ea at the indicated
doses.
Cells were left 48 hours to begin proliferation, pulsed with O.S~,Ci/well of
3H-
Thymidine in medium and harvested 16 hours later. Thyrnidine incorporation was
measured by counting in a beta-plate reader (Figure 8).
Example 7. Model Vaccine Cassette with an Allergen-Related Antigen
Using the procedures set forth above for the production of the BLP/Ea model
antigen, a vaccine cassette with an allergen-related antigen is produced using
the
pollen allergen RaSG from the giant ragweed (Ambrosia trifida). The amino acid
sequence of RaSG is as follows:
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MI~NIFMLTLF ILIITSTIKA IGSTNEVDEI KQEDDGLCYE GTNCGKVGKY
CCSPIGKYCVCYDSKAICNK NCT (SEQ ID NO: 9).
The amino acid sequence of this allergen can be fused with the BLP amino
acid sequence (SEQ ID NO: 1) to generate the BLP/RaSG fusion protein. The
resultant recombinant vaccine places the allergen in the context of an IL-12
inducing
signal, where the PAMP in this case is BLP).
When introduced into a subject, this vaccine will generate allergen-specific T-
cell responses that will be differentiated into Thl responses due to the
induction of IL-
12 by BLP in dendritic cells and macrophages.
Example 8. Model Vaccine Cassette with a Tumor-Related Antigen
Using the procedures set forth above for the production of the BLP/Ea model
antigen, a vaccine cassette with a tumor-related antigen is produced using the
model
tumor antigen, Tyrosinase-Related Protein 2 (TRP-2). The nucleic acid sequence
and
corresponding amino acid sequence of TRP-2 is provided in SEQ ID NO: 10 (shown
in Figure 20) and SEQ ID NO: 11 (shown in Figure 21), respectively. The region
used for BLP fusion includes nucleic acid number 840 through nucleic acid
number
1040 of SEQ ID NO: 10. The T-cell epitope includes nucleic acid number 945
through nucleic acid number 968 of SEQ ID NO: 10.
A region of the TRl'-2 that can be used for the vaccine construction is shown
below:
LDLAKKSIHPDYVITTQHWLGLLGPNGTQPQIANCSVYDFFVWLHYYS
VRDTLLGPGRPYKAIDFSHQ (SEQ ID NO: 12).
A T-cell epitope of SEQ ID NO: 12 is VYDFFVWL (SEQ ID NO: 13).
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Example 9: CMG Irnmunostimulation
The family of TLRs has recently been identified as an essential component of
innate immune recognition in both D~osophila and mammalian organisms
(Hoffinann
et al. (1999) Science 284:1313-1318; Imler et al. (2000) Cuy~r. Opin.
Microbiol. 3:16-
22). Drosophila Toll is required for the detection of fungal infection and the
induction of the antifungal peptide drosomycin (Lemaitre et al. (1996) Cell
86:973-
983). In the mouse, TLR2 and TLR4 were shown to mediate recognition of
bacterial
PGN and LPS, respectively (Takeuchi et al. (1999) Immunity 11:443-451). The
functions of the other members of the Drosophila and mammalian Toll families
are
currently unknown, although it is expected that at Ieast some of them are
involved in
innate immune recognition as well.
Collectively, the results described here indicate that the immunostimulatory
effect of CpG-DNA on the three types of professional antigen presenting cells-
DC,
macrophages and B-cells -- is mediated by a MyD88 signaling pathway. MyD88 is
IS involved in signal transduction by the Toll and IL-1 receptor families. The
activities
of the IL-1 family of cytokines, including IL-1 and IL-18, is dependent on
processing
by caspase-1, but in all the experiments described here, the absence of
caspase-1 had
no effect on CpG-DNA induced cellular responses (Fantuzzi et al. (1999) J.
Clin.
Immunol. 19:1-11).
We tested whether TLR2 and TLR4 are involved in the recognition of CpG-
DNA and found that they are not, at least based on the assays provided herein.
We
believe, therefore, that CpG-DNA is recognized by a Toll receptor other than
TLR2
and TLR4. Cell lines that express endogenous or transfected TLR1 through TLR6
did
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not respond to CpG-DNA (data not shown), suggesting that some other member of
the
Toll family may mediate CpG-DNA recognition.
While the identity of the Toll receptor that is responsible for CpG-DNA
recognition remains unknown at this point, the fact that CpG-DNA requires
S internalization to exert its stimulatory effect (I~rieg et al. (1995) Nature
374:546-549;
Stacey et al. (1996) J: Imf~aunol. 157:2116-2122) suggests that the TLR that
mediates
the recognition may be expressed in an intracellular compartment, such as the
late
endosome, phagosome, or lysosome.
Example 10. CpG and B-Cell Activation
B-cells from the indicated mouse strains were purified from spleen by
complement kill of CD4 +, CD8+ and macrophages. Non-adherent cells were
cultured
in the presence or absence of different amounts of stimulating CpG-DNA (5'-
TCCATGACGTTCCTGACGTT-3' (SEQ ID NO. 8), phosphorothioate modified) at
1 x lOg cells/ml. After 48 h, the cells were pulsed with [3H]thymidine
(0.5p.Ci per
well, NEN) for 16 h and processed for beta counting.
Results shown in Figure 9A are representative of three independent
experiments. B-lymphocytes derived from caspase-1 knock-out mice proliferated
in
response to CpG comparably to wild type cells (Figure 9A), suggesting that the
effect
of the MyD88 deletion is not due to a defect in IL-1/IL-18 mediated signaling.
This
result indicates that CpG-DNA signals through the receptors of the Toll
family. B-
cells from two available TLR-deficient mouse strains, the C57BL/1 OScCr strain
that
carries a spontaneous deletion of the TLR4 gene (Poltorak et al. (1998)
Science
282:2085-2088; Qureslu et al. J. Exp. Med.1999, 189:615-625) and TLR2 knock-
out
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mouse (Takeuchi et al. (1999) Immunity 11:443-451), both proliferated in
response to
CpG similar to the wild-type cells (Figure 9A). This result, together with the
normal
responses of the caspase-1 deficient cells, suggested that a members) of the
Toll
family other than TLR2 or TLR4 is involved in recognition of CpG-DNA.
Example 11. CpG and B-cell Expression of CD86 and MHC class II
The CpG-induced expression of CD86 and upregulation of MHC class-II
molecules on B-cells was tested to determine whether these processes are
mediated by
the MyD88 signaling pathway. B-lymphocytes from MyD88 knock-out mice and
wild-type littermate control mice, as well as those from TLR4-deficient mice,
were
stimulated by CpG-DNA. CD86 and MHC class -II cell surface expression were
analyzed by FAGS.
B-cells were prepared as above and cultured at 3 x 106 cells/ml with or
without
10 mM CpG for 12 h. After the stimulation, the surface expression of CD86 and
MHC class II were analyzed by flow cytometry. Results, shown in Figure 9B,
represent gated B-cells. The shaded area represents stimulated cells, whereas
the
unshaded area represents untreated controls. As shown in Figure 9B, CpG-DNA
strongly induced expression of CD86 and MHC class-II on B-cells from wild-type
and TLR4-deficient mice. By contrast, this induction was completely abrogated
in
MyD88 deficient B-lymphocytes.
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Example 12. Clonin.~ of Salmonella T~mphimurium Fla~ellin and E. coli
Fla eg llin
Full-length ,Salmonella typhirnuriurn Flagellin and E coli Flagellin were
cloned
from the respective genomic DNAs and expressed as recombinant proteins in E
coli .
Flagellin was expressed alone, or as a fusion protein with antigenic epitopes
from
ovalbumin (SIINFEKL), tyrosinase-2 protein (TRP2) cloned from marine B16
cells,
or the C-terminal fragment of I-Ea protein, which contains the Ea epitope. In
addition, all of the recombinant proteins contained a C-terminal 6x-histidine
repeat to
aid in purification.
Induced bacteria were lysed in a gentle lysis buffer containing Triton-X 100,
glycerol, imidazole, NaCI, and Tris, pH=8.0 to maintain the native
conformation of
the proteins. Fusion proteins were purified by passing filtered lysates over a
Nickel-
NTA agarose column followed by extensive washes in several buffers containing
imidazole. Purified proteins were eluted in 250mM imidazole, passed twice over
a
Polymyxin B column to remove contaminating lipopolysaccharide and then
dialyzed
extensively overnight in PBS at 4°C. The resulting purified proteins
were very stable
and retain activity at 4°C for at least a month.
Example 13. Fla~ellin In vitro Assay
In vitro assays were performed using purified Flagellin fusion proteins as
follows:
The human 293 cell line and the marine RA.W cell line were stably transfected
with a reporter gene containing two copies of the IgK NF-xB site driving
transcription
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of luciferase (this construct is referred to as "pBIIxluc"). The resulting
cell lines
(293LUC and RAWkb) were plated in 24-well dishes and treated 24 hours later
with
Flagellin fusion proteins or a control protein (lacZ) that was made in the
same vector
and purified exactly the same way as the Flagellin proteins. Cell lysates were
made
after 5 hours of treatment and were tested for luciferase activity to indicate
induction
of NF-~cB. The Flagellin proteins significantly induced NF-KB in this assay,
particularly in 293 cells whereas the control protein had no effect, as shown
in Figures
12 and 13. It is believed that this induction was not due to contamination by
LPS
since polymyxin B did not inhibit the activation in RAWxB cells, and 293LUC
cells
do not respond to LPS but do respond to Flagellin, as indicated by Figures 12
and 14.
The results of the In vitro assays demonstrate that Flagellin fusion proteins
retain their ability to stimulate Toll-Like Receptors and can therefore be
used for the
generation of recombinant Flagellin-Antigen fusion proteins for the purpose of
vaccination. In Flagellin-Antigen fusion proteins, Flagellin is believed to
stimulate
the innate immune system by triggering Toll-Like Receptors, whereas the
antigen
fused to Flagellin provides epitopes for recognition by T and B lymphocytes.
Example 14: CpG and IL-6 Production in Macrophag-eses
Adherent thioglycollate-elicited peritoneal exudate cells (PECs) from the
indicated mouse strains were treated with different stimuli for 24 h. The
release of IL-
6 into the supernatant was analyzed by specific enzyme-linked immunosorbent
assay
(ELISA) using anti-mouse IL-6 monoclonal antibodies. As CpG-DNA is also known
to have a pronounced stimulatory effect on macrophages (Stacey et al. (2000)
Curt.
Top. Microbiol. Immunol. 247: 41-58; Lipford et al. (1998) Trends Microbiol.
6: 496-
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500; Stacey et al. (1996) J. Immuraol. 157: 2116-2122), CpG-induced expression
of
IL-6 by wild-type and MyD88 was examined in deficient macrophages. Cells
derived
from caspase-1 lmock-out mice were used as a control for IL-1-mediated
induction of
IL-6. The production of IL-6 in response to CpG stimulation was completely
abolished in MyD88 -/- macrophages, but was normal in caspase-1, TLRZ- and
TLR4-deficient cells (Figure 10A). Oligonucleotides consisting of inverted CpG
sequence (GpC) were used as a control, and as expected did not induce
detectable
amounts of IL-6 (FigurelOA).
Example 15. CpG-DNA-Induced IxBa Degradation
We next tested whether activation of the NF-~cB signaling pathway is deficient
in MyD88 -/- macrophages. Peritoneal macrophages were stimulated with CpG-
DNA, or LPS as a control, for 0, 10, 20, 60, and 90 minutes and lysed
thereafter. For
each timepoint, 30 mg total protein was processed for SDS-PAGE and analyzed by
immunoblotting for IxBa protein. (Figure 10B). In wild-type cells, both LPS
and
CpG-DNA induced NF-xB activation, as evidenced by the degradation of IxB
protein
(Figure l OB). In MyD88 -/- macrophages, LPS still induced IKB degradation,
albeit
with delayed kinetics, as is consistent with published observations (Kawai et
al.
(1999) Imf~auyi.ity 11: 115-122). However, unlike LPS, CpG-DNA did not induce
IxB
degradation in MyD88 -/- macrophages (Figure 10B). Therefore, while both LPS
and
CpG-DNA signal through MyD88, the signaling pathways initiated by these
stimuli
are not identical, reflecting a possibility that different TLRs can activate
overlapping
but distinct signaling pathways.
Example 16. CpG and IL-2 Production in Dendritic Cells
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CpG-DNA has been shown to be a potent inducer of DC activation
(Sparwasser et al. (1998) Eur. J. hnrnunol. 28: 2045-2054). DC play a pivotal
role in
the initiation of the adaptive immune responses (Banchereau et al. (1998)
Nature 392:
245-252). Upon interaction with microbe-derived products (PAMPs) in peripheral
tissues, DC undergo developmental changes collectively referred to as
maturation
(Banchereau et al. (1998) Nature 392: 245-252). The hallmark of DC maturation
is
the induction of cell surface expression of CD80 and CD86 molecules, as well
as
migration into lymphoid tissues and production of cytokines such as IL-12
(Banchereau et al. (1998) Nature 392: 245-252). We tested therefore, whether
the
induction of DC maturation by CpG-DNA is mediated by the MyD88 signaling
pathway. MyD88 -/- animals produce IL-12 when stimulated with CpG
oligonucleotides. Wild-type, B 10/ScCr, and MyD88 -/- bone marrow DC, were
prepared from bone marrow suspensions cultured for 5 days in DC Growth Medium
(RPMI 5% FC + 1% GM-CSF) and stimulated with 10 mm CpG or 10 mm GpC
oligonucleotides or left untreated. Supernatants were taken 24 h and 48 h
after
stimulation and analyzed for IL-12 by ELISA using specific capture and
detection
antibodies.
The results, shown in Figure 11, are from one of three independently
performed experiments. Consistent with published reports, CpG-DNA induced
secretion of large amounts of IL-12 by DC from the wild-type mice. However, no
detectable IL-12 was produced in response to CpG stimulation by DC derived
from
MyD88 knock-out mice (Figure 11). As expected, DC from TLR4-deficient mice
produced wild-type levels of IL-12 in response to CpG-DNA (Figure 11).
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Example 17. CpG/ Ea Chimeric Construct
A non-protein PAMP, CpG, was conjugated to the characterized mouse
antigen, Ea, through a PEG polymer linker andlor copolymers of D-lysine and D-
glutamate, according to the methods described in U.S. Pat. No. 6,06,0056. A
CpG-
DNA derivative, comprising CpG4o was used as the non-protein PAMP.
All articles, patents and other materials referred to below are specifically
incorporated herein by reference.
While this invention has been disclosed with reference to specific
embodiments, it is apparent that other embodiments and variations of this
invention
may be devised by others skilled in the art without departing from the true
spirit and
scope of the invention. The appended claims are intended to be construed to
include
all such embodiments and equivalent variations.
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SEQUENCE LISTING
<110> YALE UNIVERSITY
<120> INNATE IMMUNE SYSTEM-DIRECTED VACCINES
<130> 044574-5071-WO
<140> NOT YET ASSIGNED
<141> 2001-07-31
<150> US 60/222,042
<151> 2000-07-31
<160> 13
<170> PatentIn version 3.0
<210> 1
<211> 4
<212> PRT
<213> Artificial
<220>
<223> lipidation site
<220>
<221> VARIANT
<222> (2) .. (3)
<223> X=any amino acid, preferably serine
<400> 1
Cys Xaa Xaa Asn
1
<210> 2
<211> 78
<212> PRT
<213> Escherichia coli
<220>
<221> misc feature
<223> BLP
<400> 2
Met Lys Ala Thr Lys Leu Val Leu Gly Ala Val Ile Leu Gly Ser Thr
1 5 10 15
1
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Leu Leu Ala Gly Cys Ser Ser Asn Ala Lys Ile Asp Gln Leu Ser Ser
20 25 30
Asp Val Gln Thr Leu Asn Ala Lys Va1 Asp Gln Leu Ser Asn Asp Val
35 40 45
Asn Ala Met Arg Ser Asp Val Gln Ala Ala Lys Asp Asp ATa Ala Arg
50 55 60
Ala Asn Gln Arg Leu Asp Asn Met Ala Thr Lys Tyr Arg Lys
65 70 75
<210> 3
<211> 20
<212> PRT
<213> Escherichia coli
<220>
<221> misc feature
<223> BLP leader sequence
<400> 3
Met Lys Ala Thr Lys Leu Val Leu Gly Ala Val Ile Leu Gly Ser Thr
1 5 10 15
Leu Leu Ala Gly
<210> 4
<211> 20
<212> PRT
<213> Erwinia amylovora
<220>
<221> mist feature
<223> BLP leader sequence
<400> 4
Met Asn Arg Thr Lys Leu Val Leu Gly Ala Val Ile Leu Gly Ser Thr
1 5 10 15
Leu Leu Ala Gly
<210> 5
<211> 19
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<212> PRT
<213> Serratia marcescens
<220>
<221> mist feature
<223> BLP leader sequence
<400> 5
Met Asn Arg Thr Lys Leu Val Leu Gly Ala Val Ile Leu Gly Ser His
1 5 10 15
Ser Ala Gly
<210> 6
<211> 19
<212> PRT
<213> Proteus mirabilis
<220>
<221> misc feature
<223> BLP leader sequence
<400> 6
Met Lys Ala Lys Ile Val Leu Gly Ala Val Ile Leu Ala Ser Gly Leu
l 5 10 15
Leu Ala Gly
<210> 7
<21l> 16
<212> PRT
<213> Borrelia burgdorferi
<220>
<221> misc feature
<223> Outer surface protein A
<400> 7
Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala
1 5 10 15
<210> 8
3
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<211> 20
<212> DNA
<213> Artificial
<220>
<223> CpG-DNA
<400> 8
tccatgacgt tcctgacgtt
<210> 9
<211> 73
<212> PRT
<213> Ambrosia trifida
<220>
<221> misc feature
<223> RaSG ragweed pollen allergen
<400> 9
Met Lys Asn Ile Phe Met Leu Thr Leu Phe Ile Leu Ile Ile Thr Ser
1 5 10 15
Thr Ile Lys Ala Ile Gly Ser Thr Asn Glu Val Asp Glu Ile Lys Gln
20 25 30
Glu Asp Asp Gly Leu Cys Tyr Glu Gly Thr Asn Cys Gly Lys Val Gly
35 40 45
Lys Tyr Cys Cys Ser Pro Ile Gly Lys Tyr Cys Val Cys Tyr Asp Ser
50 55 60
Lys Ala Ile Cys Asn Lys Asn Cys Thr
65 70
<210> 10
<211> 2182
<212> DNA
<213> Mus musculus
<220>
<221> misc feature
<222> (405)..(1958)
<223> Tyrosinase-Related Kinase Protein 2 (TRP-2)
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<220>
<221> misc feature
<222> (945)..(968)
<223> T-cell epitope
<220>
<221> misc recomb
<222> (840)..(1040)
<223> Region linked to BLP to form fusion protein
<400> 10
gcagcataat aagcagtatg gctggagcac tctgtaaatt aactcaatta gacagagcct
gatttaacaa ggaagactgg cgagaagctc ccctcattaa acctgatgtt agaggagctt
120
cggatgaaat taaatcagtg ttagttgttt gagtcacata aaattgcatg agcgtgtaca
180
catgtgcaca cgtgtaggct ctgtgattta ggtgggaatt ttgagaggag aggaaagggc
240
tagaactaaa cccaaagaaa aggaaagaag agaagaggaa aggaaagaaa aaagaaaagg
300
caatttgagt gagtaaaggt tccagaactc aggagtggaa gacaaggagt aaagtcagac
360
agaaaccagg tgggacgccg gccaggcctc ccaattaaga aggcatgggc cttgtgggat
420
gggggcttct gctgggttgt ctgggctgcg gaattctgct cagagctcgg gctcagtttc
480
cccgagtctg catgaccttg gatggcgtgc tgaacaagga atgctgcccg cctctgggtc
540
ccgaggcaac caacatctgt ggatttctag agggcagggg gcagtgcgca gaggtgcaaa
600
cagacaccag accctggagt ggcccttata tccttcgaaa ccaggatgac cgtgagcaat
660
ggccgagaaa attcttcaac cggacatgca aatgcacagg aaactttgct ggttataatt
720
gtggaggctg caagttcggc tggaccggcc ccgactgtaa tcggaagaag ccggccatcc
780
taagacggaa tatccattcc ctgactgccc aggagaggga gcagttcttg ggcgccttag
840
acctggccaa gaagagtatc catccagact acgtgatcac cacgcaacac tggctggggc
900
5
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tgctcggacc caacgggacc cagccccaga tcgccaactg cagcgtgtat gacttttttg
960
tgtggctcca ttattattct gttcgagaca cattattagg tccaggacgc ccctataagg
1020
ccattgattt ctctcaccaa gggcctgcct ttgtcacgtg gcacaggtac catctgttgt
1080
ggctggaaag agaactccag agactcactg gcaatgagtc ctttgcgttg ccctactgga
1140
actttgcaac cgggaagaac gagtgtgacg tgtgcacaga cgactggctt ggagcagcaa
1200
gacaagatga cccaacgctg attagtcgga actcgagatt ctctacctgg gagattgtgt
1260
gcgacagctt ggatgactac aaccgccggg tcacactgtg taatggaacc tatgaaggtt
1320
tgctgagaag aaacaaagta ggcagaaata atgagaaact gccaacctta aaaaatgtgc
1380
aagattgcct gtctctccag aagtttgaca gccctccctt cttccagaac tctaccttca
1440
gcttcaggaa tgcactggaa gggtttgata aagcagacgg aacactggac tctcaagtca
1500
tgaaccttca taacttggct cactccttcc tgaatgggac caatgccttg ccacactcag
1560
cagccaacga ccctgtgttt gtggtcctcc actcttttac agacgccatc tttgatgagt
1620
ggctgaagag aaacaaccct tccacagatg cctggcctca ggaactggca cccattggtc
1680
acaaccgaat gtataacatg gtccccttct tcccaccggt gactaatgag gagctcttcc
1740
taaccgcaga gcaacttggc tacaattacg ccgttgatct gtcagaggaa gaagctccag
1800
tttggtccac aactctctca gtggtcattg gaatcctggg agctttcgtc ttgctcttgg
1860
ggttgctggc ttttcttcaa tacagaaggc ttcgcaaagg ctatgcgccc ttaatggaga
1920
caggtctcag cagcaagaga tacacggagg aagcctagca tgctcctacc tggcctgacc
1980
tgggtagtaa ctaattacac cgtcgctcat cttgagacag gtggaactct tcagcgtgtg
2040
ctctttagta gtgatgatga tgatgcctta gcaatgacaa ttatctctag ttgctgcttt
2100
6
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gcttattgta cacagacaaa atgcttgggt cattcaccac ggtcaaagta aggtgtggct
2160
agtatatgtg acctttgatt ag
2182
<210> 11
<211> 517
<212> PRT
<213> Mus musculus
<220>
<221> mist feature
<222> (181)..(188)
<223> T-cell epitope
<220>
<221> mist recomb
<222> (146)..(212)
<223> Region linked to BLP to form fusion protein
<400> 11
Met Gly Leu Val Gly Trp Gly Leu Leu Leu Gly Cys Leu Gly Cys Gly
1 5 10 15
Ile Leu Leu Arg Ala Arg Ala Gln Phe Pro Arg Val Cys Met Thr Leu
20 25 30
Asp Gly Val Leu Asn Lys Glu Cys Cys Pro Pro Leu Gly Pro Glu Ala
35 40 45
Thr Asn Ile Cys Gly Phe Leu Glu Gly Arg Gly Gln Cys Ala Glu Val
50 55 60
Gln Thr Asp Thr Arg Pro Trp Ser Gly Pro Tyr Ile Leu Arg Asn Gln
65 70 75 80
Asp Asp Arg Glu Gln Trp Pro Arg Lys Phe Phe Asn Arg Thr Cys Lys
85 90 95
Cys Thr Gly Asn Phe Ala Gly Tyr Asn Cys Gly Gly Cys Lys Phe Gly
100 105 110
Trp Thr Gly Pro Asp Cys Asn Arg Lys Lys Pro Ala Ile Leu Arg Arg
115 120 125
Asn Ile His Ser Leu Thr Ala Gln Glu Arg Glu Gln Phe Leu Gly Ala
130 135 140
Leu Asp Leu Ala Lys Lys Ser Ile His Pro Asp Tyr Val Ile Thr Thr
145 150 155 160
7
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Gln His Trp Leu Gly Leu Leu Gly Pro Asn Gly Thr Gln Pro Gln Ile
165 170 175
Ala Asn Cys Ser Val Tyr Asp Phe Phe Val Trp Leu His Tyr Tyr Ser
180 185 190
Val Arg Asp Thr Leu Leu Gly Pro Gly Arg Pro Tyr Lys Ala Ile Asp
195 200 205
Phe Ser His Gln Gly Pro Ala Phe Val Thr Trp His Arg Tyr His Leu
210 215 220
Leu Trp Leu Glu Arg Glu Leu Gln Arg Leu Thr Gly Asn Glu Ser Phe
225 230 235 240
Ala Leu Pro Tyr Trp Asn Phe Ala Thr Gly Lys Asn Glu Cys Asp Val
245 250 255
Cys Thr Asp Asp Trp Leu Gly Ala Ala Arg Gln Asp Asp Pro Thr Leu
260 265 270
Ile Ser Arg Asn Ser Arg Phe Ser Thr Trp Glu Ile Val Cys Asp Ser
275 280 285
Leu Asp Asp Tyr Asn Arg Arg Val Thr Leu Cys Asn Gly Thr Thr Glu
290 295 300
Gly Leu Leu Arg Arg Asn Lys Val Gly Arg Asn Asn Glu Lys Leu Pro
305 310 315 320
Thr Leu Lys Asn Val Gln Asp Cys Leu Ser Leu Gln Lys Phe Asp Ser
325 330 335
Pro Pro Phe Phe Gln Asn Ser Thr Phe Ser Phe Arg Asn Ala Leu Glu
340 345 350
Gly Phe Asp Lys Ala Asp Gly Thr Leu Asp Ser Gln Val Met Asn Leu
355 360 365
His Asn Leu Ala His Ser Phe Leu Asn Gly Thr Asn Ala Leu Pro His
370 375 380
Ser Ala Ala Asn Asp Pro Val Phe Val Val Leu His Ser Phe Thr Asp
385 390 395 400
Ala Ile Phe Asp Glu Trp Leu Lys Arg Asn Asn Pro Ser Thr Asp Ala
405 410 415
Trp Pro Gln Glu Leu Ala Pro Ile Gly His Asn Arg Met Tyr Asn Met
420 425 430
Val Pro Phe Phe Pro Pro Val Thr Asn Glu Glu Leu Phe Leu Thr Ala
435 440 445
Glu Gln Leu Gly Tyr Asn Tyr Ala Val Asp Leu Ser Glu Glu Glu Ala
450 455 460
Pro Val Trp Ser Thr Thr Leu Ser Val Val Ile Gly Ile Leu Gly Ala
465 470 475 480
g
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Phe Val Leu Leu Leu Gly Leu Leu Ala Phe Leu Gln Tyr Arg Arg Leu
485 490 495
Arg Lys Gly Tyr Ala Pro Leu Met Glu Thr Gly Leu Ser Ser Lys Arg
500 505 510
Tyr Thr Glu Glu Ala
515
<210> 12
<211> 68
<212> PRT
<213> Mus musculus
<220>
<221> SITE
<222> (37) . . (44)
<223> T-cell epitope
<220>
<221> mist feature
<223> TRP-2
<400> 12
Leu Asp Leu Ala Lys Lys Ser Ile His Pro Asp Tyr Val Ile Thr Thr
1 5 10 15
Gln His Trp Leu Gly Leu Leu Gly Pro Asn Gly Thr Gln Pro Gln Ile
20 25 30
Ala Asn Cys Ser Val Tyr Asp Phe Phe Val Trp Leu His Tyr Tyr Ser
35 40 45
Val Arg Asp Thr Leu Leu Gly Pro Gly Arg Pro Tyr Lys Ala Ile Asp
50 55 60
Phe Ser His Gln
<210> 13
<211> 8
<212> PRT
<213> Mus musculus
<220>
<221> SITE
9
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<222> (1) . . (8)
<223> T-cell epitope
<400> 13
Val Tyr Asp Phe Phe Val Trp Leu
1 5
