CA2485467A1

CA2485467A1 - A method of generating pre-selected epitope-specific vaccines

Info

Publication number: CA2485467A1
Application number: CA002485467A
Authority: CA
Inventors: William Jia
Original assignee: Individual
Current assignee: Individual
Priority date: 2004-11-17
Filing date: 2004-11-17
Publication date: 2006-05-17

Description

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 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.

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 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 ..-...~r~,~~.m ,-.,x... r. .. "!~:"ks~rx~.~..,~-a.r"fir, ,._.,.~ww..x,~,,. _, .,. vs ___,r.axn, M..." ,..,.,"v""." ."..w.,mr"e,~"..:,z .mes"...x»,..."~n.;~m.,...s~.,~ -.,..".ze",a~a..~.F:.~..,a ~..-,.-...w..,...~.~.-.."."""...p..~,.,w.m"".»".,."....~ ,._ ..,...,».~,u»...,u......".., 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-. . ~ .. , w ~ .~A~ .. H , , .. ~ _ a ~. ~, r 4 a ._ . ~~~ ~rt ~ ~..~ ~.~_ ~~
Y ";' '~FV~rtCP~»--~aaN~gm-"2c",~a»a Aaun-:FNS.~a~,~avr~'Aa.t~xatea~artw~,e~c~rs».e,v,-vA_~

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 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 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.

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.

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 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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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 ..._ .uaa ~ __ ~v~.~:..~.~s~a-,..,.,......,cvc~am~a~....:ra;..."wnx,.aa~-.-, ...rm,-n~ .~.r...,..,~.n r aw.r..r..~...,~,. ..H"",~...,., ,-~".
~.~.nae.ur:MZ.. .w,..~,s.,cara~e-c-.,.-~.~,>,~...
ar,.sa°r~:: asm~~urwww.~-.~nw.~,.~,~.=..~e,.nuww~...,y:.~.=.

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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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 m,.wam,. W ,r... ..~,m.mx..;s~n..-.r.. ,..ma..,~:,:.~rrerw~a<aa..,..~," "-~.,;,~sua~asc-.;..<...~:-=:~m:«,,,~.sarn:R.~a:-a~w...,.;f_..
o '~ . ~rFP..~,~r, wfum~."aa~ssev~vpro~l~~~:~"~~s»urwniwewn.
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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 . '.7.D~. , , ,y.. _...aS;.~~ pa . a, x.pm.'sPraFj.m, ~#mF~E' r..v..rt-,~4cV~;F'p&.~,.r,-yA a ;., .. ,MM'ns..
r~'.aF~~w.~9R~5.~"~,.p::gt'~5..;,sb~'.?.:tt~9:arx~~uf4.,tA:~firr. a..:. a?!~
~,o".~~T,~.xyu,.~,.~tt~.~earwxn., .:wm.-.,..,"~.",... ...,.."r,.., 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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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 na.. , ~ , ke~~,yunr=~maa u..;a~"p,~, ~aa~r,:,wawr~,H:~:~~~n-amarc:.:~u N
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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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Claims

1. A method of generating vaccines for neutralizing antibodies to pathogens-infected diseases.

2. The method of claim1, wherein the method comprises following steps:
(1) Preparing peptides arrays;
(2) Using sera samples from patients who are recovered from a pathogens-infected disease to identify the epitopes on the proteins of the pathogen;
(3) Identifying antibody-specific peptides;
(4) Preparing affinity 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).

3. The method of claim 1, wherein the pathogens-infected disease is severe acute respiratory syndrome (SARS).

4. The method of claim 1, wherein the pathogens-infected diseases are selected from the group of bacteria and virus infected diseases.

5. Use of the method of claim 1 to generate antibodies for diagnoses of severe acute respiratory syndrome (SARS).

6. Use of the method of claim 1 to generate antibody for the treatment of severe acute respiratory syndrome (SARS).

7. Use of the method of claim 1 to generate antibody for the prevention of severe acute respiratory syndrome (SARS).

8. Uses of the method of claim 1 to generate antibodies for diagnoses of pathogen-infected diseases.

9. Uses of the method of claim 1 to generate antibody for the treatment of pathogen-infected diseases.

10. Uses of the method of claim 1 to generate antibody for the prevention of pathogen-infected diseases.

11. The uses of claim 8, 9, and 10, wherein the pathogens-infected diseases are selected from the group of bacteria and virus infected diseases.

12. A method of generating vaccines for non-pathogens-infected diseases.

13. The method of claim 12, wherein the method comprises following steps:
(1) Preparing peptides arrays;
(2) Using sera samples from patients who are recovered from a non-pathogens-infected disease to identify the epitopes on the proteins of the pathogen;
(3) Identifying antibody-specific peptides;
(4) Preparing affinity 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).

14. The method of claim 12, wherein the non-pathogens-infected diseases are selected from the group of cancer, diabetes, cardio-vascular disease, endocrinological diseases.

15. Uses of the method of claim 12 to generate epitope specific peptides for diagnoses of non-pathogen-infected diseases.

16. Uses of the method of claim 12 to generate epitope specific peptides for diagnoses of non-pathogen-infected diseases.

17. Uses of the method of claim 12 to generate epitope specific peptides for diagnoses of non-pathogen-infected diseases.

18. The uses of claim 15, 16, and 17, wherein the non-pathogens-infected diseases are selected from the group of cancer, diabetes, cardio-vascular disease, endocrinological diseases.