Epitope profiles of SARS coronavirus

Abstract

The present invention relates to the identification of SARS corona virus epitopes specific to anti-SARS corona virus antibodies found in the sera of SARS patients. Epitope profiles can be used to detect and characterize SARS-CoV infection. The epitope polypeptides can also be used as immunogenic peptides to create polyclonal and/or monoclonal antibodies against SARS coronavirus.

Description

FIELD OF THE INVENTION

The invention relates to the identification of epitopes of SARS-associated coronavirus, and more specifically, a time-dependent epitope profile of the SARS coronavirus infected patients.

BACKGROUND OF THE INVENTION

In 2003, a severe febrile respiratory disease was reported in China, Vietnam, Canada, Hong Kong and Taiwan. Within months of initial reports, the illness had spread from infected persons to healthcare workers and household members. The syndrome was named “severe acute respiratory syndrome” (SARS) in March 2003. Also, in March 2003, a novel coronavirus from SARS patients was isolated (SARS-CoV).

Most coronaviruses cause disease in only one host species. All known coronaviruses are found in three different groups. Two of them can infect mammalian animals and one can infect poultry. SARS-COV was not similar to other human coronaviruses (HCoV-229E and HCoV-OC43), and researchers suggested that it be classified into a new group.

This type of coronavirus has a genome of more than 29,700 nucleotides, and it has a complex two-step replication mechanism. Generally, for viral replications, many RNA virus genomes contain a gene that is translated by the host's system to produce all viral proteins. This gene has been called the replicase gene. The structural proteins of coronaviruses (spike (S or E2), small envelope (sE or E), matrix (M), and nucleocapsid (N)) function during host cell entry and virion morphogenesis and release. The SARS-CoV has several small open reading frames (ORFs) that are found between the S and sE genes and between the M and N genes. The functions of these small ORFs are still unknown, and the complete mechanism of SARS-CoV is yet to be determined.

The SARS-CoV's complete genomic sequences and encoded protein sequences have been available on the web at GeneBank.

SUMMARY OF THE INVENTION

The present invention provides the relevant epitopes of the SARS-COV and various epitope profiles, i.e. a synopsis of epitopes and anti-SARS-CoV sera cross reactions. The invention also provides peptide immunogens used to produce polyclonal and monoclonal antibodies against SARS-CoV. The availability of SARS-CoV epitope sequence profiles would be useful in controlling the disease by making it possible to develop diagnostic tests, vaccines, and antiviral agents.

Specifically, the invention provides polypeptide(s), which encompass epitopes specific to anti-SARS-CoV sera (sera containing antibodies against SARS-CoV), comprising any one or more of the amino acid sequence(s) selected from SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 112, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 183, and SEQ ID NO: 187 and degenerate variants thereof. These epitopes are also referred to as the SARS-COV specific epitopes.

The invention further provides polypeptides which encompass epitopes not specific to anti-SARS-CoV sera comprising any one or more of the amino acid sequences selected from SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 25, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 81, SEQ ID NO: 88, and SEQ ID NO: 118 and degenerate variants thereof. These epitopes are referred to as the SARS-COV non-specific epitopes.

Also provided are polypeptides which encompass inflammation epitopes of SARS-COV (SARS-COV specific and non-specific epitopes having stronger antibody binding activities (AT>1) during hospitalization than in post-hospitalization period), comprising any one or more of the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 25, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 88, and SEQ ID NO: 140 and degenerate variants thereof.

Moreover, the invention provides for novel polypeptides, used as a control in the assays, with sequences of any one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

In addition, the invention provides an immunogenic composition that comprises one or more polypeptides comprising amino acid sequences selected from SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 44, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 76, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 90, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99 and degenerate variants thereof.

The polypeptides described by the invention may be in a linear or branched form.

The invention further provides an apparatus bearing one or more polypeptide(s) comprising amino acid sequence(s) selected from SEQ ID NO: 7-SEQ ID NO: 195 and degenerate variants thereof. The invention also provides for an apparatus bearing one or more polypeptide(s) comprising the amino acid sequence(s) selected from SEQ ID NO: 44-SEQ ID NO: 62 (polypeptides related to the N1 protein); or in the alternative SEQ ID NO: 63-SEQ ID NO: 82 (polypeptides related to the N2 protein); SEQ ID NO: 124-SEQ ID NO: 142 (polypeptides related to the X2 protein); SEQ ID NO: 83-SEQ ID NO: 89 and SEQ ID NO: 112-SEQ ID NO: 123 (polypeptides related to the M protein); or SEQ ID NO: 7-SEQ ID NO: 43 (polypeptides related to the S protein).

In addition, the invention provides an apparatus bearing one or more polypeptide(s) comprising the amino acid sequence(s) selected from, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 79; or in the alternative SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 112, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 183, and SEQ ID NO: 187 (the immediate early epitopes identified during 1-6 days of hospitalization); or SEQ ID NO: 71 (the early epitope identified in the following 7-29 days after the initial 1-6 days).

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated herein and constitute a part of this specification, illustrate one (several) embodiment(s) of the invention and together with the description, serve to explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pictorial diagram of the SARS-COV epitope related polypeptides. The polypeptides were synthesized in the form of multiple antigenic peptide (eight branched) or linear polypeptide, as listed on Table 1. These polypeptides span the SARS-COV spike (S1: aa 421-˜520, S2: 1021˜1120, 1116˜1200), nucleocapsid (N1: 70˜169, N2: 300˜399), matrix (M: 1˜20, 61˜85, 95˜203), small envelope (sE: 1˜20, 16˜76), protein X1 (1˜40, 93˜102), protein X2 (24˜98, 119˜149), protein X3 (1˜63), protein X4 (70˜119), and protein X5 (1˜84) regions. The adjacent polypeptides are overlapping in 4˜6 amino acids in continuous sequences.

FIG. 2 is the ELISA result of serum from five groups of subjects (group 0 consists of Genesis employees outside of hospital, group 1 consists of hospital employees, group 2 consists of SARS suspected patients, group 3 consists of SARS probable patients, and group 4 consists of recovering patients from groups 2 and 3) using the control polypeptides.

FIG. 3 is the ELISA result of serum from the five groups of subjects using the designated SARS-COV S1 polypeptides.

FIG. 4 is the ELISA result of serum from the five groups of subjects using the designated SARS-COV S2 polypeptides.

FIG. 5 is the ELISA result of serum from the five groups of subjects using the designated SARS-COV N1 polypeptides.

FIG. 6 is the ELISA result of serum from the five groups of subjects using the designated SARS-CoV N2 polypeptides.

FIG. 7 is the ELISA result of serum from the five groups of subjects using the designated SARS-COV M polypeptides.

FIG. 8 is the ELISA result of serum from the five groups of subjects using additional designated SARS-CoV M polypeptides.

FIG. 9 is the ELISA result of serum from the five groups of subjects using the designated SARS-COV sE polypeptides.

FIG. 10 is the ELISA result of serum from the five groups of subjects using additional designated SARS-CoV sE polypeptides.

FIG. 11 is the ELISA result of serum from the five groups of subjects using the designated SARS-COV X1 polypeptides.

FIG. 12 is the ELISA result of serum from the five groups of subjects using the designated SARS-COV X2 polypeptides.

FIG. 13 is the ELISA result of serum from the five groups of subjects using the designated SARS-COV X3 polypeptides.

FIG. 14 is the ELISA result of serum from the five groups of subjects using the designated SARS-COV X4 polypeptides.

FIG. 15 is the ELISA result of serum from the five groups of subjects using the designated SARS-CoV X5 polypeptides.

FIG. 16 is the epitope profiles of control subjects' sera IgG immune response in view of epitopes N1: GA151, GA152 and N2: GA162, GA168, GA170.

FIG. 17 is the epitope profiles of time course of SARS probable patient #51's sera IgG immune response in view of epitopes N1: GA51, GA152 and N2: GA162, GA168, GA170.

FIG. 18 is the epitope profiles of time course of SARS probable patient #50's sera IgG immune response in view of epitopes N1: GA151, GA152 and N2: GA162, GA168, GA170.

FIG. 19 is the epitope profiles of time course of SARS probable patient #47's sera IgG immune response in view of epitopes N1: GA151, GA152 and N2: GA162, GA168, GA170.

FIG. 20 is the epitope profiles of control subjects' sera IgG immune response in view of epitopes M: GA203, X2: GA230, GA231, and S2: GA287, GA291.

FIG. 21 is the epitope profiles of time course of SARS probable patient #51's sera IgG immune response in view of epitopes M: GA203, X2: GA230, GA231, and S2: GA287, GA291.

FIG. 22 is the epitope profiles of time course of SARS probable patient #50's sera IgG immune response in view of epitopes M: GA203, X2: GA230, GA231, and S2: GA287, GA291.

FIG. 23 is the epitope profiles of time course of SARS probable patient #47's sera IgG immune response in view of epitopes M: GA203, X2: GA230, GA231, and S2: GA287.

FIG. 24 is the epitope profiles of cross reactions between control polypeptides and chicken anti-avian infectious bronchitis virus (IBV) sera.

FIG. 25 is the epitope profiles of cross reactions between S1 polypeptides and chicken anti-avian infectious bronchitis virus (IBV) sera.

FIG. 26 is the epitope profiles of cross reactions between S2 polypeptides and chicken anti-avian infectious bronchitis virus (IBV) sera.

FIG. 27 is the epitope profiles of cross reactions between N1 polypeptides and chicken anti-avian infectious bronchitis virus (IBV) sera.

FIG. 28 is the epitope profiles of cross reactions between N2 polypeptides and chicken anti-avian infectious bronchitis virus (IBV) sera.

FIG. 29 is the epitope profiles of cross reactions between M polypeptides and chicken anti-avian infectious bronchitis virus (IBV) sera.

FIG. 30 is the epitope profiles of cross reactions between sE polypeptides and chicken anti-avian infectious bronchitis virus (IBV) sera.

FIG. 31 is the epitope profiles of cross reactions between X1 polypeptides and chicken anti-avian infectious bronchitis virus (IBV) sera.

FIG. 32 is the epitope profiles of cross reactions between control polypeptides and cat anti-feline coronavirus sera.

FIG. 33 is the epitope profiles of cross reactions between S1 polypeptides and cat anti-feline coronavirus sera.

FIG. 34 is the epitope profiles of cross reactions between S2 polypeptides and cat anti-feline coronavirus sera.

FIG. 35 is the epitope profiles of cross reactions between N1 polypeptides and cat anti-feline coronavirus sera.

FIG. 36 is the epitope profiles of cross reactions between N2 polypeptides and cat anti-feline coronavirus sera.

FIG. 37 is the epitope profiles of cross reactions between M polypeptides and cat anti-feline coronavirus sera.

FIG. 38 is the epitope profiles of cross reactions between sE polypeptides and cat anti-feline coronavirus sera.

FIG. 39 is the epitope profiles of cross reactions between X1 polypeptides and cat anti-feline coronavirus sera.

FIG. 40 shows the immune response of serum from ducks immunized with S1 epitopes.

FIG. 41 shows the immune response of serum from ducks immunized with S2 epitopes.

FIG. 42 shows the immune response of serum from ducks immunized with N1 epitopes.

FIG. 43 shows the immune response of serum from ducks immunized with N2 epitopes.

FIG. 44 shows the immune response of serum from ducks immunized with M epitopes.

FIG. 45 shows the immune response of serum from ducks immunized with sE epitopes.

FIG. 46 shows the immune response of serum from ducks immunized with X1 epitopes.

FIG. 47 shows epitope profiles of time course of SARS probable patient #51's serum IgM response in view of N1: GA151, GA152, GA153, and GA154. The #51 patients' serum is diluted 300×.

Table 1 lists the zone, SEQ ID NO., peptide, sequence, location, annotation, format and notes of the synthetic SARS-COV related polypeptides.

DESCRIPTION OF THE EMBODIMENTS

Definitions

The term “degenerate variant” as used herein refers to a polypeptide that has the same function but with one or more different amino acid(s) from mutation, substitution, addition or deletion or that is at least 90% identical to the original amino acid sequence.

The term “immunogenic composition” as used herein refers to a composition that provokes an immune response.

The term “immune response” as used herein refers to bodily response to an antigen that occurs when lymphocytes identify the antigenic molecule as foreign and induce the formation of antibodies and lymphocytes capable of reacting with it and rendering it harmless.

The term “linear form” as used herein refers to a single chain of amino acids.

The term “branched form” as used herein refers to the Multiple Antigenic Peptides (MAP), having at least two branches; it can be four, eight or more branches that result in a molecule which has a high molar ratio of peptide antigen to core molecule and, typically, will elicit a stronger anti-peptide antibody response.

SARS-COV Specific Epitopes

The present invention relates to a collection of one or more polypeptides from the 189 SARS-COV related polypeptides that permit an analysis of the epitope profiles of SARS-COV infected and non-infected human and animal sera. The collection is set forth in Table 1. The SARS-CoV specific epitopes were identified by comparison of each peptide's antibody binding activity in parallel in 5 human groups' sera (group 0 being Genesis' employees, group 1 being hospital employees, group 2 being SARS suspected patients, group 3 being SARS probable patients, and group 4 being recovering patients from groups 2 and 3). The specific epitopes identified are further categorized into two groups, i.e. the most specific epitopes and the less specific epitopes, also referred to as the second specific epitopes.

The most specific epitopes are the SARS-CoV nucleocapsid protein N1: GA137 (SEQ ID NO: 46), GA139 (SEQ ID NO: 47), GA142 (SEQ ID NO: 50), GA146 (SEQ ID NO: 54), GA147 (SEQ ID NO: 55), GA151˜GA153 (SEQ ID NO: 59-SEQ ID NO: 61); N2: GA156 (SEQ ID NO: 65), GA 160 (SEQ ID NO: 69), GA164 (SEQ ID NO: 73), GA166 (SEQ ID NO: 75), GA 167 (SEQ ID NO: 76), GA169 (SEQ ID NO: 78), GA170 (SEQ ID NO: 79); and protein X2: GA231 (SEQ ID NO: 140), which polypeptides bound with SARS probable patients' serum (group 3) more strongly (usually AT>2.5) than other non-SARS sera (group 0 and group 1).

The second specific epitopes are the SARS-COV nucleocapsid protein N1: GA149 (SEQ ID NO: 57), GA150 (SEQ ID NO: 58), GA154 (SEQ ID NO: 62); N2: GA161 (SEQ ID NO: 70), GA162 (SEQ ID NO: 71), GA165 (SEQ ID NO: 74), GA 168 (SEQ ID NO: 77); matrix protein (M): GA203 (SEQ ID NO: 112); spike protein (S): GA132 (SEQ ID NO: 40), GA134 (SEQ ID NO: 42), GA287 (SEQ ID NO: 183), GA291 (SEQ ID NO: 187); protein X2: GA230 (SEQ ID NO: 139); and protein X4: GA247 (SEQ ID NO: 156), which polypeptides bound with SARS probable patients' serum (group 3) more strongly (1<AT<2.5) than the other non-SARS sera (group 0, group 1).

An analysis, such as a serum ELISA, using an epitope profile containing SARS-COV S1, S2, N1, N1, M, sE, X1, X2, X3, X4, and X5 proteins (SEQ ID NO: 7-SEQ ID NO: 195) can provide information on the status of SARS-CoV infection. Also useful is the epitope profiles of N1 proteins (SEQ ID NO: 44-SEQ ID NO: 62), N2 proteins (SEQ ID NO: 63-SEQ ID NO: 82), or X2 proteins (SEQ ID NO: 124-SEQ ID NO: 142) containing the most specific epitopes and epitope profiles of M proteins (SEQ ID NO: 83-SEQ ID NO: 89 and SEQ ID NO: 112-SEQ ID NO: 123) and S proteins (SEQ ID NO: 7-SEQ ID NO: 43) containing the second specific epitopes.

Specifically, using the most specific epitope profile of the GA151, GA152, and GA170 polypeptides, one can differentiate the 23 SARS suspected patients (group 2) to be real SARS-COV infected (17 cases) or SARS-COV non-infected cases (6 cases) (data set forth below). Moreover, by using the most specific epitopes profile ELISA method, the anti-SARS-CoV immune response can be detected two days earlier than the RT-PCR method of virus RNA detection (data set forth below).

Moreover, among the specific epitopes, certain epitopes have immediate early antibody binding activities (days 1-6) and early antibody binding activities (days 7-29). These were identified in parallel analysis of the epitope profiles of serum taken from different time points. The SARS-COV infected patients' serum bound to the following immediate early epitopes: X2: GA230 (SEQ ID NO: 139), GA231 (SEQ ID NO: 140); S2: GA287 (SEQ ID NO: 183); M: GA203 (SEQ ID NO: 112); N1: GA151 (SEQ ID NO: 59), GA152 (SEQ ID NO: 60); N2: GA168 (SEQ ID NO:77), GA170 (SEQ ID NO: 79); and S2: GA291 (SEQ ID NO:187), and to the following early epitope: N2: GA162 (SEQ ID NO: 71).

Each of these epitopes and the epitope profiles have research, diagnostic, and therapeutic values.

SARS-COV Non-Specific Epitopes

The invention also describes SARS-COV epitopes that are not specific to SARS-CoV antibody containing sera. These non-specific epitopes have antibody binding activities in all five groups of human sera, and they are S1: GA 101 (SEQ ID NO: 11), GA102 (SEQ ID NO: 11), GA102 (SEQ ID NO: 12), S2: GA117 (SEQ ID NO: 25), N2: GA155 (SEQ ID NO: 64), GA158 (SEQ ID NO: 67), GA172 (SEQ ID NO: 81), M: GA179 (SEQ ID NO: 88), GA209 (SEQ ID NO: 118). These non-specific epitopes may be used in affinity purification and or preabsorption of antisera as is routinely performed by one skilled in the art.

SARS-CoV Inflammation Epitopes

Some inflammation epitopes have been identified. These represent polypeptides that frequently bound with SARS probable patients' serum more strongly during the treatment period than during the recovery period after discharge from hospital. They include both SARS-COV specific and non-specific epitopes, i.e. SARS-CoV spike protein: GA01 (SEQ ID NO: 11), GA102 (SEQ ID NO: 12), GA117 (SEQ ID NO: 25), GA132 (SEQ ID NO: 40), GA134 (SEQ ID NO: 42), nucleocapsid protein: GA137 (SEQ ID NO: 46), GA139 (SEQ ID NO: 49), GA142 (SEQ ID NO: 50), GA143 (SEQ ID NO: 51), GA146 (SEQ ID NO: 54), GA147 (SEQ ID NO: 55), GA149˜GA154 (SEQ ID NO: 57-SEQ ID NO: 62), GA155 (SEQ ID NO: 64), GA156 (SEQ ID NO: 65), GA158 (SEQ ID NO: 67), GA160-GA162 (SEQ ID NO: 69-SEQ ID NO: 71), GA164˜GA170 (SEQ ID NO: 73-SEQ ID NO: 79), matrix protein: GA179 (SEQ ID NO: 88) and protein X2: GA231 (SEQ ID NO: 140).

The use of inflammation epitopes that show strong SARS sera antibody binding activities only during the treatment phase in the hospital can, by subtraction, lead to the identification of SARS-CoV specific epitopes that bind strongly even after hospitalization, i.e. subtracting SARS-CoV specific inflammation epitopes from the total SARS-COV specific epitopes. The identification of SARS-CoV specific epitopes that bind strongly even after hospitalization allow identification of people who were infected with SARS-COV but are in the recovery period.

Immunogenic Peptides

Certain polypeptides can be used as immunogens (immunogenic peptides) to raise corresponding antibody in hosts such as mice, rabbits, and ducks. There is a high immune response when compared with pre-immunization (pre-bleed) serum when using the following peptide immunogens: S1: GA100˜GA102 (SEQ ID NO: 10-SEQ ID NO: 12), GA91 (SEQ ID NO: 7), GA109 (SEQ ID NO: 17), GA111 (SEQ ID NO: 19), GA113 (SEQ ID NO: 21), S2: GA117 (SEQ ID NO: 25), GA119 (SEQ ID NO: 27), GA128˜GA130 (SEQ ID NO: 36-SEQ ID NO: 38), N1: GA143 (SEQ ID NO: 51), GA145 (SEQ ID NO: 53), GA95 (SEQ ID NO:44), GA150˜GA154 (SEQ ID NO: 58-SEQ ID NO: 62), N2: GA155 (SEQ ID NO:64), GA156 (SEQ ID NO: 65), GA158˜GA161 (SEQ ID NO: 67-SEQ ID NO: 70), GA96 (SEQ ID NO: 63), GA167 (SEQ ID NO: 76), GA173 (SEQ ID NO: 82), M: GA174 (SEQ ID NO: 84), GA93 (SEQ ID NO: 83), sE: GA181 (SEQ ID NO: 90), and protein X1: GA186 (SEQ ID NO: 95), GA188˜GA190 (SEQ ID NO:97-SEQ ID NO: 99).

The availability of SARS-CoV epitope sequences profiles should be useful in controlling the disease by making it possible to develop diagnostic tests, vaccines, and antiviral agents.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Unless defined otherwise, the meanings of all technical and scientific terms used herein are those commonly understood by one of ordinary skill in the art to which this invention belongs. One of ordinary skill in the art will also appreciate that any methods and materials similar or equivalent to those described herein can also be used to practice or test the invention. Further, all publications mentioned herein are incorporated by reference.

With respect to ranges of values, the invention encompasses each intervening value between the upper and lower limits of the range to at least a tenth of the lower limit's unit, unless the context clearly indicates otherwise. Further, the invention encompasses any other stated intervening values. Moreover, the invention also encompasses ranges excluding either or both of the upper and lower limits of the range, unless specifically excluded from the stated range.

Further, all numbers expressing quantities of ingredients, reaction conditions, % purity, polypeptide and polynucleotide lengths, and so forth, used in the specification and claims, are modified by the term “about,” unless otherwise indicated. Accordingly, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties of the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits, applying ordinary rounding techniques. Nonetheless, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors from the standard deviation of its experimental measurement.

It must be noted that, as used herein and in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a subject polypeptide” includes a plurality of such polypeptides and reference to “the agent” includes reference to one or more agents and equivalents thereof known to those skilled in the art, and so forth.

The following examples further illustrate the invention. They are merely illustrative of the invention and disclose various beneficial properties of certain embodiments of the invention. The following examples should not be construed as limiting the invention.

EXAMPLES

The following examples illustrate the identification, design and production of SARS-COV epitope related synthetic polypeptides and production of polyclonal antibodies using these polypeptides.

Example 1

Peptide Synthesis

Polypeptides were synthesized by using solid phase peptide synthesis strategy. The standard F-moc chemistry was performed on an Advanced ChemTech's Peptide Synthesizer, Model Apex 396, according to manufacturer's instructions. The branched multiple antigenic peptides (GA98˜GA191, GA283˜GA298) were synthesized from the K_core wang resin (heptalysyl core K₄K₂K) which was purchased from Novabiochem. For linear polypeptide synthesis (GA91˜GA96 and GA192˜GA269), we used wang resin coupled with the first amino acid of the C-terminal as solid support. After complete synthesis of the peptide, the resin was treated with cleavage cocktails [TFA(trifluoroacetic acid), TIS(triisipropyl silane), EDT(ethanol dithiol)] according to standard procedures used to cleave the peptide from the resin and deprotect the protecting groups on the amino acid side chains. For quality control of the polypeptides synthesis, each peptide was analyzed with HPLC and MALDI-TOF Mass spectrum methods.

Example 2

Peptide Epitope Annotation

Different polypeptides were synthesized based on the published genome sequence of SARS-CoV and were given the following annotations.

Six non-SARS-CoV polypeptides (GA6, GA53, GA64, GA81, GA83, GA84) were used as control polypeptides.

The synthetic polypeptides of SARS-COV spike (S) protein included amino acids (aa) sequence 421-520 (GA91, GA98˜GA116), within the S1 region. Also included were aa 1021˜1120 (GA117˜GA135) and aa 1116˜1200 (GA283˜GA298), both of which were within the S2 region.

The synthetic polypeptides of SARS-CoV nucleocapsid (N) protein included aa 70-169 (GA95, GA136˜GA154), defined as the N1 region and aa 329-350, 300-399 (GA 96, GA155˜GA173), defined as the N2 region.

The synthetic polypeptides of SARS-COV matrix membrane (M) protein included aa 1-20 (GA93, GA174˜GA176), aa 61˜85 (GA177˜GA180), aa 95˜124 (GA203˜GA207), and aa 164˜203 (GA208˜GA214) which were defined as M.

The synthetic polypeptides of SARS-COV small envelope (sE) protein included aa 1˜20 (GA181˜GA183) and aa 16˜76 (GA192˜GA202), which were defined as sE.

The synthetic polypeptides of SARS-COV protein X1 (X1) included aa 1˜40 (GA184˜GA190) and aa 93˜102 (GA191), which were defined as X1.

The synthetic polypeptides of SARS-COV protein X2 (X2) included aa 24-98, 119˜149 (GA215˜GA233), which were defined as X2.

The synthetic polypeptides of SARS-COV protein X3 (X3) included aa 163 (GA234˜GA244), defined as X3.

The synthetic polypeptides of SARS-COV protein X4 (X4) included aa 70-119 (GA245˜GA253), defined as X4.

The synthetic polypeptides of SARS-COV protein X5 (X5) included aa 1-84 (GA254˜GA269), defined as X5.

Genes encoding protein X1 and X2 are found within the small open reading frames (ORFs) between the S and sE genes. Genes encoding proteins X3, X4, and X5 are found within the ORFs between the M and N genes.

A table of the synthetic polypeptides is shown in Table 1.

Example 3

ELISA

The enzyme linked immunosorbent assay (ELISA) was used to screen for the existence of anti-SARS-CoV antibody in human and animal sera. The ELISA was conducted by coating polystyrene 96 well plates with 1 μg/well of polypeptides. For comparison of results from each group in parallel, experiments were done at the same time. The antibody titer (AT) was defined as (O.D.₄₀₅ value of Target —O.D.₄₀₅ value of Blank)/O.D.₄₀₅ value of Blank) to reduce the variation of different time performance to the least extent. The primary sera were all diluted 3000× in 1% BSA/PBS, and the secondary antibody was HRP labeled goat anti-human IgG(H+L) (Pierce Biotechnology Inc.) diluted 5000× or Donkey anti-chicken IgY (IgG) (H+L) (Research Diagnostics, Inc.) diluted 5000× or goat anti-cat IgG (H+L) (Research Diagnostics, Inc.) diluted 5000×. Using TMB (3,3′, 5, 5′ tetramethyl benzidine) as the substrate, generally, the immune response was defined as elevated significantly when the antibody titer (AT) was more than 1 in human serum and more than 0.8 in animal serum.

Example 4

Grouping of Human and Animal Sera Samples

Human Sera

There were 74 human sera samples divided into five groups. The groups were group 0: the sera of five to seven normal humans sampled from employees of Genesis Biotech Inc. who didn't work in the hospital; group 1: the sera of eighteen humans who worked in the hospital and may have came in contact with SARS patients or related specimens (healthcare workers) (#1˜#18); group 2: the sera of twenty-three “SARS suspected patients”, identified according to the WHO case definition standard (#19˜#41); group 3: the sera of ten “SARS probable patients”, identified according to the WHO case definition standard (#42˜#51); group 4: the sera of eighteen humans, originally from group 2 and group 3 (#52˜#69), who had been discharged from the hospital and had recovered from the therapy of “SARS suspected” or “SARS probable”cases.

Blood samples were also collected from patients at different time points of the infection to examine putative time-dependent epitopes of SARS-CoV found in the infected patients. Three SARS probable patients' (#51, #50, and #47 patients) sera were collected at several time points.

Probable SARS patient #51: male. 35 years old

The patient record is as follows:

• • May 8, 2003—hospitalization
  • 5/9—chest radiograph shows interstitial infiltrate
  • 5/10—fever, diarrhea twice
  • 5/11-fever
  • 5/13—RT-PCR diagnosis of SARS-COV was positive, SD rapid test
  • (Standard Diagnostics Inc.) of SARS-COV antibody was negative
  • 5/15—fever
  • 5/16—body temperature back to normal
  • 5/18—chest radiograph shows ok
  • 6/5—clinical syndrome recovered
  • 6/12—bleed serum after recovery Blood samples from patient #51 were collected at various time points as follows:
• #51A: day 0, May 11, 2003, fever, SD rapid test of SARS-COV antibody was negative
• #51B: day 2E (“early”, in the mornings), May 13, 2003, SD rapid test of SARS-CoV antibody was negative
• #51C: day 2L (“late”, in the afternoons), May 13, 2003, RT-PCR diagnosis of SARS-CoV was positive, SD rapid test of SARS-COV antibody was negative
• #51D: day 4, May 15, 2003, fever, SD rapid test of SARS-COV antibody was negative
• #51E: day 5E, May 16, 2003, body temperature back to normal, SD rapid test of SARS-CoV antibody Was negative
• #51F: day 5L, May 16, 2003, SD rapid test of SARS-COV antibody was negative
• #51G: day 8, May 19, 2003, SD rapid test of SARS-COV antibody was positive
• #51H: day 12, May 23, 2003, SD rapid test of SARS-COV antibody was positive
• #51K: day 25, Jun. 5, 2003, clinical syndrome recovered, SD rapid test of SARS-CoV antibody was positive
• #51L: day 32, Jun. 12, 2003, discharged from hospital, bleed serum after recovery
• #51M: day 41, Jun. 21, 2003, bleed serum after recovery Probable SARS patient #50: female, 58 years old Patient record is as follows:
  • May 3, 2003—transferred from another hospital, had fever prior to transfer, hospitalization, body temperature was normal
  • 5/6—chest radiograph showed recovering
  • 5/13—bleed serum during hospitalization, RT-PCR diagnosis negative of SARS-CoV, SD rapid test of SARS-CoV antibody positive
  • 5/15—body temperature normal
  • 5/20—bleed serum after recovery
  • 5/25—body temperature normal Blood samples from patient #50 were collected at various time points as follows:
• #50A: day 0, May 10, 2003, SD rapid test of SARS-CoV antibody was positive
• #50B: day 2, May 12, 2003, SD rapid test of SARS-COV antibody was positive
• #50C: day 3, May 13, 2003, bleed serum during hospitalization
• #50D: day 10, May 20, 2003, discharged from hospital, bleed serum after recovery
• #50E: day 16, May 26, 2003, bleed serum after recovery
• #50F: day 27, Jun. 6, 2003, bleed serum after recovery
• #50G: day 55, Jul. 4, 2003, bleed serum after recovery Probable SARS patient #47: male, 35 years old The patient record is as follows:
  • May 22, 2003—hospitalization
  • 5/23—fever
  • 5/24—fever, SD rapid test of SARS-COV antibody was positive
  • 5/25—body temperature was normal, chest radiograph showed recovering Blood samples from patient #47 were collected at various time points as follows:
• #47A: day 0, May 26, 2003, SD rapid test of SARS-CoV antibody was positive
• #47B: day 2, May 28, 2003, bleed serum during hospitalization, RT-PCR of SARS-COV was negative, SD rapid test of SARS-COV antibody was positive
• #47C: day 7, Jun. 2, 2003, discharged from hospital, bleed serum after recovery
• #47D: day 22, Jun. 17, 2003, discharged from hospital, bleed serum after recovery
• #47E: day 28, Jun. 23, 2003, discharged from hospital, SD rapid test of SARS-CoV antibody was positive Animal Sera

The cross reaction of animal coronavirus (infectious bronchitis virus and feline coronavirus antibody positive and negative sera with the SARS-CoV peptide epitopes were examined in 3 chicken samples and 8 cat samples. The three chicken samples were one chicken positive control (CPC) of anti-avian infectious bronchitis virus (IBV) and two chicken negative controls (CNC 1 and CNC 2) using specific pathogen free (SPF) chicken sera. The eight cat samples include four sera samples from 4 cats with feline coronavirus (FCV) diagnosis-RT-PCR positive and antibody rapid test positive: #5-4, #5-16, #3-12, #10-3; one sample from 1 cat with FCV-RT-PCR negative and antibody rapid test positive: #5-15; one sample from 1 cat with FCV-RT-PCR positive and antibody rapid test negative: #5-14; one sample from 1 cat with FCV-RT-PCR positive and antibody rapid test not performed: #922305; one sample from 1 cat with FCV-RT-PCR negative and antibody rapid test negative: #3-2. These sera were analyzed by standard ELISA method.

Example 5

Identification of SARS Related Epitopes

To identify potential epitopes that would be recognized by the SARS-CoV antibodies, sera samples from Groups 0-4 were analyzed with ELISA plates coated with synthetic SARS-COV polypeptides spanning the regions of S, N, M, sE, X1, X2, X3, X4, and X5 and control polypeptides.

Peptide array was used to detect the potential epitope binding activity of human sera of normal and infected subjects. The peptide array covered the sequential peptide sequences with 4˜6 amino acids overlapping in adjacent wells. These branched or linear polypeptides included the following SARS-CoV proteins:

• spike glycoprotein (S): S1: 437th aa˜461th aa (GA91), aa 421-520 (GA98˜GA116), S2: 1021˜1120 (GA117˜GA135), 1116˜1200 (GA283˜GA298)
• nucleocapsid phosprotein (N): N1: aa 107˜126 (GA95), 70˜169 (GA136˜GA154) N2: aa 329-350, 300˜399 (GA 96, GA155˜GA173)
• matrix protein (M): aa 1˜20 (GA93, GA174˜176), 61 ˜85 (GA177˜GA180), 95˜124 (GA203˜GA207), 164˜203 (GA208˜GA214)
• small envelope protein (sE): aa 1˜20 (GA181˜GA183), 16˜76 (GA192˜GA202)
• protein X1 (X1): aa 1˜40 (GA184˜GA190), 93˜102 (GA191)
• protein X2 (X2): aa 24˜98 (GA215˜GA228), 119˜149 (GA229˜GA233)
• protein X3 (X3): aa 1˜63 (GA234˜GA244)
• protein X4 (X4): aa 70˜119 (GA245˜GA53)
• protein X5 (X5): aa 1˜84 (GA254˜GA269)
• control polypeptides: GA6 (28 kDa structural protein of VP28 of shrimp white spot syndrome virus (WSSV) 52th aa ˜66th aa), GA53 (hemagglutinin of Influenza A virus (H3N2) 306th aa ˜313th aa fused with VP1 of enterovirus 714th aa ˜10th aa), GA64 (coat protein of fish nervous necrosis virus (NNV) 274th aa ˜289th aa), GA81 (spike glycoprotein of rabies virus 355th aa ˜364th aa fused with cyclic 157th aa ˜172th aa of peptide sequence of latent membrane protein LMP-1 of human herpesvirus 4/Epstein-Barr Virus), GA83 (4th aa ˜30th aa of membrane matrix protein M1 of Influenza A virus), and GA84 (25th aa ˜41th aa of membrane matrix protein M1 of Influenza A virus).

The B cells immune response (antibody) to multiple SARS-COV synthetic polypeptides (S, N, M, sE, X1, X2, X3, X4, and X5) and control polypeptides were analyzed with peptide array-ELISA and serum from group 0 to group 4 in parallel. When the antibody titer index (AT) was more than 1, it suggested significant epitope (or peptide) binding activity of the serum examined.

SARS-COV Specific Epitopes

In the peptide array-serum interaction, the SARS specifically related epitopes, also referred to as SARS-CoV specific epitopes, were defined by having both a positive signal in SARS-probable and or SARS-suspected cases and a negative signal in the other non-SARS-CoV infected groups (group 0 and group 1). Generally, AT>1 is positive, AT<1 is negative; however, for some epitopes a value of AT<2 was designated as a negative response. Many SARS-COV specific eiptopes were identified and they were further categorized as the most specific epitopes or the second specific epitopes according to their anti-SARS-CoV antibody binding activities in different groups.

Most Specific

The most specific epitopes reacted with sera of SARS-probable patients (group 3) more strongly (usually AT>2.5) than with other non-SARS sera. They were: SARS-COV nucleocapsid protein (N): aa sequence N1: 75-94 (GA137, GA139), 100-109 (GA142), 120-134 (GA146, GA147), 145-164 (GA151GA153), N2: 305-314 (GA156), 325-334 (GA160), 345-354 (GA164), 355-364 (GA166), 360-369 (GA167), 370-379 (GA169), 375-384 (GA170) and protein X2: 129-138 (GA231) (see FIGS. 5, 6, and 12).

Second Specific Epitopes

The second specific epitopes were the nucleocapsid protein N1: GA149, GA150, GA154; N2: GA161, GA162, GA165, GA168; matrix protein (M): GA203; spike protein (S): GA132, GA134, GA287, GA291; protein X2: GA230; and protein X4: GA247, which polypeptides bound with SARS probable patients' serum (group 3) more strongly (1<AT<2.5) than the other non-SARS sera (group 0, group 1) (see FIGS. 4, 5, 6, 8, 12, and 14).

Using the most specific epitope profile containing GA151, GA152, GA170, one can differentiate the 23 SARS suspected patients (group 2, #19-#41) to be real SARS-COV infected (17 cases: #19, #21, #22, #23, #24, #25, #26, #27, #28, #30, #31, #32, #36, #38 #39, #40, #41) or SARS-COV non-infected cases (6 cases: #20, #29, #33, #34, #35, #37). In other words, 74% of group 2 can be regarded as probable SARS patients, and 26% of group 2 can be regarded as non-probable SARS patients. The Responder (AT>1, positive)/Total samples (R/T) examined in group 3 (SARS probable patients, #42˜#51) was 10/10=100% when using the same epitope profile (GA151, GA152, GA170) as an anti-SARS-CoV antibody binding markers. In contrast, the R/T value=0 (0/18) in group 1 (normal human control of hospital employees) and group 0 (normal human control subjects outside of the hospital).

SARS-CoV Non-Specific Epitopes

Epitopes that had binding activities (AT>1) in all five groups of sera, sera from normal, infected, and recovering subjects, were designated as non-specific epitopes. These epitopes were GA101, GA102, GA117, GA155, GA158, GA172, GA179, and GA209.

Inflammation Epitopes

Certain immune response elevated (inflammation) epitopes were also identified (including both SARS-CoV specific and non-specific epitopes), which polypeptides frequently bound with SARS probable and suspected patients' serum more strongly during the treatment than during the recovery period after discharge from the hospital. They include SARS-COV spike protein: GA101, GA102, GA117, GA132, GA134, nucleocapsid protein: GA137, GA139, GA142, GA143, GA146, GA147, GA149˜GA154, GA155, GA156, GA158, GA160˜GA162, GA164˜GA170, matrix protein: GA179 and protein X2: GA231. The antibody titer for these polypeptides was higher during hospital therapy in contrast to the lower antibody titer in the recovery period after discharge from the hospital (patient #19 (hospitalization period) vs. #61 (post-hospitalization period), #22 vs. #62, #24 vs. #67, #25 vs. #70, #26 vs. #71, #28 vs. #60, #29 vs. #72, #30 vs. #63, #31 vs. #68, #32 vs. #65, #36 vs. #69, #37 vs. #52, #41 vs. #59, #42 vs. #73, #47 vs. #66, #50 vs. #58, #51 vs. #55).

In sum, ELISA analysis of SARS-infected human sera with SARS-COV polypeptides, certain epitopes were identified as SARS-specifically related epitopes, non-specific epitopes, and inflammation epitopes.

Example 6

Determination of Time-Dependent Epitope Profiles of SARS-COV

SARS-CoV specific epitopes' time dependency was investigated.

The sera of three probable SARS patients (#51, #50, and #47) were analyzed at multiple time points using the most specific epitopes (N11: GA151, GA152, N2: GA162, GA168, GA170, M: GA203, X2: GA230, GA231, S2: GA287, GA291) as markers. Then, the time-dependent epitope profiles of probable SARS patients' anti-SARS-CoV sera (see FIGS. 17, 18, 19, 21, 22, and 23) were ascertained. First, there were quick and fluctuating immune responses to SARS-CoV within the same day in the probable SARS patients (#51B and #51C, #51E and #51F) (see FIGS. 17 and 21). Also, the phenomena of higher antibody titer during the hospital treatment period, in contrast to the lower antibody titer in the recovery period after discharge from the hospital, was significant in the time-dependent epitope profile analysis, #51K versus #51L, #50C versus #50D, #47B versus #47C (see FIGS. 17, 18, 19, 21, 22, and 23). Moreover, by using the most specific epitope profile in ELISA method, the anti-SARS-CoV immune response can be detected two days earlier than the RT-PCR method of virus RNA detection, used with sample #51C (see patient #51's patient record in Example 4). For example, using ELISA with epitopes GA151, GA152, GA168, GA170, GA203. GA230, and GA231, SARS-COV antibody can be detected in the SARS probable patient #51 on day 0 (sample #51A) (see FIGS. 17 and 21).

In a comparative analysis of SARS-probable patient's IgM response to SARS-COV infection from early to late stage (other analysis were of patients' IgG response), we used the most specific epitopes (N1: GA151, GA152, GA153, GA154) as markers to analyze one probable SARS patients' sera (#51) at multiple time points. The higher AT of IgM specific to SARS-COV N1 was detected only in the early stage (#51 day 0 (R/T=4/4), day 2E (R/T=3/4), day 2L (R/T=414), and day 4 (R/T=1/4)), but not in the later stage of hospitalization (day 5˜day 25) nor during the recovery period after discharge from hospital (day 32) (see FIG. 47). The phenomena of higher antibody titer in the late hospital therapeutic period, in contrast to lower antibody titer in the recovery period after discharge from the hospital, was not observed in the IgM response but was present in the corresponding IgG response. Again, by using the specific epitope profile in the ELISA method, the anti-SARS-COV immune response of IgM can be detected two days earlier than the RT-PCR method of virus RNA detection used with sample #51C. For example, using the ELISA with epitopes GA151, GA152, GA153, and GA154, SARS-COV antibody can be detected in the SARS-probable patient #51 on day 0 (sample #51A) (see FIG. 47). The antibody titer of human IgM specific to SARS-COV protein (such as the nucleocapsid) is at least 10 times less than that of the corresponding IgG counterpart. The assay of IgM has to be carried out with AT at 300× dilution, as AT would be negative at 3000× dilution. On the other hand, IgG immune response assay can be carried with AT at 3000× dilution (see FIG. 17 and FIG. 47).

In a parallel analysis of epitope profile of serum obtained at different time points, the time dependent epitopes were identified. The SARS-COV infected patients' antibody bound the immediate early (days 1-6) epitopes: X2: 124-138 (GA230, GA231); S2: 1136-1145, 1156-1165 (GA287, GA291); M: 95-104 (GA203); N1: 145-154 (GA151), 150-159 (GA152); N2: 365-374 (GA168), 375-384 (GA170); and S2: 1156-1165 (GA291) and early (days 7-29) epitope: N2: 335-344 (GA162), (see FIGS. 17, 18, 19, 21, 22, and 23).

By ELISA analysis of SARS patients' serum at different time points, the SARS-CoV specific epitopes were further characterized by their time dependency in the period of infection and recovery.

Example 7

Cross Reaction of Epitope Profiles with Chicken Anti-Avian Infectious Bronchitis Virus (IBV) and Cat Anti-Feline Coronavirus Sera

To confirm the profile of the SARS-CoV epitopes profile established based on the use of SARS-COV infected human sera, ELISAs were performed with chicken anti-avian infectious bronchitis virus sera and cat anti-feline coronavirus sera.

Three chicken sera samples were used in ELISA (2 SPF sera (CNC1 and CNC2) and 1 anti-IBV positive serum (CPC)). The chicken anti-IBV antiserum cross reacted only but weakly with GA115 (S1), GA170 (N2), GA173 (N2), and GA186 (protein X1), but did not cross react with the GA151 and GA152 epitopes which were good markers for detecting SARS-COV infected sera. The non-specific epitopes frequently cross reacted with the chicken sera. Epitope GA6 (WSSV) had a response ratio of R/T=1/3, with a binding result in the CPC serum (see FIG. 24). Epitope GA101 (S1) had a response ratio of R/T=3/3, with the strongest binding activity in the CNC1 serum (see FIG. 25). Epitope GA102 (S1) had a response ratio of R/T=2/3, with a binding activity in CPC and CNC1 sera (see FIG. 25) Epitope GA117 (S2) had a response ratio of R/T=1/3, with a binding activity in the CNC1 serum (see FIG. 26). Epitope GA147 (N2) had a response ratio of R/T=1/3, with a binding activity in the CPC serum (see FIG. 27). GA147 is a cross-species coronavirus specific epitope that is recognized by antibodies against human coronavirus as well as IBV. Epitope GA155 (N2) had a response ratio of R/T=2/3, with a binding activity in the CNC1, CNC2 and CPC sera (see FIG. 28). Epitope GA158 (N2) had a response ratio of R/T=3/3, with binding activities in all three chicken sera (see FIG. 28). Epitope GA172 (N2) had a response ratio of R/T=3/3, with binding activities in all three chicken sera (see FIG. 28). Epitope GA179 (M2) had a response ratio of R/T=3/3, with binding activities in all three chicken sera (see FIG. 29).

The cat anti-feline coronavirus (FCV) sera interaction with these polypeptides was also analyzed using 8 sera samples (2 negative FCV antibody rapid test sera #5-14, #3-2; 5 positive FCV antibody rapid test sera #54, #5-16, #3-12, #10-3, #5-15; and 1 sera with not test performed #922305). The result was R/T=0 (AbT<1) in all 8 sera with the SARS related specific epitopes GA152 (R/T=0/8) (see FIG. 35) and GA170 (R/T=0/8) (see FIG. 36). Only #5-4 cat serum cross reacted with the specific epitope of GA151 (AbT=2.9) (R/T=1/8) (see FIG. 35).

Sera samples (where FCV antibody rapid test was positive and where rapid test was not performed), cross reacted with SARS-CoV epitope GA129 (AbT>1) except for the #5-4 cat sample. The response ratio was R/T 5/6. The non-specific epitopes frequently cross reacted with the cat sera. Epitope GA6 (WSSV) had a response ratio of R/T=8/8, with binding activities in all eight sera samples (see FIG. 32). Epitope GA91 (S1) had a response ratio of R/T=6/8 (see FIG. 39). Epitope GA101 (S1) had a response ratio of R/T=8/8, with binding activities in all eight sera samples (see FIG. 33). Epitope GA102 (S1) had a response ratio of R/T=8/8, with binding activities in all eight sera samples (see FIG. 33). Epitope GA117 (S2) had a response ratio of R/T=6/8 (see FIG. 34). Epitope GA155 (N2) had a response ratio of R/T=8/8 (see FIG. 36). Epitope GA158 (N2) had a response ratio of R/T=8/8 (see FIG. 36). Epitope GA172 (N2) had a response ratio of R/T=8/8 (see FIG. 36). Epitope GA179 (M2) had a response ratio of R/T=7/8, with negative response in sample #5-4) (see FIG. 37).

The SARS-COV related specific epitopes did not interact with the chicken IBV nor the FCV sera, confirming the specificity of these epitopes to SARS-Coronavirus.

Example 8

Production of Antibodies Specific to Epitopes

Branched multiple antigenic peptides were used as immunogens to raise corresponding antibodies in animal hosts. Each peptide of GA91˜GA96, GA98˜GA191 was used as an immunogen to raise corresponding antibody in mice, rabbits, and ducks. Four doses were administered in 50 days.

The same immunogenic peptides were used to test immune response. In ducks, a high immune response was detected using epitopes of S1: 431-450 (GA100˜GA102), 437-461 (GA91), 476-505 (GA109, GA111, GA13), as shown in FIG. 40; S2: 1021-1040 (GA117, GA119), 1076-1095 (GA128-GA130), as shown in FIG. 41; N1: 105-124 (GA143, GA145), 107-126 (GA95), 140-169 (GA150˜GA154), as shown in 5C; N2: 300-344 (GA155, GA156, GA158˜GA161), 329-350 (GA96), 360-369 (GA167), 390-399 (GA173), as shown in FIG. 43; M: 1-10 (GA174), 4-20 (GA93), as shown in FIG. 44; sE: 1-10 (GA181), as shown in FIG. 45; and protein X1: 11-40 (GA186, GA188˜GA190), as shown in FIG. 46.

Thus, polyclonal antibodies can be produced by immunizing animal hosts with these SARS-CoV epitopes.

TABLE 1
	SEQ ID
Zone	NO.	Peptide	Sequence	Location	Annotation	Format	Notes
Control	1	GA6	(LRIPVTAEVGSSYFK)8K4K2K-βA	52-66	WSSV VP28	M8	8 branch MAP
	2	GA53	(PKYVKQNTVADVIES)8K4K2K-βA	306-313/	Influenza HA1/	M8	8 branch MAP
				410	EV71 VP1
	3	GA64	(VDRAVYWHLKKFAGNA)4K2K-βA	274-289	GNNV coat	M4	4 branch MAP
					protein
	4	GA81	NEIIPSKGCLALYLQQNWWTLLVDLLC—NH2	355-364/	Rabies virus	cyclic	C—C
				157-172	spike/EBV LMP1		disulfide
							bridge
	5	GA83	LTEVETYVLSIVPSGPLKAEIAQRLEDVF	4-30	Matrix protein	linear	linear
					M1 of Influenza	(L)	peptide
					A virus
	6	GA84	AQRLEDVFAGKNTDLEAYQKRMGVQMQRFK	25-54	Matrix protein	L
					M1 of Influenza
					A virus
S1	7	GA91	NYKYRYLRHGKLRPFERDISNVPFS	C437-461	SARS spike (S)	L
					protein
	8	GA98	(LAWNTRNIDA)8K4K2K-βA	421-430		M8
	9	GA99	(RNIDATSTGN)8K4K2K-βA	426-435		M8
	10	GA100	(TSTGNYNYKY)8K4K2K-βA	431-440		M8
	11	GA101	(YNYKYRYLRH)8K4K2K-βA	436-445		M8
	12	GA102	(RYLRHGKLRP)8K4K2K-βA	441-450		M8
	13	GA104	(FERDISNVPF)8K4K2K-βA	451-460		M8
	14	GA105	(SNVPFSPDGK)8K4K2K-βA	456-465		M8
	15	GA106	(SPDGKPCTPP)8K4K2K-βA	461-470		M8
	16	GA107	(PCTPPALNCY)8K4K2K-βA	466-475		M8
	17	GA109	(WPLNDYGFYT)8K4K2K-βA	476-485		M8
	18	GA110	(YGFYTTTGIG)8K4K2K-βA	481-490		M8
	19	GA111	(TTGIGYQPYR)8K4K2K-βA	486-495		M8
	20	GA112	(YQPYRVVVLS)8K4K2K-βA	491-500		M8
	21	GA113	(VVVLSFELLN)8K4K2K-βA	496-505		M8
	22	GA114	(FELLNAPATV)8K4K2K-βA	501-510		M8
	23	GA115	(APATVCGPKL)8K4K2K-βA	506-515		M8
	24	GA116	(GPKLSTDLI)8K4K2K-βA	511-520		M8
S2	25	GA117	(RVDFCGKGYH)8K4K2K-βA	1021-1030		M8
	26	GA118	(GKGYHLMSFP)8K4K2K-βA	1026-1035		M8
	27	GA119	(LMSFPQAAPH)8K4K2K-βA	1031-1040		M8
	28	GA120	(QAAPHGVVFL)8K4K2K-βA	1036-1045		M8
	29	GA121	(GVVFLHVTYV)8K4K2K-βA	1041-1050		M8
	30	GA122	(HVTYVPSQER)8K4K2K-βA	1046-1055		M8
	31	GA123	(PSQERNFTTA)8K4K2K-βA	1051-1060		M8
	32	GA124	(NFTTAPAICH)8K4K2K-βA	1056-1065		M8
	33	GA125	(PAICHEGKAY)8K4K2K-βA	1061-1070		M8
	34	GA126	(EGKAYFPREG)8K4K2K-βA	1066-1075		M8
	35	GA127	(FPREGVFVFN)8K4K2K-βA	1071-1080		M8
	36	GA128	(VFVFNGTSWF)8K4K2K-βA	1076-1085		M8
	37	GA129	(GTSWFITQRN)8K4K2K-βA	1081-1090		M8
	38	GA130	(ITQRNFFSPQ)8K4K2K-βA	1086-1095		M8
	39	GA131	(FFSPQIITTD)8K4K2K-βA	1091-1100		M8
	40	GA132	(IITTDNTFVS)8K4K2K-βA	1096-1105		M8
	41	GA133	(NTFVSGNCDV)8K4K2K-βA	1101-1110		M8
	42	GA134	(GNCDVVIGII)8K4K2K-βA	1106-1115		M8
	43	GA135	(VIGIINNTVY)8K4K2K-βA	1111-1120		M8
N1	44	GA95	PRWYFYYLGTGPEASLPYGA	C107-126	SARS nucleo-	L
					capsid (N)
					protein
	45	GA136	(GQGVPINTNS)8K4K2K-βA	70-79		M8
	46	GA137	(INTNSGPDDQ)8K4K2K-βA	75-84		M8
	195	GA138	(GPDDQIGYYR)8K4K2K-βA	80-89		M8
	47	GA139	(IGYYRRATRR)8K4K2K-βA	85-94		M8
	48	GA140	(RATRRVRGGD)8K4K2K-βA	90-99		M8
	49	GA141	(VRGGDGKMKE)8K4K2K-βA	95-104		M8
	50	GA142	(GKMKELSPRW)8K4K2K-βA	100-109		M8
	51	GA143	(LSPRWYFYYL)8K4K2K-βA	105-114		M8
	52	GA144	(YFYYLGTGPE)8K4K2K-βA	110-119		M8
	53	GA145	(GTGPEASLPY)8K4K2K-βA	115-124		M8
	54	GA146	(ASLPYGANKE)8K4K2K-βA	120-129		M8
	55	GA147	(GANKEGIVWV)8K4K2K-βA	125-134		M8
	56	GA148	(GIVWVATEGA)8K4K2K-βA	130-139		M8
	57	GA149	(ATEGALNTPK)8K4K2K-βA	135-144		M8
	58	GA150	(LNTPKDHIGT)8K4K2K-βA	140-149		M8
	59	GA151	(DHIGTRNPNN)8K4K2K-βA	145-154		M8
	60	GA152	(RNPNNNAATV)8K4K2K-βA	150-159		M8
	61	GA153	(NAATVLQLPQ)8K4K2K-βA	155-164		M8
	62	GA154	(LQLPQGTTLP)8K4K2K-βA	160-169		M8
	63	GA96	GTWLTYHGAIKLDDKDPQFKDN	C329-350
N2	64	GA155	(KHWPQIAQFA)8K4K2K-βA	300-309		M8
	65	GA156	(IAQFAPSASA)8K4K2K-βA	305-314		M8
	66	GA157	(PSASAFFGMS)8K4K2K-βA	310-319		M8
	67	GA158	(FFGMSRIGME)8K4K2K-βA	315-324		M8
	68	GA159	(RIGMEVTPSG)8K4K2K-βA	320-329		M8
	69	GA160	(VTPSGTWLTY)8K4K2K-βA	325-334		M8
	70	GA161	(TWLTYHGAIK)8K4K2K-βA	330-339		M8
	71	GA162	(HGAIKLDDKD)8K4K2K-βA	335-344		M8
	72	GA163	(LDDKDPQFKD)8K4K2K-βA	340-349		M8
	73	GA164	(PQFKDNVILL)8K4K2K-βA	345-354		M8
	74	GA165	(NVILLNKHID)8K4K2K-βA	350-359		M8
	75	GA166	(NKHIDAYKTF)8K4K2K-βA	355-364		M8
	76	GA167	(AYKTFPPTEP)8K4K2K-βA	360-369		M8
	77	GA168	(PPTEPKKDKK)8K4K2K-βA	365-374		M8
	78	GA169	(KKDKKKKTDE)8K4K2K-βA	370-379		M8
	79	GA170	(KKTDEAQPLP)8K4K2K-βA	375-384		M8
	80	GA171	(AQPLPQRQKK)8K4K2K-βA	380-389		M8
	81	GA172	(QRQKKQPTVT)8K4K2K-βA	385-394		M8
	82	GA173	(QPTVTLLPAA)8K4K2K-βA	390-399		M8
M	83	GA93	NGTITVEELKQLLEQWN	C4-20	SARS matrix (M)	L
					protein
	84	GA174	(MADNGTITVE)8K4K2K-βA	1-10		M8
	85	GA175	(TITVEELKQL)8K4K2K-βA	6-15		M8
	86	GA176	(ELKQLLEQWN)8K4K2K-βA	11-20		M8
	87	GA177	(LACFVLAAVY)8K4K2K-βA	61-70		M8
	88	GA179	(RINWVTGGIA)8K4K2K-βA	71-80		M8
	89	GA180	(TGGIAIAMAC)8K4K2K-βA	76-85		M8
sE	90	GA181	(MYSFVSEETG)8K4K2K-βA	1-10	SARS small	M8
					envelope (sE)
					protein
	91	GA182	(SEETGTLIVN)	6-15		M8
	92	GA183	(TLIVNSVLLF)8K4K2K-βA	11-20		M8
X1	93	GA184	(MDLFMRFFTL)8K4K2K-βA	1-10	SARS protein X1	M8
					(X1)
	94	GA185	(RFFTLGSITA)8K4K2K-βA	6-15		M8
	95	GA186	(GSITAQPVKI)8K4K2K-βA	11-20		M8
	96	GA187	(QPVKIDNASP)8K4K2K-βA	16-25		M8
	97	GA188	(DNASPASTVH)8K4K2K-βA	21-30		M8
	98	GA189	(ASTVHATATI)8K4K2K-βA	26-35		M8
	99	GA190	(ATATIPLQAS)8K4K2K-βA	31-40		M8
	100	GA191	(HLLLVAAGME)8K4K2K-βA	93-102		M8
sE	101	GA192	SVLLFLAFVV	16-25	SARS small	L
					envelope (sE)
					protein
	102	GA193	LAFVVFLLVT	21-30		L
	103	GA194	FLLVTLAILT	26-35		L
	104	GA195	LAILTALRLC	31-40		L
	105	GA196	ALRLCAYCCN	36-45		L
	106	GA197	AYCCNIVNVS	41-50		L
	107	GA198	IVNVSLVKPT	46-55		L
	108	GA199	LVKPTVYVYS	51-60		L
	109	GA200	VYVYSRVKNL	56-65		L
	110	GA201	RVKNLNSSEG	61-70		L
	111	GA202	SSEGVPDLLV	67-76		L
M	112	GA203	FVASFRLFAR	95-104	SARS matrix (M)	L
					protein
	113	GA204	RLFARTRSMW	100-109		L
	114	GA205	TRSMWSFNPE	105-114		L
	115	GA206	SFNPETNILL	110-119		L
	116	GA207	TNILLNVPLR	115-124		L
	117	GA208	PKEITVATSR	164-173		L
	118	GA209	VATSRTLSYY	169-178		L
	119	GA210	TLSYYKLGAS	174-183		L
	120	GA211	KLGASQRVGT	179-188		L
	121	GA212	QRVGTDSGFA	184-193		L
	122	GA213	DSGFAAYNRY	189-198		L
	123	GA214	AYNRYRIGNY	194-203		L
X2	124	GA215	QIQLSLLKVT	24-33	SARS protein X2	L
					(X2)
	125	GA216	LLKVTAFQHQ	29-38		L
	126	GA217	AFQHQNSKKT	34-43		L
	127	GA218	NSKKTTKLVV	39-48		L
	128	GA219	TKLVVILRIG	44-53		L
	129	GA220	ILRIGTQVLK	49-58		L
	130	GA221	TQVLKTMSLY	54-63		L
	131	GA222	TMSLYMAISP	59-68		L
	132	GA223	MAISPKFTTS	64-73		L
	133	GA224	KFTTSLSLHK	69-78		L
	134	GA225	LSLHKLLQTL	74-83		L
	135	GA226	LLQTLVLKML	79-88		L
	136	GA227	VLKMLHSSSL	84-93		L
	137	GA228	HSSSLTSLLK	89-98		L
	138	GA229	WIQFMMSRRR	119-128		L
	139	GA230	MSRRRLLACL	124-133		L
	140	GA231	LLACLCKHKK	129-138		L
	141	GA232	KHKKVSTNL	134-143		L
	142	GA233	STNLCTHSFR	140-149		L
X3	143	GA234	MFHLVDFQVT	1-10	SARS protein X3	L
					(X3)
	144	GA235	DFQVTIAEIL	6-15		L
	145	GA236	IAEILIIIMR	11-20		L
	146	GA237	IIIMRTFRIA	16-25		L
	147	GA238	TFRIAIWNLD	21-30		L
	148	GA239	IWNLDVIISS	26-35		L
	149	GA240	VIISSIVRQL	31-40		L
	150	GA241	IVRQLFKPLT	36-45		L
	151	GA242	FKPLTKKNYS	41-50		L
	152	GA243	KKNYSELDDE	46-55		L
	153	GA244	DEEPMELDYP	54-63		L
X4	154	GA245	GTRHTYQLRA	70-79	SARS protein X4	L
					(X4)
	155	GA246	YQLRARSVSP	75-84		L
	156	GA247	RSVSPKLFIR	80-89		L
	157	GA248	KLFIRQEEVQ	85-94		L
	158	GA249	QEEVQQELYS	90-99		L
	159	GA250	QELYSPLFLI	95-104		L
	160	GA251	PLFLIVAALV	100-109		L
	161	GA252	VAALVFLILC	105-114		L
	162	GA253	FLILCFTIKR	110-119		L
X5	163	GA254	MCLKILVRYN	1-10	SARS protein X5	L
					(X5)
	164	GA255	LVRYNTRGNT	6-15		L
	165	GA256	TRGNTYSTAW	11-20		L
	166	GA257	YSTAWLCALG	16-25		L
	167	GA258	LCALGKVLPF	21-30		L
	168	GA259	KVLPFHRWHT	26-35		L
	169	GA260	HRWHTMVQTC	31-40		L
	170	GA261	MVQTCTPNVT	36-45		L
	171	GA262	TPNVTINCQD	41-50		L
	172	GA263	INCQDPAGGA	46-55		L
	173	GA264	PAGGALIARC	51-60		L
	174	GA265	LIARCWYLHE	56-65		L
	175	GA266	WYLHEGHQTA	61-70		L
	176	GA267	GHQTAAFRDV	66-75		L
	177	GA268	AFRDVLVVLN	71-80		L
	178	GA269	VLVVLNKRTN	75-84		L
S2	179	GA283	(NNTVYDPLQP)8K4K2K-βA	1116-1125	SARS spike (S)	M8
					protein
	180	GA284	(DPLQPELDSF)8K4K2K-βA	1121-1130		M8
	181	GA285	(ELDSFKEELD)8K4K2K-βA	1126-1135		M8
	182	GA286	(KEELDKYFKN)8K4K2K-βA	1131-1140		M8
	183	GA287	(KYFKNHTSPD)8K4K2K-βA	1136-1145		M8
	184	GA288	(HTSPDVDLGD)8K4K2K-βA	1141-1150		M8
	185	GA289	(VDLGDISGIN)8K4K2K-βA	1146-1155		M8
	186	GA290	(ISGINASVVN)8K4K2K-βA	1151-1160		M8
	187	GA291	(ASVVNIQKEI)8K4K2K-βA	1156-1165		M8
	188	GA292	(IQKEIDRLNE)8K4K2K-βA	1161-1170		M8
	189	GA293	(DRLNEVAKNL)8K4K2K-βA	1166-1175		M8
	190	GA294	(VAKNLNESLI)8K4K2K-βA	1171-1180		M8
	191	GA295	(NESLIDLQEL)8K4K2K-βA	1176-1185		M8
	192	GA296	(DLQELGKYEQ)8K4K2K-βA	1181-1190		M8
	193	GA297	(GKYEQYIKWP)8K4K2K-βA	1186-1195		M8
	194	GA298	(YIKWPWYVWL)8K4K2K-βA	1191-1200		M8

Claims

1. One or more polypeptide(s) comprising amino acid sequence(s) selected from SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO:50, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 112, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 183, and SEQ ID NO: 187 and degenerate variant(s) of thereof.

2. One or more polypeptide(s) comprising amino acid sequence(s) selected from SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 25, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 81, SEQ ID NO: 88, and SEQ ID NO: 118 and degenerate variant(s) thereof.

3. One or more polypeptide(s) comprising amino acid sequence(s) selected from SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 25, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 88, and SEQ ID NO: 140 and degenerate variant(s) thereof.

4. One or more polypeptide(s) comprising amino acid sequence(s) selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 and degenerate variant(s) thereof.

5. An immunogenic composition that comprises one or more polypeptides comprising amino acid sequences selected from SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 44, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 76, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 90, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99 and degenerate variant(s) thereof.

6. The polypeptide of any of claims 1, 2, 3, 4, or 5, wherein the polypeptide is in a linear form.

7. The polypeptide of any of claims 1, 2, 3, 4, or 5, wherein the polypeptide is in a branched form.

8. An apparatus bearing one or more polypeptide(s) comprising amino acid sequence(s) selected from SEQ ID NO: 7-SEQ ID NO: 195 and the degenerate variants thereof.

9. An apparatus bearing one or more polypeptide(s) comprising amino acid sequence(s) selected from SEQ ID NO: 44-SEQ ID NO: 62 and the degenerate variants thereof.

10. An apparatus bearing one or more polypeptide(s) comprising amino acid sequence(s) selected from SEQ ID NO: 63-SEQ ID NO: 82 and the degenerate variants thereof.

11. An apparatus bearing one or more polypeptide(s) comprising amino acid sequence(s) selected from SEQ ID NO: 124-SEQ ID NO: 142 and the degenerate variants thereof.

12. An apparatus bearing one or more polypeptide(s) comprising amino acid sequence(s) selected from SEQ ID NO: 83-SEQ ID NO: 89 and SEQ ID NO: 112-SEQ ID NO: 123 and the degenerate variants thereof.

13. An apparatus bearing one or more polypeptide(s) comprising amino acid sequence(s) selected from SEQ ID NO: 7-SEQ ID NO: 43 and the degenerate variants thereof.

14. An apparatus bearing one or more polypeptide(s) comprising amino acid sequence(s) selected from SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 79 and the degenerate variants thereof.

15. An apparatus bearing one or more polypeptide(s) comprising amino acid sequence(s) selected from SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 112, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 183, and SEQ ID NO: 187 and the degenerate variants thereof.

16. An apparatus bearing a polypeptide comprising amino acid sequence of SEQ ID NO: 71 and the degenerate variants thereof.

17. The degenerate variant(s) of claims 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 13, 14, 15 or 16, wherein the one or more different amino acid(s) in the degenerate variant(s) have the same hydrophobic/hydrophilic nature as the original amino acid(s).

18. The degenerate variant(s) of claims 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 13, 14, 15 or 16, wherein the one or more different amino acid(s) in the degenerate variant(s) have the same +/−charge(s) as the original amino acid(s).