CA 02485467 2004-11-17
A Method of generating Pre-selected Epitope-specific Vaccines
BACKGROUND
Peptide array has been used in many studies and applications. Those arrays are
mainly
used to study or identify interaction of proteins of interests. Antigen and
antibody reaction
has been used to determine the presence of a pathogen for dliagnosis. However,
the
antigens currently employed for detecting antibodies are either the pathogen
itself, or one
or more proteins of the pathogen. Since an antibody only recognizes a fragment
of peptide
of 3-6 amino acids, named epitope, pathogen or protein based antigens are
frequently
cross reacted with antibodies that are not related to the partiicular pathogen
due to
homologues of amino acid sequences in many regions of the antigen to unrelated
proteins.
This cross-reaction causes frequent false positive results.
A vaccine is an antigen that can be delivered to human body to stimulate the
immune
system to produce specific antibodies for the purpose of protecting body from
a particular
pathogen. 'The antigens most frequently used for vaccines are attenuated or
denatured
pathogens. Those antigens bear significant risks of adverse effects associated
with the
pathogen. To reduce the risk, recently, fragments of proteins from a pathogen
of interest
were used as vaccines. However, since immune systems of conventional
experimental
animals are quite different from that of human being, it is necessary to
inject the
polypeptide into healthy human volunteers and test whethea- there are any
neutralizing
antibodies generated in serum samples from those people. As not all the
protein fragment
can generate antibodies that can neutralize the pathogen, development of a
good peptide
vaccine is basically a process of trial and failure, which not only time
consuming but also
expensive. Thus, there is a need in the art for an accurate, efficient, and
inexpensive method of making vaccines.
Current methods for making vaccines include: a) using viral nucleotides as
vaccines, b)
using live attenuated or inactivated viruses as vaccines, c) using viral
proteins to make
CA 02485467 2004-11-17
vaccines. After these vaccines are introduced into the body, they can
stimulate the
immune system to generate specific antibodies to protect the human body. A
good
vaccine is characterized by the following three aspects, namely, possessing
high
specificity to a particular virus, possessing strong immune stimulation and
possessing no
toxicity or side effects. The immune stimulation of nucleotide vaccines is
usually not
strong enough and their specificities are not optimal either. Although
attenuated or
inactivated viruses can stimulate the production of antibodies, many viruses
carry proteins
that are toxic, which makes vaccination very risky. One other hand, vaccines
derived
from virus proteins are highly effective and safe. Nevertheless, it is very
difficult to
identify the virus proteins among a variety of virus proteins that have both
strong immune
stimulation and no toxicity to the body. Currently, almost all of the methods
for screening
vaccines are through random tests based on previous experiience, which is both
time-
consuming and less effective.
Current methods of developing vaccine using peptides can be summarized as
following
steps: (1) Select potential vaccine peptides; (2) Vaccinate animal with the
peptides; (3)
Collect antibodies from the animal; (4) Verify whether the antibodies can
neutralize the
pathogen; (5) Clinical trial: test toxicity; (6) Verify whether the human
antibody can
neutralize the pathogen; (7) Finding the Vaccine and Marketing. These methods
reduire
two necessary steps before identifying antibody candidates: animal test and
human test,
which involve tremendous time and cost to complete, but not promising (Figure
1 ).
This background information is provided for the purpose o:E'making known
information
believed by the applicant to be of possible relevance to the present
invention. No
admission is necessarily intended, nor should be construed, that any of the
preceding
information constitutes prior art against the present invention.
SUM1VIARY OF THE INYEN1'ION
An object of the present invention is to provide a new metrrod for generating
vaccines that
can be tested for its function prior to human trial.
CA 02485467 2004-11-17
The present invention consists of the following steps (Figure 2)e (1)
Preparing peptides
arrays;(2) Using recovered patients' serum samples to identify the epitopes on
the
proteins of the pathogen; (3) Identifying antibody-specific peptides; (4)
Preparing afFnity
column; (5) Isolating antibodies bound to specific epitopes; (6) Testing
functions of the
isolated antibodies and selecting the epitopes corresponding to the antibodies
with desired
functions; (7) Generating a polypeptide vaccine containing the pre-selected
epitope(s).
In accordance with one embodiment of the present invention, the present
invention
provides a method of generating vaccines to pathogens infected and non-
pathogens
infected diseases.
In accordance with one embodiment of the present invention, the serum samples
are those
from patients recovered from pathogens infected and non-pathogens infected
diseases.
In accordance with one embodiment of the present invention, the present
invention
provides uses of diagnoses, treatments, and preventions to pathogens infected
and non-
pathogens infected diseases.
BRIEF DESCRIPTION OF THE DItAV~IIhTGS
Figure 1 is a diagram of current methods of making vaccines
Figure 2 is a diagram of current invented method of making vaccines
Figure 3 is a diagrammatic representation of peptide libran,~
Figure 4 is an example of an epitope mapping for BARS
Figure 5 is an example of epitope-specific diagnosis for SARS
DETAILED DESCRIPTION OF THE INVENTION
The following terms and abbreviations are used throughout the specification
and in the
CA 02485467 2004-11-17
ClalmS:
The terms "polypeptide" and "peptide," as used herein, refer to a sequence of
amino acid
residues linked together by peptide bonds and may be used interchangeably.
The term "antigen" as used herein denotes any molecule capable of eliciting an
immune
response in an animal.
The term "vaccine" as used herein refers to a preparation of"material capable
of
stimulating an immune response in an animal without inducing disease.
The present invention consists of the following steps:
1. Preparation of Peptides Array (Peptide Library)
In accordance with the present invention, the peptides can be peptide
sequences derived
from the nucleotide sequence of viruses or diseases (available on publicly
available
databases) or they can be recombinant peptide sequences that have been
specifically
engineered using genetic engineering techniques known in the art.
These peptides are candidate peptides which can be organized into a peptide
library by
specifically being bound to a solid support. Solid supports utilized in the
preparation of
peptide libraries include, but are not limited to, nitrocellulose membrane,
polymer sheets,
microplates, nylon sheets, peptide microchips, such as Pepscan System, Ink jet
peptide
arrays, and Xeotron's peptide microchips. In one embodiment, the peptide
fragments of
the present invention are bound to a piece of specially treated nitrocellulose
membrane.
Chemical approaches to building peptide libraries are known in the art. For
example, for
selection of those peptides that bind to antibodies in serum derived from
patients of
SARS, a peptide library can be generated using standard finoc chemistry as
described by
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Merrifield. Fmoc beta alanine is coupled via an ester bond to the hydroxy
groups of a
Whatman cellulose membrane. After coupling, the fmoc group is removed with
pipiridine
and the first fmoc protected amino acid is chemically coupled following
activation with
HOBT and DIC, both standard chemicals for activating th.e carboxy group of the
amino
acid for attachment of the free amino group of beta alanine to it forming a
peptide bond.
Each amino acid starting from the C terminal of the peptide, is added
sequentially using a
robotic spotter until the full-length peptide is assembled., A program is
written using
Intavis software to generate all peptides in any protein, in this case, all
peptides encoded
by all open reading frames of the SAl~S virus genome. The; program is written
so that the
robot assembles all overlapping peptides, frame shifted by i;wo amino acids in
all proteins
of the virus. Each cycle of the synthesis requires deprotec;tion of the N
terminal amino
group to remove frnoc with pipiridine. After washing the membranes with
dirnethyl
formamide and methanol, the next round of synthesis begins adding the
appropriate finoc
amino acid to its position in the sequence. The whole procedure is repeated 10
times to
generate 10 mer peptides. After the final deprotection step., side chain
protecting groups
are removed with trifluoroacetic acid and other scavengers.
In one embodiment, the peptide library comprises peptidE; fragments of 10
amino acid
residues, and the N-terminal of the peptide and the C-tenminal of its previous
peptide
fragment overlap by an 8-residue sequence (each displaced by two amino acids).
The
required numbers of peptide fragments for peptides array vary from making
different
vaccines for pathogens infected and non-pathogens infected diseases.
2. Screening patients serum samples
In accordance with the present invention, the peptide fragments are used to
react with
serum samples from two group of human: a group of patients who are recovered
from a
pathogens-infected or non-pathogens-infected disease and a group of health
human. By
washing these peptides fragments in O.1M phosphate buffered saline (PBS) 3
times, the
array then is incubated for 2 hours at room temperature with 1:1000 diluted
HRP-
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Y ";' '~FV~rtCP~»--~aaN~gm-"2c",~a»a Aaun-
:FNS.~a~,~avr~'Aa.t~xatea~artw~,e~c~rs».e,v,-vA_~
CA 02485467 2004-11-17
conjugated antibodies against human IgG, or IgM or IgA, respectively. After
another 3-
time wash in PBS, by comparing the results from serum samples of two groups of
human,
the antibody-specific epitopes of those samples can be identified by HRP-
substrate
chemiluminescence reaction (ECL). The luminescence images are captured with
either x-
ray films or a digital camera. The images are then input into a computer for
analysis.
3.Identi, fying Antibody-Specific Epitope Peptides
Images of positively reacted peptide spots of the array are converted to the
corresponding
peptide sequences with a custom made software. The software also compares the
positive
sequences between the viruses or diseases samples vs. control group (from
health human)
and identifies the peptide sequences (epitopes) that were bound by virus or
disease
specific antibodies.
In accordance with the present invention, the peptide fragments are
characterized by
binding to antibodies in sera of patients who are recovered i~om a pathogens-
infected or
non-pathogens-infected disease. Candidate peptides can be screened for binding
to
antibodies found in such sera. For example, a peptide library comprising the
candidate
peptides can be exposed to control sera and sera of SARS infected individuals
using
methods known in the art.
In one embodiment of the present invention, the sera samplc;s are derived from
patients
recovered from a pathogens infected or non-pathogens infecaed disease.
In another embodiment, the peptides are on a solid support. In a further
embodiment, the
solid support is a nitrocellulose membrane. In another embodiment, the
peptides are in
solution.
4. Preparing Affinity Column
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One ml of packed beads (Act. Ultrogel 22 AcA) be prepared by washing antibody-
specific epitope peptides 4 times in 10 ml of d-H20 in a 15 ml tube. The beads
are further
washed 4 times in 10 ml of O.1M NaBorate (pH 8.5) + 0.01% SDS. 2-5 mg of each
peptide selected from step 3 is added to 1 ml of above prepared beads
contained in each
tube and rotate overnight at room temperature on a nutator. Uncoupled peptides
are
washed away any by 4 washes of 10 ml in PBS (pH 7.4) followed by incubation
with 2 ml
of O.1M lysine in PBS for 5 hours at room temperature wit~~ rotation.
Excessive lysine is
washed away by 4 washes of 10 ml each of PBS. The beads are load to plastic
columns
with PBS + lOmM NaN3 and stored at 4°C.
S. Isolating antibodies bound to specific epitopes
~5 ml of diluted sera from patients who are recovered from a pathogens-
infected or non-
pathogens-infected disease are loaded to the column and let it continuously
flow through
the column for at least 2 hours with a pump to make a circular flow from the
bottom to
the top of the column. Any contaminating proteins are washed away with PBS
until the
A280nm= or < 0.005. The antibodies bound were eluted from the columns with 2
ml
0.2M glycine-HCl (pH 2.8) at 4°C and collect in tubes with 0.3 ml of 1M
K2HP04. The
collected antibodies are dialyzed against 500 ml of 50% glycerol in PBS at
4°C overnight
with stirring.
6. Testing functions of the isolated antibodies and selecting the epitopes
corresponding to
the antibodies with desired functions
Each of the epitope-specific antibodies isolated from step 5 will be serially
diluted
and incubated with DMEM medium containing 106pfu/ml viruses for 30min at
37°C before added to cultured VERO cells at multiplicity of infection
(M~I)=0.1.
The infected cell cultures will be fixed with 10% formallin for 30 min at RT
followed by staining with Neutral Red for visualizing plaques. The epitope
peptides corresponding to antibodies with neutralizing effect on the virus
will be
CA 02485467 2004-11-17
selected.
7. Generating a polypeptide vaccine containing the pre-selected epitope(s)
If one of the epitope antibodies is capable of neutralizing virus, the
corresponding epitope
peptide will be selected as a candidate of vaccine for furthers tests. If more
than one
epitope antibodies are found to be able to neutralizing virus, the multiple
epitope peptides
can be fused as a polypeptide or mixed as a cocktail to generate a rnulti-
antigenic vaccine.
The epitope peptides corresponding to antibodies with neutralizing effect
demonstrated in
step 6 will be selected as vaccine candidates for further toxicity test on
animals and
human trials.
The novelty of this invention is that this procedure allows a pre-selection of
epitope
peptides with the capability of generating neutralizing antibodies prior to
human or
animal tests. The pre-selection is based on the screening of antibodies from
sera of
patients who are recovered from a pathogens-infected or non-pathogens-infected
disease
using specific epitope peptides with affinity column or by other affinity
binding methods.
Since the sera of patients recovered from a pathogens-infecl;ed or non-
pathogens-infected
disease contain neutralizing antibodies for the pathogen. In vitro viral
neutralization tests
on individual antibodies isolated according to their specific corresponding
epitopes
identified with epitope mapping can predict which epitopes can generate
neutralizing
antibodies by human immune system. Thus, a vaccines madle of the epitope
peptides pre-
selected through the above procedure has a much higher likelihood of success
when used
to immunize human population for neutralizing the targeted. pathogen.
The present invention can be used for generating vaccines to pathogens
infected
and non-pathogens infected disease. The pathogens infected diseases include
bacteria and virus infected diseases; the non-pathogens infected diseases
include cancer, diabetes, cardio-vascular disease, and endocrinological
diseases.
CA 02485467 2004-11-17
The antibodies identified using present invention can be produced in large
quantity by
methods known in the art in order to apply to diagnosis, treat, and prevent
pathogens
infected and non-pathogens infected disease.
Diagnostic compositions
The instant invention also provides for the use of the present invention, for
the
manufacture of diagnostic compositions and reagents. By using the peptides,
for example
in an array, a skilled person in the art is able to find the viral peptide
fragments that can
bind to specific antibodies through the reaction of the antiserum of patients
recovered
from a pathogens-infected or a non-pathogens-infected disease with the peptide
array.
The reagents containing these specific immune-reactive peptides can be used to
screen
and diagnose the infected patients.
Pharmaceutical Compositions, Medicaments and Drugs
In accordance with the invention, use of the present invention, for the
manufacture of
pharmaceutical compositions, medicaments and drugs is provided. In one
embodiment,
the epitope-specific antibodies of the present invention are used to design
drugs.
The pharmaceutical compositions, medicaments or drugs may be administered
orally,
topically, parenterally, by inhalation or spray or rectally in fosage unit
formulations
containing conventional non-toxic pharmaceutically acceptable carriers,
adjuvants and
vehicles. The term parenteral as used herein includes subcutaneous injections,
intravenous, intramuscular, intrasternal injection or infusion techniques. The
pharmaceutically active compound or salts thereof may be present in
association with one
or more non-toxic pharmaceutically acceptable carriers andl'or diluents and/or
adjuvants
and, if desired, other active ingredients.
The pharmaceutical compositions, medicaments or drugs may be in a form
suitable for
oral use, for example, as tablets, troches, lozenges, aqueous or oily
suspensions,
dispersible powders or granules, emulsion hard or soft capsules, or syrups or
elixirs.
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pharmaceutical compositions, medicaments or drugs intended for oral use may be
prepared according to methods known to the art for the marmfacture of
pharmaceutical
compositions, medicaments or drugs and may contain one or more agents selected
from
the group of sweetening agents, flavouring agents, colouring agents and
preserving agents
in order to provide pharmaceutically elegant and palatable preparations.
Tablets contain
the active ingredient in admixture with suitable non-toxic pharmaceutically
acceptable
excipients including, for example, inert diluents, such as ca'Lcium carbonate,
sodium
carbonate, lactose, calcium phosphate or sodium phosphate:; granulating and
disintegrating agents, such as corn starch, or alginic acid; binding agents,
such as starch,
gelatine or acacia, and lubricating agents, such as magnesium stearate,
stearic acid or talc.
The tablets can be uncoated, or they may be coated by known techniques in
order to delay
disintegration and absorption in the gastrointestinal tract and thereby
provide a sustained
action over a longer period. For example, a time delay material such as
glyceryl
monosterate or glyceryl distearate may be employed.
Pharmaceutical compositions, medicaments or drugs for or<~1 use may also be
presented as
hard gelatine capsules wherein the active ingredient is mixed with an inert
solid diluent,
for example, calcium carbonate, calcium phosphate or kaolin, or as soft
gelatine capsules
wherein the active ingredient is mixed with water or an oil medium such as
peanut oil,
liquid paraffin or olive oil.
Aqueous suspensions contain the active compound in admi:Kture with suitable
excipients
including, for example, suspending agents, such as sodium
carboxymethylcellulose,
methyl cellulose, hydropropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone,
gum tragacanth and gum acacia; dispersing or wetting agents such as a
naturally-
occurnng phosphatide, for example, lecithin, or condensation products of an
alkylene
oxide with fatty acids, for example, polyoxyethyene stearate, or condensation
products of
ethylene oxide with long chain aliphatic alcohols, for example, hepta-
decaethyleneoxycetanol, or condensation products of ethylene oxide with
partial esters
derived from fatty acids and a hexitol for example, polyoxyethylene sorbitol
monooleate,
or condensation products of ethylene oxide with partial esters derived from
fatty acids
CA 02485467 2004-11-17
and hexitol anhydrides, for example, polyethylene sorbitan ~monooleate. The
aqueous
suspensions may also contain one or more preservatives, for example ethyl, or
n-propyl p-
hydroxy-benzoate, one or mare colouring agents, one or more flavouring agents
or one or
more sweetening agents, such as sucrose or saccharin.
Oily suspensions may be formulated by suspending the active ingredients in a
vegetable
oil, for example, arachis oil, olive oil; sesame oil or coconut oil, or in a
mineral oil such
as liquid paraffin. The oily suspensions may contain a thickening agent, for
example,
beeswax, hard paraffin or cetyl alcohol. Sweetening agents ouch as those set
forth above,
and/or flavouring agents may be added to provide palatable oral preparations.
These
compositions can be preserved by the addition of an anti-oxidant such as
ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous
suspension by
the addition of water provide the active ingredient in admixture with a
dispersing or
wetting agent, suspending agent and one or more preservatives. Suitable
dispersing or
wetting agents and suspending agents are exemplified by those already
mentioned above.
Additional excipients, for example sweetening, flavouring and colouring
agents, may also
be present.
Pharmaceutical compositions, medicaments or drugs of the invention may also be
in the
form of oil-in-water emulsions. The oil phase may be a vegetable oil, for
example, olive
oil or arachis oil, or a mineral oil, for example, liquid paraffin, or it may
be mixtures of
these oils. Suitable emulsifying agents may be naturally occurring gums, for
example,
gum acacia or gum tragacanth; naturally-occurring phosphatides, for example,
soy bean,
lecithin; or esters or partial esters derived from fatty acids and hexitol,
anhydrides, for
example, sorbitan monoleate, and condensation products of the said partial
esters with
ethylene oxide, for example, polyoxyethylene sorbitan monoleate. The emulsions
may
also contain sweetening and flavouring agents.
Syrups and elixirs may be formulated with sweetening agents, for example
glycerol,
propylene glycol, sorbitol or sucrose. Such formulations may also contain a
demulcent, a
preservative and flavouring and colouring agents.
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CA 02485467 2004-11-17
The pharmaceutical compositions, medicaments or drugs may be in the form of a
sterile
injectable aqueous or oleaginous suspension. This suspension may be formulated
according to known art using suitable dispersing or wetting agents and
suspending agents
such as those mentioned above. The sterile injectable prep~~ration may also be
sterile
injectable solution or suspension in a non-toxic parentally acceptable diluent
or solvent,
for example, as a solution in 1,3-butanediol. Acceptable vehicles and solvents
that may
be employed include, but are not limited to, water, Ringer's solution,
lactated Ringer's
solution and isotonic sodium chloride solution. Other examples are, sterile,
fixed oils
which are conventionally employed as a solvent or suspending medium, and a
variety of
bland fixed oils including, for example, synthetic mono- or diglycerides. In
addition, fatty
acids such as oleic acid find use in the preparation of injectables.
Other pharmaceutical compositions, medicaments or drugs and methods of
preparing
pharmaceutical compositions, medicaments or drugs are known in the art and are
described, for example, in "Remington: The Science and Practice of Pharmacy"
(formerly "Remingtons Pharmaceutical Sciences"); Gennaro, A., Lippincott,
Williams &
Wilkins, Philidelphia, PA (2UUU).
Pharmaceutical Kits
The present invention additionally provides for therapeutic kits containing a
pharmaceutical composition comprising epitope-specific antibodies for use in
the
prevention and treatment of pathogens-infected and/or non-pathogens-infected
diseases.
Individual components of the kit would be packaged in separate containers and,
associated with such containers, can be a notice in the form prescribed by a
governmental
agency regulating the manufacture, use or sale of pharmaceuticals or
biological products,
which notice reflects approval by the agency of ma~nufactur~e, use or sale for
human
administration.
When the components of the kit are provided in one or more liquid solutions,
the liquid
solution can be an aqueous solution, for example a sterile aqueous solution.
In this case
the container means may itself be an inhalant, syringe, pipette, eye dropper,
or other such
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CA 02485467 2004-11-17
like apparatus, from which the composition may be administered to a patient or
applied to
and mixed with the other components of the kit.
The components of the kit may also be provided in dried or lyophilised form
and the kit
can additionally contain a suitable solvent for reconstitution of the
lyophilised
components. Irrespective of the number or type of containws, the kits of the
invention
also may comprise an instrument for assisting with the administration of the
composition
to a patient. Such an instrument may be an inhalant, syringe, pipette,
forceps, measured
spoon, eye dropper or any such medically approved deliverr~ vehicle.
Diagnostic Kits
The present invention additionally provides for diagnostic kits containing
diagnostic
compositions comprising epitope-specific antibodies, peptides, peptide
analogues, and
nucleic acid seduences of the present invention, for use in diagnosing or
identifying
individuals who have been infected with pathogens diseases or non pathogens
diseases.
The kit may alternatively comprise epitope-specific antibodies, peptide or
nucleic acid
arrays for diagnostic screening of patients. The contents of ahe kit can be
lyophilized and
the kit can additionally contain a suitable solvent for reconstitution of the
lyophilized
components. Individual components of the kit would be packaged in separate
containers
and, associated with such containers, can be a notice in the form prescribed
by a
governmental agency regulating the manufacture, use or sale of pharmaceuticals
or
biological products, which notice reflects approval by the agency of
manufacture, use or
sale for human administration.
The invention now being generally described, it will be more readily
understood by
references to the following example, which are included for purposes of
illustration only
and are not intended to limit the invention unless so stated.
EXAMPLES
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CA 02485467 2004-11-17
Example 1: An example is a vaccine development for Severe Acute Respiratory
Syndrome (BARS) related virus.
Methods and Materials:
1. Generate a peptide array. The array comprises peptides selected from the
group of
peptides SARS viral proteins. Each peptide comprises a. sequence of 8-20 amino
acids
or at least 10 amino acids. The peptide array can be designed in such that a
series of
peptides with 5-12 or more .amino acids each are attached to a matrix with
their C-
termini or N-termini. The peptides are arranged on the matrix in such order
that each
peptide is adjacent to the next with 80-90% a.a. sequence identical but shift
towards
N-terminus or C-terminus a few a.a. in accordance to the sequence of the
proteins of
interest.
The peptide arrays we use are generated using standard fmoc chemistry as
described
by Merrifield. We are not using conventional coupling to a fixed resin but
carry out
the following procedure. Fmoc beta alanine is coupled via an ester bond to the
hydroxy groups of a Whatman cellulose membrane. After coupling, the finoc
group is
removed with pipiridine and the first finoc protected amino acid is chemically
coupled following activation with HOBT and DIC, both standard chemicals for
activating the carboxy group of the amino acid for attachment of the free
amino group
of beta alanine to it forming a peptide bond. Each amino acid starting from
the C
terminal of the peptides is added sequentially using a robotic spotter until
the full-
length peptide is assembled. A program is written using Intavis software to
generate
all peptides in any protein, in this case, all peptides encoded by all open
reading
frames of the SARS virus genome. The program is written so that the robot
assembles
all overlapping peptides, frame shifted by two amino acids in alI proteins of
the virus.
Each cycle of the synthesis requires deprotection of the N terminal amino
group to
remove finoc with pipiridine. After washing the membranes with dimethyl
formamide
and methanol, the next round of synthesis begins adding the appropriate frnoc
amino
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CA 02485467 2004-11-17
acid to its position in the sequence. The whole procedure is repeated 10 times
to
generate I O mer peptides. After the final deprotection step, side chain
protecting
groups are removed with trifluoroacetic acid and other scavengers. The
membranes
are rinsed, air-dried and then can be used for probing with normal control
sera and
sera of SARS infected individuals from both the acute phase and convalescent
phase
as described below.
The dried membranes are exposed to control sera and sera of SARS infected
individuals diluted I00 fold in phosphate buffered saline. These membranes are
then
incubated at 37 C for two hours and rinsed with buffer several times, leaving
only
antibodies attached to specific peptide spots. The membranes are then probed
with a
secondary antibody, either anti human IgG or anti human IgM antibodies
conjugated
to horseradish peroxidase. Wherever human IgG or IgM antibodies have bound to
the
spots, these secondary antibodies will also bind. The spots are developed with
standard visualization methodology using the ECL cherniluminescence kit.
Chemiluminescence of the spots is measured quantitati~rely with a Biorad Image
Scanner. Spots, which appear for SARS sera treated membranes compared to
control
treated membranes are taken as specifically having reacted with antibodies in
SARS
sera.
Membranes are probed with acute phase and convalescent sera to determine
antibodies specifically illicited by SARS infection itself and not previously
developed
cross-reacting antibodies to other antigens. The spots binding specific
antibodies are
resynthesized in larger numbers and reprobed with the same positive sera.
Antibodies
are elut~l from these spots and used to measure their efficiency in terms of
neutralizing virus in a standard virus-neutralizing assay. Antibodies
discovered in this
way will confirm that they are directed to peptides that can be used as
antigens in
design of a specific anti viral vaccine.
2. Screen SARS patient serum samples: The above array was used to react with
1:100
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CA 02485467 2004-11-17
diluted serum samples from. 97 SARS patients and 88 non-SARS patients for 2
hours
at room temperature. Following 3 times brief wash in 0..1 M phosphate buffered
saline
(PBS), the array was incubated for 2 hours at room temperature with 1:1000
diluted
HRP-conjugated antibodies against human IgG, or IgM or IgA, respectively.
After
another 3-time wash in PBS, peptide fragments (epitopE;s) bound by antibodies
of
those samples were identified by HRP-substrate chemilvminescence reaction.
(ECL).
The luminescence images were captured with either x-r,ay films or a digital
camera.
The images were then input into a computer for analysis.
3. Select SARS antibody-specific epitope peptides: Images of positively
reacted peptide
spots of the array were converted to the corresponding peptide sequences with
a
custom made software. The software also compares the positive sequences
between
the BARS samples vs. controls and identifies the peptide sequences (epitopes)
that
were bound by SARS specific antibodies.
4. Prepare affinity column: One ml of packed beads (Act. Ultrogel 22 AcA) was
prepared by washing them 4 times in 10 ml of d-H20 ire a 15 ml tube. The beads
were
further washed 4 times in 10 ml of O.IM NaBorate (pH 8.5) + 0.01% SDS. 2-5 rng
of
each peptide selected from step 3 was added to 1 ml of .above prepared beads
contained in each tube and rotate overnight at room temperature on a nutator.
Uncoupled peptides were washed away any by 4 washes of 10 mI in PBS (pH 7.4)
followed by incubation with 2 ml of 0.1 M lysine in PB S for 5 hours at room
temperature with rotation. Excessive lysine was washed. away by 4 washes of 10
ml
each of PBS. The beads were load to plastic columns with PBS + lOmM NaN3 and
stored at 4oC.
5. Purify epitope-specific antibodies: ~S ml of diluted seru~.m from recovered
BARS
patients were loaded to the column and let it continuously flow through the
columnfor
at least 2 hours with a pump to make a circular flow from the bottom to the
top of the
column. any contaminating proteins were washed away with PBS until the A280nm=
or < 0.005. The antibodies bound were eluted from the s;olumns with 2 ml 0.2M
glycine-HCl (pH 2.8) at 4C and collected in tubes with 0.3 ml of 1 M K2HP04.
The
collected antibodies were dialyzed against 500 ml of 50'% glycerol in PBS at
4oC
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CA 02485467 2004-11-17
overnight with stirnng.
6. Identification of neutralizing antibodies: Each of the antibodies isolated
from step 5
will be serially diluted and incubated with DMEM medium containing 106pfu/ml
SARS virus for 30min at 37C before added to cultured VERO cells at
multiplicity of
infection (MOI)=0.1. The infected cell cultures will be i:ixed with 10%
formalin for
30 min at RT followed by staining with Neutral Red for plaques.
7. Selection of peptides as vaccine. The epitope peptides corresponding to
antibodies
with neutralizing effect on the BARS virus demonstrated in step 6 will be
selected as
vaccine candidates for further toxicity test on animals and human trials.
Results:
Through comparison with the reaction to serum samples from non-BARS patients,
the
epitope map of SARS viral proteins was generated. Figure 4 shows an example of
an
epitope map. The x-axis is epitopes coded by numbers and the y-axis is the
frequency of
positive reaction on those epitopes in a patient population. It is obvious
that epitope No.6,
9 and I2 are more frequent in BARS patients comparing to control population,
indicating
these epitopes are specifically related to SARS virus.
Three affinity columns will be loaded with peptides corresponding to Epitope
No.d, 9 and
12, respectively, and sera from late stage or recovered SARS patients will be
passed
through the columns. Antibodies specifically bound to the <;olumns will be
eluted and
collected followed by in vitro viral neutralization tests. If one of the
antibodies is capable
of neutralizing SARS virus, the corresponding epitope peptide will be selected
as a
candidate of vaccine for further tests. If more than one antibodies are found
to be able to
neutralizing SARS virus, the multiple epitope peptides can be fused as a
polypeptide or
mixed as a cocktail to generate a multi-antigenic vaccine.
Example 2: An example is an epitope-specific diagnostic device
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CA 02485467 2004-11-17
An epitope-specific diagnostic device can be developed on the base of above
epitope
mapping. The peptide fragments (i.e. epitopes) that are specific to antibodies
for a
particular pathogen can be identified using the above epitope mapping
technique. These
are epitope No.6, 9 and 12 in figure 4: The individual selected epitapes can
then be Yaid
on a solid base such as the bottom of a multiple well dish or on a membrane.
Patient
serum samples can be used to react with the multiple well dish or the membrane
followed
by a detecting process such as EL1SA. Probability of the presence of the
pathogen can be
determined by measuring the number of positive epitopes and the intensity of
the
antibody binding on each epitope as shown in Fig.S. In this figure, serums of
three
probable patients and one normal person were reacted with the device and the
intensities
of reactions to each epitope selected by above (figure 4) were measured and
plotted. Since
patient A showed positive to all of the three epitopes, this person definitely
contains the
pathogen. Patient B is a highly suspected for the pathogen since he had 2 out
of 3 positive
epitopes. Patient C is likely to have the pathogen but need further
confirmation while
healthy control has no positivity in any of the three epitopes. The number of
epitopes used
for diagnostic device can be from 1 to 100 or even mare depending on the
disease to be
diagnosed.
Example 3: An example is for the treatment of type 1 diabetes.
By using the same methodology described in Example 1, The epitope mapping can
be
used. on development of a therapy for certain diseases, especially autoimmune
diseases.
Peptides of cell surface proteins on insulin secreting cells can be put on the
above array.
Serum from diabetes patients can be used to identify epitopes that are bound
by
antibodies of the patients. Since the cause of type 1 diabetes is the
destnzction of insulin
secreting cells that are attacked by antibodies generated by the
malfunctioning immune
system of the patients, identification of those epitopes can help to design a
drug that will
block the binding the antibody to that specific epitope. One of the above
drugs can be the
same epitope peptide. The peptide injected into the patients will neutralize
the antibody
by specific binding to the antibody that other wise attacks the insulin
secreting cells.
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