WO2022148455A1

WO2022148455A1 - Identification and uses of peptide sequences of sars-cov-2 t cell and b cell epitopes

Info

Publication number: WO2022148455A1
Application number: PCT/CN2022/070948
Authority: WO
Inventors: Matthew Robert MCKAY; Ahmed Abdul Quadeer; Syed Faraz Ahmed; Muhammad Saqib Sohail
Original assignee: Hong Kong University of Science and Technology
Current assignee: Hong Kong University of Science and Technology
Priority date: 2021-01-11
Filing date: 2022-01-10
Publication date: 2022-07-14
Anticipated expiration: 2023-07-11
Also published as: US20250000965A1

Abstract

The present disclosure identifies a set of T cell and B cell epitopes derived from Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-CoV-1) and their use in designing vaccines against SARS-CoV-2. A method for eliciting an immune response for prophylactic or therapeutic application using the T cell and/or B cell epitopes is described. Further disclosed are SARS-CoV-2-specific polypeptides, nucleic acids, host cells, and corresponding compositions for eliciting or measuring an immune response to SARS-CoV-2 and/or other coronaviruses.

Description

IDENTIFICATION AND USES OF PEPTIDE SEQUENCES OF SARS-COV-2 T CELL AND B CELL EPITOPES

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/229,063, filed August 3, 2021, and U.S. Provisional Patent Application No. 63/136,175, filed January 11, 2021, the contents of both are hereby incorporated by reference in the entirety for all purposes.

BACKGROUND

COVID-19 is caused by a novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) . Worldwide collaborative efforts from scientists are working on this disease and SARS-CoV-2 to develop effective interventions for controlling and preventing it [6–9] .

Currently, treatment options for COVID-19, which is caused by a novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) , are limited. The Food and Drug Authorization (FDA) has approved the antiviral drug Veklury (remdesivir) for adults and certain pediatric patients with COVID-19 who are sick enough to need hospitalization. By Emergency Use Authorization (EUA) , the FDA may authorize the use of unapproved drugs or unapproved uses of approved drugs under certain conditions. For example, the FDA has issued EUAs for several monoclonal antibody treatments for COVID-19 for the treatment, and in some cases prevention (prophylaxis) , of COVID-19 in adults and pediatric patients. Monoclonal antibodies are laboratory-made molecules that act as substitute antibodies. They can help the immune system recognize and respond more effectively to the virus, making it more difficult for the virus to reproduce and cause harm. Like other infectious organisms, SARS-CoV-2 can mutate over time, resulting in genetic variation in the population of circulating viral strains. The risk-benefit assessment for using certain monoclonal antibodies may not be favorable due to the increased frequency of resistant variants. Global efforts to combat COVID-19 have led to the rapid development of multiple vaccines. As the virus continues to circulate worldwide, virus variants have emerged in several regions, raising concerns about their potential to escape vaccine-induced antibody responses.

As such, there is an imminent need to improve efficacy of COVID-19 treatments and provide protection against severe disease and hospitalization. While vaccines have been pursued with success, there is an unmet need for developing vaccines that may induce a robust neutralizing antibody response to combat the COVID-19 pandemic.

SUMMARY

The summary is provide to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.

This present disclosure provides new compositions and methods useful for eliciting an immune response to SARS-CoV-2 and/or other coronavirus infections, based on the discovery that SARS-CoV-2 and/or other coronavirus infections may elicit T-cell and/or B cell response in a subject. Thus, in one aspect, the present disclosure is related to a peptide of no more than 500 amino acids, comprising (1) at least one T cell epitope set forth in Table 3 or Tables 15-47 or (2) at least one B cell epitope set forth in Table 4, the peptide optionally further comprising at least one heterologous amino acid sequence.

In some embodiments, the peptide comprises at least one T cell epitope set forth in Table 3 and at least one T cell epitope set forth in Tables 15-47. In some embodiments, the peptide comprises at least one T cell epitope set forth in Table 3 and at least one B cell epitope set forth in Table 4. In some embodiments, the peptide comprises at least one T cell epitope set forth in Tables 15-47 and at least one B cell epitope set forth in Table 4. In some embodiments, the peptide comprises at least one T cell epitope set forth in Table 3 and at least one T cell epitope set forth in Tables 15-47 and at least one B cell epitope set forth in Table 4. In any cases, the peptide may optionally further comprise at least one heterologous amino acid sequence. In some embodiments, the peptide comprises or consists of at least one of the T cell epitopes. In some embodiments, the peptide comprises or consists of at least one of the B cell epitopes. In some embodiments, the peptide comprises or consists of at least one of the T cell epitopes and at least one heterologous amino acid sequence. In some embodiments, the peptide comprises or consists of at least one of the B cell epitopes and at least one heterologous amino acid sequence.

In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises of no more than 500, 450, 400, 350, 300, 250, 200, 150, 120, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 amino acids. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises between about 5-500, about 20-100, about 40-200, about 50-450, about 80-350, about 100-400, or about 150-350 amino acids. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises about 20 amino acids. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises about 100 amino acids. In any cases, the peptide may further comprise at least one heterologous amino acid sequence.

The second aspect of the present disclosure provides a nucleic acid comprising a polynucleotide sequence encoding the peptide of no more than 500 amino acids, comprising (1) at least one T cell epitope set forth in Table 3 or Tables 15-47 or (2) at least one B cell epitope set forth in Table 4, the peptide optionally further comprising at least one heterologous amino acid sequence, or a peptide comprising or consisting of at least one of the T cell epitopes, or a peptide comprising or consisting of at least one of the B cell epitopes, or a peptide comprising or consisting of at least one of the T cell epitopes and at least one heterologous amino acid sequence, or a peptide comprising or consisting of at least one of the B cell epitopes and at least one heterologous amino acid sequence. In some embodiments, the polynucleotides encodes a peptide of at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids. In some embodiments, the polynucleotides encodes a peptide of between about 5-500, about 20-100, about 40-200, about 50-450, about 80-350, about 100-400, or about 150-350 amino acids. In some embodiments, the polynucleotides encodes a peptide of about 20 amino acids. In some embodiments, the polynucleotides encodes a peptide of about 100 amino acids. In some embodiments, the polynucleotide sequence comprises deoxyribonucleic acid (DNA) . In some embodiments, the polyneucleotide sequence comprises ribonucleic acid (RNA) such as messenger RNA (mRNA) . In some embodiments, the present disclosure provides an expression cassette comprises any of the peptide as described herein and the peptide is operably linked to a promoter. In some embodiments, the present disclosure provides a vector comprising the expression cassette comprising any of the peptides as described herein and the peptide is operably linked to a promoter. In some embodiments, the present disclosure provides a host cell comprising the vector comprising the expression cassette.

In a third aspect, the present disclosure provides a composition comprising (1) a peptide of no more than 500 amino acids, comprising (1) at least one T cell epitope set forth in Table 3 or Tables 15-47 or (2) at least one B cell epitope set forth in Table 4, the peptide optionally further comprising at least one heterologous amino acid sequence, or a peptide comprising or consisting of at least one of the T cell epitopes, or a peptide comprising or consisting of at least one of the B cell epitopes, or a peptide comprising or consisting of at least one of the T cell epitopes and at least one heterologous amino acid sequence, or a peptide comprising or consisting of at least one of the B cell epitopes and at least one heterologous amino acid sequence, the nucleic acid encoding any of the peptides, the expression cassette comprising any of the peptides, the vector comprising the expression cassette, or the host cell; and (2) a pharmaceutically acceptable excipient. In some embodiments, the composition further comprises an adjuvant. In some embodiments, the composition comprises a plurality of peptides each comprising a T cell epitope set forth in Table 3 or Tables 15-47. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises of no more than 500, 450, 400, 350, 300, 250, 200, 150, 120, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 amino acids. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises between about 5-500, about 20-100, about 40-200, about 50-450, about 80-350, about 100-400, or about 150-350 amino acids. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises about 20 amino acids. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises about 100 amino acids.

In a fourth aspect, the present disclosure provides a method of eliciting an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising (1) a peptide of no more than 500 amino acids, comprising (1) at least one T cell epitope set forth in Table 3 or Tables 15-47 or (2) at least one B cell epitope set forth in Table 4, the peptide optionally further comprising at least one heterologous amino acid sequence, or a peptide comprising or consisting of at least one of the T cell epitopes, or a peptide comprising or consisting of at least one of the B cell epitopes, or a peptide comprising or consisting of at least one of the T cell epitopes and at least one heterologous amino acid sequence, or a peptide comprising or consisting of at least one of the B cell epitopes and at least one heterologous amino acid sequence, the nucleic acid encoding any of the peptides, the expression cassette comprising any of the peptides, or the vector comprising the expression cassette. In some embodiments, the method comprises administering the composition to the subject by a route selected from the group consisting of, subcutaneous, intramuscular, and oral. In some embodiments, the subject is at risk of exposure to SARS-CoV, SARS-CoV-2 or other coronavirus infections. In some embodiments, the subject is at risk of exposure to SARS-CoV-2 infections. In some embodiments, the subject is at risk of developing severe illnesses when contracted with SARS-CoV-2 (e.g., COVID-19) . In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises of no more than 500, 450, 400, 350, 300, 250, 200, 150, 120, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 amino acids. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises between about 5-500, about 20-100, about 40-200, about 50-450, about 80-350, about 100-400, or about 150-350 amino acids. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises about 20 amino acids. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises about 100 amino acids.

In a fifth aspect, the present disclosure provides a kit for eliciting an immune response in a subject in need thereof, the kit comprising a first container containing the composition comprising (1) a peptide of no more than 500 amino acids, comprising (1) at least one T cell epitope set forth in Table 3 or Tables 15-47 or (2) at least one B cell epitope set forth in Table 4, the peptide optionally further comprising at least one heterologous amino acid sequence, or a peptide comprising or consisting of at least one of the T cell epitopes, or a peptide comprising or consisting of at least one of the B cell epitopes, or a peptide comprising or consisting of at least one of the T cell epitopes and at least one heterologous amino acid sequence, the nucleic acid encoding any of the peptides, the expression cassette comprising any of the peptides, or the vector comprising the expression cassette. In some embodiments, the kit optionally contains an additional container containing a therapeutic agent against SARS-CoV-2. In some embodiments, the kit further comprises at least a second container each containing at least one different composition. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises of no more than 500, 450, 400, 350, 300, 250, 200, 150, 120, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 amino acids. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises between about 5-500, about 20-100, about 40-200, about 50-450, about 80-350, about 100-400, or about 150-350 amino acids. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises about 20 amino acids. In some embodiments, the peptide comprising the one or more T cell epitope set forth in Table 3 and/or Tables 15-47, and/or one or more B cell epitopes set forth in Table 4 comprises about 100 amino acids.

In a sixth aspect, the present disclosure provides a method for detecting T cell immunity against SARS-CoV-2 in a subject, comprising: (1) contacting T cells obtained from the subject with a T cell epitope set forth in Table 3 or Tables 15-47 and antigen-presenting cells having an HLA allele associated with the epitope; and (2) detecting activation of the T cells, thereby detecting presence of T cell immunity against SARS-CoV-2 in the subject. In some embodiments, step (2) of the method comprises detection of T cell proliferation or T cell secretion of one or more cytokines. In some embodiments, step (2) of the method comprises T cell proliferation assay, flow cytometry, ELISPOT, or ELISA. In some embodiments, step (1) of the method comprises contacting T cells obtained from the subject with a composition comprising a plurality of peptides each comprising a T cell epitope set forth in Table 3 or Tables 15-47 and antigen-presenting cells having HLA alleles associated with each of the plurality of epitopes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B show comparison of the similarity of structural proteins of SARS-CoV-2 with the corresponding proteins of SARS-CoV and MERS (Middle East Respiratory Syndrome) -CoV. FIG. 1A shows the percentage genetic similarity of the individual structural proteins of SARS-CoV-2 with those of SARS-CoV and MERS-CoV. The reference sequence of each coronavirus (Materials and Methods) was used to calculate the percentage genetic similarity. FIG. 1B is a circular phylogram of the phylogenetic trees of the four structural proteins. All trees were constructed based on the available unique sequences using PASTA and rooted with the outgroup Zaria Bat CoV strain (accession ID: HQ166910.1) .

FIGS. 2A-C depict location of SARS-CoV S protein subunits and SARS-CoV-derived B cell epitopes on the protein structure (PDB ID: 5XLR) . FIG. 2A shows subunits S1 and S2 are indicated in medium and light grey color, respectively. The receptor binding motif lies within the S1 subunit and is indicated in dark grey color. FIG. 2B shows residues of the linear B cell epitopes, that were identical in SARS-CoV-2 (Table 4) , are shown as shaded. The black and grey colors reflect the surface and buried residues, respectively. FIG. 2C shows locations of discontinuous B cell epitopes that share at least one identical residue with corresponding SARS-CoV-2 sites (Table 5) . Identical epitope residues are shown in dark grey color, while the remaining epitope residues are shown in medium grey color. Both the side view (left panel) and the top view (right panel) of the structure are shown.

FIG. 3 shows fraction of mutations in the observed sequences of the structural proteins of the three coronaviruses. Mutation is defined here as an amino acid difference from the reference sequence of the respective coronavirus; accession IDs: NC_045512.2 (2019-nCoV) , NC_004718.3 (SARS-CoV) , and NC_019843.3 (MERS-CoV) .

FIG. 4 illustrates location of identified T cell epitopes on the SARS-CoV S protein structure (PDB ID: 5XLR) . Residues of the SARS-CoV-derived T cell epitopes (determined using positive MHC binding assays and that were identical in SARS-CoV-2) are shown with filled color. The dark and light shade reflect the surface and buried residues, respectively.

FIG. 5 shows pairwise sequence alignment of the reference sequences of the S proteins of SARS-CoV and SARS-CoV-2 (accession ID: NP_828851.1 and YP_009724390.1, respectively) . Identical residues are indicated by *.

FIGS. 6A-C show global diversity of HLA class I alleles and the corresponding T cell responses, and summary of the experimentally-determined SARS-CoV-2 CD8+ T cell epitope data. FIG. 6A shows different HLA class I alleles are prevalent in different geographical regions (left panel) ; heatmaps depicting the diverse HLA allele distribution in North America, North East Asia, and Australia are shown as examples (middle panel) . Each square in the heatmap represents a distinct HLA class I allele, and its color shade represents the frequency of the allele in the geographical region with a dark shade representing high frequency and vice versa; and different HLA alleles present different SARS-CoV-2 peptides, and consequently the T cell responses elicited against SARS-CoV-2 are expected to differ among geographical regions (right panel) . Thus, peptide pools to test for SARS-CoV-2 CD8+ T cell responses need to be designed specific to the HLA alleles prevalent in the population being tested. This figure was created with BioRender. com. FIG. 6B shows the number of HLA alleles in each geographical region for which at least one SARS-CoV-2 experimentally-determined CD8+ T cell epitope is known. FIG. 6C shows the number of experimentally-determined epitopes within spike and other SARS-CoV-2 proteins determined so far for class I HLA alleles (left panel) and the frequencies of these HLA alleles in different geographical regions (right panel) (for details of experimentally-determined epitope data, see Methods) .

FIGS . 7A-B show an optimized in silico strategy to predict SARS-CoV-2 CD8 ⁺ T cell epitopes. FIG. 7A shows a performance comparison of in silico HLA class I epitope prediction methods based on predicting experimentally-determined SARS-CoV-2 CD8 ⁺ T cell epitopes. Intersection of the top predictions of the analysed 12 in silico methods showed that MHCflurry2.0P predicts the highest number of epitopes not predicted by any other method. For each method, the set of top-ranked predicted peptides consisted of n = 200 predictions, with 20 predictions for each of the 10 HLA alleles having the most experimental data available (see Methods) . FIG. 7B shows the set of top-ranked predictions (n = 341) obtained from the proposed approach, union of MHCflurry2.0P and NetMHCpan4.1BA (see Methods) , comprised of more experimentally-determined epitopes than the sets of top-ranked predictions of the individual methods

FIGS. 8A-B show a schematic of SARS2TPools framework and snapshot of the platform’s interface for a use case. FIG. 8A is a schematic showing how the SARS2TPools, based on user-selected options (protein, geographical region, specific HLA alleles) , combines experimentally-determined epitope data, in silico predictions, and information of prevalent HLA alleles across regions to obtain optimized peptide pools for assessing vaccine-induced T cell responses. This figure was created with BioRender. com. FIG. 8B is a snapshot of the SARS2TPools interface for the “Region-specific” tab with selected options (Region: Oceania; Protein: NSP12; Pool-size of peptides: small; Threshold for in silico predictions: Default; and Length of peptides: 9) .

FIGS. 9A-C are summary of region-specific CD8+ T cell peptide pools provided by SARS2TPools. FIG. 9A shows the number of peptides in region specific pools from experimental studies and in silico predictions. FIG. 9B shows overlap between pairs of region-specific pools. FIG. 9C shows a source of peptides for each region-specific pool. Each circle represents one of the top 30 prevalent HLAs for a specific region and the color of the circle indicates whether the peptides were obtained from only experimental studies, or only from in silico predictions, or from both.

FIG. 10 shows histograms of ranks assigned by different in silico epitope prediction methods to the experimentally-determined SARS-CoV-2 CD8 ⁺ T cell epitopes associated with the 10 HLA alleles with the most experimental data (see Methods) . Each bin here represents a set of peptides within the specified rank range of all 10 HLA alleles.

FIGS. 11A-D show the observation that predictions of MHCflurry2.0P are distinct from those of other in silico methods (FIG. 12) was robust to the size of the set of top-ranked predictions compared. Distinct sets of predicted epitopes ranked by the considered 12 in silico methods in their (FIG. 11A) top 10, (FIG. 11B) top 15, (FIG. 11C) top 20, and (FIG. 11D) top 25 predictions corresponding to each of the 10 HLA alleles having the most experimental data (see Methods) .

FIG. 12 shows the strategy of combining top-ranked predictions of MHCflurry2.0P with any of the other 11 in silico methods considered in this study had a higher hit-rate on experimentally-determined SARS-CoV-2 CD8 ⁺ epitope data than either of the individual methods. Here, hit-rate represents the fraction of experimentally known epitopes present in the set of top-ranked predicted peptides.

FIG. 13 shows hit-rates of union methods comprising of MHCflurry2.0P and one of the other 11 in silico epitope prediction methods. While none of these clearly outperformed the rest, the 4 union methods comprising of MHCflurry2.0P and either NetMHCpan4.1BA, NetMHCpan4.0BA, NetMHC4.0, or NetMHCpan4.1EL were among the top 4 performing methods (also see Table 13) . Here, hit-rate represents the fraction of experimentally known epitopes present in the set of top-ranked predicted peptides.

FIGS. 14A-B illustrate COVIDep providing an up-to-date set of B-cell and T-cell epitopes that can serve as potential vaccine targets for SARS-CoV-2. FIG. 14A shows the identified epitopes are experimentally-derived from SARS-CoV and have a close genetic match with the available SARS-CoV-2 sequences. FIG. 14B is an example of the T-cell epitopes reported by COVIDep (as of 20 May 2020) for the spike protein of SARS-CoV-2. Here, the Search box (in the top right) was used to select only the HLA-A*02: 01-restricted epitopes. (An explanation of all interactive COVIDep visualizations is incorporated in the “How to use COVIDep page” of the platform. ) Of the 14 epitopes listed in the display, 9 of them (

IEDB IDs

36724, 54507, 54725, 69657, 71663, 2801, 54680, 16156, and 37289) overlap with epitopes against which cytotoxic CD8+ T cell responses have been observed in peripheral blood mononuclear cells isolated from COVID-19 patients. T cell responses were also recorded against protein regions overlapping with the epitope with IEDB ID 71663 in a pre-clinical trial of a DNA vaccine candidate.

FIG. 15 is an epitope screening protocol used by COVIDep for providing vaccine target recommendations for SARS-CoV-2. COVIDep periodically pools SARS-CoV-2 sequence data from the GISAID database (www. gisaid. org) and compares with experimentally-determined T cell and B cell epitopes of SARS-CoV, obtained from the ViPR database (www. viprbrc. org) . The T cell epitopes were determined based on either positive T cell assays or positive MHC binding assays for SARS-CoV. For the B cell epitopes, both linear and discontinuous epitopes were considered. The system outputs those epitopes that are genetically similar in SARS-CoV-2, based on an epitope screening parameter. This user-defined parameter allows the user to select epitopes based on their conservation in the SARS-CoV-2 sequence data, where conservation is defined as the fraction of SARS-CoV-2 sequences with the exact epitope sequence. The value of this parameter is set to 0.95 as default; however, the user may change this value to adjust the stringency of the screening criterion. For example, reducing the value of the parameter will allow for the consideration of epitopes with greater genetic variation, potentially increasing the set of recommended SARS-CoV-2 vaccine targets. For the identified T cell epitopes, the population coverage analysis tool available at IEDB (www. iedb. org) is used to estimate the percentage of a specified population that can elicit a response against them.

DEFINITIONS

The term "isolated, " when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term "purified" denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 60%, 70%, 80%, 85%, 90%, 95%, or 99%pure, more preferably at least 95%pure, and most preferably at least 99%pure.

In this application, the term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Unnatural (non-naturally occurring) amino acids include, without limitation, amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids in either the L-or D-configuration that function in a manner similar to the naturally-occurring amino acids. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure different from the general chemical structure of an amino acid, but capable of functioning in a manner similar to a naturally occurring amino acid.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991) ; Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985) ; and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994) ) . The term nucleic acid is used interchangeably with gene, cDNA, or mRNA encoded by a gene.

When the relative locations of elements in a polynucleotide sequence are concerned, a "downstream" location is one at the 3' side of a reference point, and an "upstream" location is one at the 5' side of a reference point.

The terms “polypeptide, ” “peptide, ” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens) , wherein the amino acid residues are linked by covalent peptide bonds. In this application, the amino acid sequence of a polypeptide is presented from the N-terminus to the C-terminus. In other words, when describing an amino acid sequence of a peptide, the first amino acid from the N-terminus is referred to as the “first amino acid. ”

When used in the context of describing partners of a fusion peptide, the term "heterologous" refers to the relationship of one peptide fusion partner to the another peptide fusion partner: the manner in which the fusion partners are present in the fusion peptide is not one that can be found a naturally occurring protein. For instance, a "heterologous polypeptide" fused with a T cell or B cell epitope to form a fusion peptide may be one that is originated from a protein other than the antigen from which the T cell or B cell epitope is derived, such as a granulocyte-macrophage colony-stimulating factor (GM-CSF) . On the other hand, a "heterologous polypeptide" may be one derived from another portion of the T cell or B cell protein that is not immediately contiguous to the T cell or B cell epitope. A "heterologous polypeptide" may contain modifications of a naturally occurring protein sequence or a portion thereof, such as deletions, additions, or substitutions of one or more amino acid residues. Regardless of the origin of the "heterologous polypeptide" (i.e., whether it is derived from the antigen from which the T cell or B cell epitope is derived or another protein) , the fusion peptide should not contain a subsequence of the human T cell or B cell that encompasses the amino acid sequences in Table 3 and Tables 15-47 and have more than 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or more amino acids in length. In some exemplary embodiments, the fusion peptide should not contain a subsequence of the human T cell or B cell that encompasses the amino acid sequences in Table 3 and Tables 15-47 and have more than 500 amino acids in length. In some exemplary embodiments, a "heterologous polypeptide" for use in the present disclosure has no more than 15-20 amino acids in length; in other embodiments, a "heterologous polypeptide" has at least 100 amino acids in length. In some exemplary embodiments, a "heterologous polypeptide" is a recombinant polynucleotide (or a copy or complement of a recombinant polynucleotide) that has been manipulated using well known methods. For example, a "heterologous polypeptide" can comprise a recombinant expression cassette comprising a promoter operably linked to a second polynucleotide (e.g., a coding sequence of the antigen from which the T cell or B cell epitope is derived) . In this context, the promoter is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning -A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley &Sons, Inc. (1994-1998) ) . As another example, the "heterologous polypeptide" may comprise a promoter that is heterologous to a second polynucleotide encoding the polypeptide of interest (e.g., a T cell and/or a B cell epitope) or a fragment thereof, and a third polynucleotide encoding a detecting (e.g., a tag or identifier) molecule. The detecting molecule can be a fluorescent protein, e.g., a green fluorescent protein (GFP) or a variant or the like, such as DsRed and other red fluorescent protein.

The word "fuse" or "fused, " as used in the context of describing a peptide of this disclosure that comprises a T cell or B cell epitope joined with a heterologous polypeptide, refers to a connection between the epitope and the heterologous polypeptide by any covalent bond, including a peptide bond.

The phrase "a nucleic acid sequence encoding" refers to a nucleic acid which contains sequence information for a structural RNA such as rRNA, a tRNA, or the primary amino acid sequence of a specific protein or peptide, or a binding site for a trans-acting regulatory agent. This phrase specifically encompasses degenerate codons (i.e., different codons which encode a single amino acid) of the native sequence or sequences that may be introduced to conform to codon preference in a specific host cell.

An “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter.

The term “recombinant, ” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a nucleic acid or protein from an outside source or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all.

The term "administration" or "administering" refers to various methods of contacting a substance with a mammal, especially a human. Modes of administration may include, but are not limited to, methods that involve contacting the substance intravenously, intraperitoneally, intranasally, transdermally, topically, subcutaneously, parentally, intramuscularly, orally, or systemically, and via injection, ingestion, inhalation, implantation, or adsorption by any other means. One exemplary means of administration of a T cell and/or B cell peptide of this disclosure or a fusion peptide comprising a T cell and/or B cell epitope (e.g., a T cell or B cell epitope derived from the S protein, N protein, or full length of SARS-CoV and/or SARS-CoV-2) and a heterologous polypeptide is via intramuscular delivery, where the peptide or fusion peptide can be formulated as a pharmaceutical composition in the form suitable for intramuscular injection, such as an aqueous solution, a suspension, or an emulsion, etc. Other means for delivering a T cell and/or B cell epitope or a fusion peptide of this disclosure includes intradermal injection, subcutaneous injection, intravenous injection, or transdermal application as with a patch.

An "effective amount" of a certain substance refers to an amount of the substance that is sufficient to effectuate a desired result. For instance, an effective amount of a composition comprising a peptide of this disclosure that is intended to induce an anti-SARS-CoV or SARS-CoV-2 immunity is an amount sufficient to achieve the goal of inducing the immunity when administered to a subject. The effect to be achieved may include the prevention, correction, or inhibition of progression of the symptoms of a disease/condition and related complications to any detectable extent. The exact quantity of an "effective amount" will depend on the purpose of the administration, and can be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992) ; Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999) ; and Pickar, Dosage Calculations (1999) ) .

A "therapeutically effective amount" of a substance/molecule of the disclosure may vary depending on factors such as: the disease state, age, sex and weight of the individual, and the ability of the substance/molecule to induce a desired response in the individual. A therapeutically effective amount is also an amount that has a therapeutically beneficial effect over any toxic or detrimental effect of the substance/molecule.

A "prophylactically effective amount" refers to an amount of dosage and time necessary to effectively achieve the desired prophylactic result. Since prophylactic doses are used in individuals prior to or early in the disease, the prophylactically effective amount is typically (but not necessarily) less than the therapeutically effective amount.

A "physiologically or pharmaceutically acceptable excipient" is an inert ingredient used in the formulation of a composition of this disclosure, which contains the active ingredient (s) of a T cell and/or B cell peptide or a fusion peptide comprising a T cell and/or B cell peptide and a heterologous polypeptide and is suitable for use, e.g., by injection into a patient in need thereof. This inert ingredient may be a substance that, when included in a composition of this disclosure, provides a desired pH, consistency, color, smell, or flavor of the composition.

As used herein, the term "T cell immune response" refers to activation of antigen specific T cells as measured by proliferation or expression of molecules on the cell surface or secretion of proteins such as cytokines.

As used herein, the term “B cell immune response” refers to either a T cell-independent immune response or a T cell-dependent immune response. In a T cell-independent response, B cells respond directly to the antigen. In a T cell-dependent immune response, B cells rely on the assistance from T cells to respond. Activated B cells may express IgA, IgE, IgG or retain IgM expression. Cytokines produced by T cells and others may determine what isotype the B cells express. Several general techniques are commonly used to identify antigen-specific B cells. Non-limiting examples are B cell enzyme linked immunospot (ELISPOT) , limiting dilution, flow cytometry, adoptive transfer, microscopy, and B cell receptor (BCR) transgenic mice.

As used herein, the term “immune response” generally refers to the immune system recognizing the antigens, e.g., proteins, on the surface of substances or microorganisms, such as bacteria or virus, and attacks and destroys, or tries to destroy, them. As described herein, the term “immune response” refers to a cell-mediated (T-cell) immune response and/or an antibody (B-cell) immune response.

As described herein, the terms “immunogenic composition, ” or “vaccine” are used interchangeably and refer to a composition that elicits an immune response in a subject, especially a human. An immunogenic composition or vaccine can be used prophylactically to prevent COVID-19 (SARS-CoV-2) or other coronavirus diseases (e.g., SARS-CoV) .

As described herein, the term “immunity” refers to protection from an infectious disease. For instance, if a subject is immune to a disease, the object can be exposed to it without becoming infected.

As described herein, the term “vaccine” generally refers to a preparation that is used to stimulate the body’s immune system to generate immunity for a disease. For instance, it refers to an antigen-containing formulation consisting of whole pathogenic organisms (killed or attenuated) or components of these organisms (such as proteins, peptides or polysaccharides) for conferring immunity against disease caused by these organisms. Vaccine formulations may be natural, synthetic or obtained by recombinant DNA techniques. A vaccine may be administered in any route known in the field.

As described herein, the terms “vaccination” or “immunization” or “inoculation” are used interchangeably. They refer to a process by which a person becomes protected against a disease by introducing a vaccine into the body to produce protection from the specific disease. For example, vaccines are usually administered intramuscularly through needle injections. As another example, vaccines can be administered by mouth (oral) or sprayed into the nose (intranasal) .

As described herein, the term "immunogenic" refers to the ability of an immunogen, antigen or vaccine to stimulate an immune response.

As used herein, the term “antigen" is defined as any substance capable of eliciting an immune response. For example, an “antigen” may be a small molecule or a macromolecule such as a protein, a peptide, a polysaccharide, a nucleic acid, a lipid, or a biomolecule.

As used herein, the term " antigen-specific" refers to a property of a population of cells such that the provision of a particular antigen or antigen fragment causes the proliferation of a specific cell.

The phrase “specifically (or selectively) binds” to an antibody or “specifically (or selectively) immunoreactive with, ” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to fusion proteins can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with fusion protein and not with individual components of the fusion proteins. This selection may be achieved by subtracting out antibodies that cross-react with the individual antigens. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow &Lane, Antibodies, A Laboratory Manual (1988) , for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity) . Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.

The terms "antibody" and "immunoglobulin" are used interchangeably in a broad sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies) , polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity) , and may also include certain antibody fragments (as described in more detail herein) . The antibody may be a chimeric antibody, a human antibody, a humanized antibody, and/or an affinity matured antibody.

The terms “a, ” “an, ” and “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a, ” “an, ” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the agent” includes reference to one or more agents known to those skilled in the art, and so forth.

As used herein, the term “about” denotes a range of ±10%of a specified value. For instance, “about 10” denotes a range of 9-11.

The term “subject” or "subject in need of treatment" refers to an individual who seeks medical attention due to risk of, or actual sufferance from, a condition involving undesirable inflammation (e.g., pneumonia or an infection that inflames air sacs) or a condition involving infection of the respiratory tract (e.g., the upper respiratory tract, the lungs) . Subjects or individuals in need of treatment include those that demonstrate symptoms of infection of the respiratory tract or those are at risk of later developing the disease or disorder and/or its symptoms. For example, the subject may experience or is at risk of sufferance from a SARS-CoV infection. The subject may have one or more symptoms of SARS-CoV infection as defined by the Centers for Disease Control and Prevention (www. cdc. gov/coronavirus/2019-ncov/symptoms-testing/symptoms. html) . The subject may experience or have a wide range of symptoms ranging from mild to severe illness. Exemplary symptoms are fever or chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, new loss of taste or smell, sore throat, congestion or running nose, nausea or vomiting, or diarrhea, trouble breathing, persistent pain or pressure in the chest, new confusion, inability to wake or stay awake, and/or pale, gray, or blue-colored skin, lips, or nail beds, depending on skin tone, or combinations thereof. Symptoms may appear 0-60 days, 1-30 days, or 2-14 days after exposure to the source of infection (e.g., SARS-CoV or SARS-CoV-2 viruses) . The term subject can include both animals, especially mammals, and humans.

DETAILED DESCRIPTION

I. INTRODUCTION

Efforts have been made to develop vaccines against the novel SARS-CoV-2. Described herein are peptides, their related compositions, and methods of uses thereof. The disclosed peptides comprise at least one T cell epitope and/or at least one B cell epitope for eliciting an immune response in a subject against SARS-CoV-2. Epitopes that provide broad coverage broadly and region-specific are also explored.

We sought to gain insights for vaccine design against SARS-CoV-2 by considering the high genetic similarity between SARS-CoV-2 and SARS-CoV, which caused the outbreak in 2003, and leveraging existing immunological studies of SARS-CoV. By screening the experimentally-determined SARS-CoV-derived B cell and T cell epitopes in the immunogenic structural proteins of SARS-CoV, we identified a set of B cell and T cell epitopes derived from the spike (S) and nucleocapsid (N) proteins that map identically to SARS-CoV-2 proteins. As no mutation has been observed in these identified epitopes among the 120 available SARS-CoV-2 sequences (as of 21 February 2020) , immune targeting of these epitopes may potentially offer protection against this novel virus. For the T cell epitopes, we performed a population coverage analysis of the associated MHC alleles and proposed a set of epitopes that is estimated to provide broad coverage globally, as well as in China. The findings provide a screened set of epitopes that can help guide experimental efforts towards the development of vaccines against SARS-CoV-2.

Unless specifically stated, SARS-CoV-2 is referred to the original SARS-CoV-2 (GenBank accession no. NC_045512) or any variants thereof. A variant has one or more mutations that differentiate it from other variants of the SARS-CoV-2 viruses. According to The Center for Disease Control and Prevention (CDC) , current known variants include, but are not limited to, Alpha (B. 1.1.7 and Q lineages) , Beta (B. 1.351 and descendent lineages) , Gamma (P. 1 and descendent lineages) , Epsilon (B. 1.427 and B. 1.429) , Eta (B. 1.525) , Iota (B. 1.526) , Kappa (B. 1.617.1) , 1.617.3, Mu (B. 1.621, B. 1.621.1) , Zeta (P. 2) , Delta (B. 1.617.2 and AY lineages) , and Omicron (B. 1.1.529 and BA lineages) . In the context of this disclosure, a polynucleotide of the full length SARS-CoV-2 (GenBank accession no. NC_045512) , a variant, or a fragment thereof may activates a T cell and/or B cell response. The polynucleotides may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more sequence identity with the full length SARS-CoV-2 (GenBank accession no. NC_045512) , a variant, or a fragment thereof.

II. CHEMICAL SYNTHESIS OF PEPTIDES

The peptides of the present disclosure, particularly those of relatively short length (e.g., no more than 50-100 amino acids, no more than 10-500 amino acids) , may be synthesized chemically using conventional peptide synthesis or other protocols well known in the art. In some embodiments, the peptides are of no more than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids, or in the range of from about 10 to about 50, 100, 200, 300, 400 or 500 amino acids, from about 20 to about 50, 100, 200, 300, 400 or 500 amino acids, from about 50 to about 100, 150, 200, 300, 400 or 500 amino acids, from about 100 to about 150, 200, 250, 300, 400 or 500 amino acids, or from about 200 to about 250, 300, 350, 400 or 500 amino acids.

Peptides may be synthesized by solid-phase peptide synthesis methods using procedures similar to those described by Merrifield et al., J. Am. Chem. Soc., 85: 2149-2156 (1963) ; Barany and Merrifield, Solid-Phase Peptide Synthesis, in The Peptides: Analysis, Synthesis, Biology Gross and Meienhofer (eds. ) , Academic Press, N.Y., vol. 2, pp. 3-284 (1980) ; and Stewart et al., Solid Phase Peptide Synthesis 2nd ed., Pierce Chem. Co., Rockford, Ill. (1984) . During synthesis, N-α- protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to a solid support, i.e., polystyrene beads. The peptides are synthesized by linking an amino group of an N-α-deprotected amino acid to an α-carboxy group of an N-α-protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide. The attachment of a free amino group to the activated carboxyl leads to peptide bond formation. The most commonly used N-α-protecting groups include Boc, which is acid labile, and Fmoc, which is base labile.

Materials suitable for use as the solid support are well known to those of skill in the art and include, but are not limited to, the following: halomethyl resins, such as chloromethyl resin or bromomethyl resin; hydroxymethyl resins; phenol resins, such as 4- (α- [2, 4-dimethoxyphenyl] -Fmoc-aminomethyl) phenoxy resin; tert-alkyloxycarbonyl-hydrazidated resins, and the like. Such resins are commercially available and their methods of preparation are known by those of ordinary skill in the art.

Briefly, the C-terminal N-α-protected amino acid is first attached to the solid support. The N-α-protecting group is then removed. The deprotected α-amino group is coupled to the activated α-carboxylate group of the next N-α-protected amino acid. The process is repeated until the desired peptide is synthesized. The resulting peptides are then cleaved from the insoluble polymer support and the amino acid side chains deprotected. Longer peptides can be derived by condensation of protected peptide fragments. Details of appropriate chemistries, resins, protecting groups, protected amino acids and reagents are well known in the art and so are not discussed in detail herein (See, e.g., Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press (1989) , and Bodanszky, Peptide Chemistry, A Practical Textbook, 2nd Ed., Springer-Verlag (1993) ) .

III. RECOMBINANT PRODUCTION OF PEPTIDES

A. General Recombinant Technology

Basic texts disclosing general methods and techniques in the field of recombinant genetics include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001) ; Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990) ; and Ausubel et al., eds., Current Protocols in Molecular Biology (1994) .

For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp) . These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemically synthesized, e.g., according to the solid phase phosphoramidite triester method first described by Beaucage &Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981) , using an automated synthesizer, as described in Van Devanter et. al., Nucleic Acids Res. 12: 6159-6168 (1984) . Purification of oligonucleotides is performed using any art-recognized strategy, e.g., native acrylamide gel electrophoresis or anion-exchange HPLC as described in Pearson &Reanier, J. Chrom. 255: 137-149 (1983) .

Recombinant production is an effective means to obtain peptides of this disclosure, particularly those of relatively large molecular weight, for example, a fusion peptide of a HER-2/Neu epitope and a GM-CSF. The sequence of a polynucleotide encoding a peptide of this disclosure, and synthetic oligonucleotides can be verified after cloning or subcloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al., Gene 16: 21-26 (1981) .

B. Construction of an Expression Cassette

i. Obtaining a Polynucleotide Sequence Encoding a Peptide

A polynucleotide sequence encoding a peptide of this disclosure can be obtained by chemical synthesis, or can be purchased from a commercial supplier, which may then be further manipulated using standard techniques of molecular cloning.

ii. Modification of Nucleic Acids for Preferred Codon Usage in Host Organism

The polynucleotide sequence encoding a peptide of this disclosure can be optionally altered to coincide with the preferred codon usage of a particular host. For example, the preferred codon usage of one strain of bacterial cells can be used to derive a polynucleotide that encodes a peptide of the disclosure and includes the codons favored by this strain. The frequency of preferred codon usage exhibited by a host cell can be calculated by averaging frequency of preferred codon usage in a large number of genes expressed by the host cell (e.g., calculation service is available from web site of the Kazusa DNA Research Institute, Japan) . This analysis is preferably limited to genes that are highly expressed by the host cell.

At the completion of modification, the coding sequences are verified by sequencing and are then subcloned into an appropriate expression vector for recombinant production of the peptides of this disclosure.

Following verification of the coding sequence, the peptide of the present disclosure can be produced using routine techniques in the field of recombinant genetics.

C. Expression Systems

To obtain high level expression of a nucleic acid encoding a peptide of the present disclosure, one typically subclones a polynucleotide encoding the peptide into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator and a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook and Russell, supra, and Ausubel et al., supra. Bacterial expression systems for expressing a peptide of this disclosure are available in, e.g., E. coli, Bacillus sp., Salmonella, and Caulobacter. Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.

In one embodiment, the eukaryotic expression vector is an adenoviral vector, an adeno-associated vector, a retroviral vector, or a yeast artificial chromosome (YAC) . In some embodiments, the eukaryotic (e.g., mammalian) expression vector is a pEAK10, pEAK12, pEAK13, pCDNA3.0, pCDNA4.0, pCDM7, pCDM8, pCDM10, pCDM12. In some embodiments, the promoter is CMV, EF1α, or SV40.

For instance, the expression system comprises a promoter operably linked to the peptide nucleic acid (e.g., cDNA) sequence such that it is under control of a promoter. The term "promoter" describes the combination of the promoter (RNA polymerase binding site) and operators. The promoter may function in vivo or in a cell-free system. Any number of promoters may be used depending on the needs and preferences of the practitioner. Promoters for controlling recombinant protein, (e.g., peptide) expression can be any promoter for any DNA dependent RNA polymerase, for example. Generally a promoter (e.g., T7, T3, and SP6 RNA promoters and compatible RNA polymerases) is selected for in vitro expression for producing recombinant protein in a bacterial system, e.g., E. coli, which usually requires the molecular inducer isopropyl-β-D-thiogalactoside (IPTG) for regulating the promoter’s transcriptional activity. In some cases, a modified Self-Inducible Expression system that utilizes lactose as an inducer may be used, as described in Briand et al, A self-inducible heterologous protein expression system in Escherichia coli. Sci Rep 6, 33037 (2016) .

The promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter is optionally positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically includes a transcription unit or expression cassette that contains all the additional elements required for the expression of a peptide of this disclosure in host cells. A typical expression cassette thus contains a promoter operably linked to the polynucleotide sequence encoding the peptide and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. The nucleic acid sequence encoding the peptide is typically linked to a cleavable signal peptide sequence to promote secretion of the peptide by the transformed cell. Such signal peptides include, among others, the signal peptides from tissue plasminogen activator, insulin, and neuron growth factor, and juvenile hormone esterase of Heliothis virescens. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene (e.g., encoding the heterologous polypeptide) , introns with functional splice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.

The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as GST and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc.

Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A ⁺, pMTO10/A ⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as a baculovirus vector in insect cells, with a polynucleotide sequence encoding the peptide of this disclosure under the direction of the polyhedrin promoter or other strong baculovirus promoters.

The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences. The particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable. The prokaryotic sequences are optionally chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary. Similar to antibiotic resistance selection markers, metabolic selection markers based on known metabolic pathways may also be used as a means for selecting transformed host cells.

When periplasmic expression of a recombinant protein (e.g., a peptide of the present disclosure) is desired, the expression vector further comprises a sequence encoding a secretion signal, such as the E. coli OppA (Periplasmic Oligopeptide Binding Protein) secretion signal or a modified version thereof, which is directly connected to 5'of the coding sequence of the protein to be expressed. This signal sequence directs the recombinant protein produced in cytoplasm through the cell membrane into the periplasmic space. The expression vector may further comprise a coding sequence for signal peptidase 1, which is capable of enzymatically cleaving the signal sequence when the recombinant protein is entering the periplasmic space. More detailed description for periplasmic production of a recombinant protein can be found in, e.g., Gray et al., Gene 39: 247-254 (1985) , U.S. Patent Nos. 6,160,089 and 6,436,674.

D. Transfection Methods

Standard transfection methods are used to produce bacterial, mammalian, yeast, insect, or plant cell lines that express large quantities of a peptide of this disclosure, which are then purified using standard techniques (see, e.g., Colley et al., J. Biol. Chem. 264: 17619-17622 (1989) ; Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990) ) . Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J. Bact. 132: 349-351 (1977) ; Clark-Curtiss &Curtiss, Methods in Enzymology 101: 347-362 (Wu et al., eds, 1983) .

Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell (see, e.g., Sambrook and Russell, supra) . It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the peptide of this disclosure.

E. Detection of Recombinant Expression of a Peptide in Host Cells

After the expression vector is introduced into appropriate host cells, the transfected cells are cultured under conditions favoring expression of the peptide of this disclosure. The cells are then screened for the expression of the recombinant peptide, which is subsequently recovered from the culture using standard techniques (see, e.g., Scopes, Protein Purification: Principles and Practice (1982) ; U.S. Patent No. 4,673,641; Ausubel et al., supra; and Sambrook and Russell, supra) .

Several general methods for screening gene expression are well known among those skilled in the art. First, gene expression can be detected at the nucleic acid level. A variety of methods of specific DNA and RNA measurement using nucleic acid hybridization techniques are commonly used (e.g., Sambrook and Russell, supra) . Some methods involve an electrophoretic separation (e.g., Southern blot for detecting DNA and Northern blot for detecting RNA) , but detection of DNA or RNA can be carried out without electrophoresis as well (such as by dot blot) . The presence of nucleic acid encoding a peptide of this disclosure in transfected cells can also be detected by PCR or RT-PCR using sequence-specific primers.

Second, gene expression can be detected at the polypeptide level. Various immunological assays are routinely used by those skilled in the art to measure the level of a gene product, particularly using polyclonal or monoclonal antibodies that react specifically with a peptide of the present disclosure, particularly one containing a sufficiently large heterolougs polypeptide (e.g., Harlow and Lane, Antibodies, A Laboratory Manual, Chapter 14, Cold Spring Harbor, 1988; Kohler and Milstein, Nature, 256: 495-497 (1975) ) . Such techniques require antibody preparation by selecting antibodies with high specificity against the peptide or an antigenic portion thereof. The methods of raising polyclonal and monoclonal antibodies are well established and their descriptions can be found in the literature, see, e.g., Harlow and Lane, supra; Kohler and Milstein, Eur. J. Immunol., 6: 511-519 (1976) .

F. Purification of Peptides

i. Purification of Chemically Synthesized Peptides

Purification of synthetic peptides is accomplished using various methods of chromatography, such as reverse phase HPLC, gel permeation, ion exchange, size exclusion, affinity, partition, or countercurrent distribution. The choices of appropriate matrices and buffers are well known in the art.

ii. Purification of Chemically Synthesized Peptides

1. Purification of Peptides from Bacterial Inclusion Bodies

a. Solubility Fractionation

Often as an initial step, and if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest, e.g., a peptide of the present disclosure. The preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol is to add saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This will precipitate the most hydrophobic proteins. The precipitate is discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, through either dialysis or diafiltration. Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.

b. Size Differential Filtration

Based on a calculated molecular weight, a protein of greater and lesser size can be isolated using ultrafiltration through membranes of different pore sizes (for example, Amicon or Millipore membranes) . As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of a protein of interest, e.g., a peptide of the present disclosure. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the peptide of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed as described below.

c. Column Chromatography

A protein of interest (such as a peptide of the present disclosure) can also be separated from other proteins on the basis of its size, net surface charge, hydrophobicity, or affinity for ligands. In addition, antibodies raised against a peptide of this disclosure can be conjugated to column matrices and the peptide immunopurified. All of these methods are well known in the art.

It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech) .

When a peptide of the present disclosure is produced recombinantly by transformed bacteria in large amounts, typically after promoter induction, although expression can be constitutive, the peptides may form insoluble aggregates. There are several protocols that are suitable for purification of protein inclusion bodies. For example, purification of aggregate proteins (hereinafter referred to as inclusion bodies) typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells, e.g., by incubation in a buffer of about 100-150 μg/ml lysozyme and 0.1%Nonidet P40, a non-ionic detergent. The cell suspension can be ground using a Polytron grinder (Brinkman Instruments, Westbury, NY) . Alternatively, the cells can be sonicated on ice. Alternate methods of lysing bacteria are described in Ausubel et al. and Sambrook and Russell, both supra, and will be apparent to those of skill in the art.

The cell suspension is generally centrifuged and the pellet containing the inclusion bodies resuspended in buffer which does not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2) , l mM EDTA, 150 mM NaCl and 2%Triton-X 100, a non-ionic detergent. It may be necessary to repeat the wash step to remove as much cellular debris as possible. The remaining pellet of inclusion bodies may be resuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl) . Other appropriate buffers will be apparent to those of skill in the art.

Following the washing step, the inclusion bodies are solubilized by the addition of a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each having one of these properties) . The proteins that formed the inclusion bodies may then be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to, urea (from about 4 M to about 8 M) , formamide (at least about 80%, volume/volume basis) , and guanidine hydrochloride (from about 4 M to about 8 M) . Some solvents that are capable of solubilizing aggregate-forming proteins, such as SDS (sodium dodecyl sulfate) and 70%formic acid, may be inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity. Although guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of the immunologically and/or biologically active protein of interest. After solubilization, the protein can be separated from other bacterial proteins by standard separation techniques. For further description of purifying recombinant polypeptides from bacterial inclusion body, see, e.g., Patra et al., Protein Expression and Purification 18: 182-190 (2000) .

Alternatively, it is possible to purify recombinant polypeptides, e.g., a peptide of this disclosure, from bacterial periplasm. Where the recombinant polypeptide is exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those of skill in the art (see e.g., Ausubel et al., supra) . To isolate recombinant peptides from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20%sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO ₄ and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged and the supernatant decanted and saved. The recombinant peptides present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.

2. Standard Protein Separation Techniques for Purification

When a recombinant polypeptide, e.g., a peptide of the present disclosure, is expressed in host cells in a soluble form, its purification can follow the standard protein purification procedure described below. This standard purification procedure is also suitable for purifying peptides obtained from chemical synthesis.

G. Confirmation of Peptide Sequence

The amino acid sequence of a peptide of this disclosure can be confirmed by a number of well established methods. For example, the conventional method of Edman degradation can be used to determine the amino acid sequence of a peptide. Several variations of sequencing methods based on Edman degradation, including microsequencing, and methods based on mass spectrometry are also frequently used for this purpose.

H. Modification of Peptides

The peptides of the present disclosure can be modified to achieve more desirable properties. The design of chemically modified peptides and peptide mimics that are resistant to degradation by proteolytic enzymes or have improved solubility or binding ability is well known.

Modified amino acids or chemical derivatives of the T cell or B cell peptides or fusion peptides of this disclosure may contain additional chemical moieties of modified amino acids not normally a part of the T cell or B cell protein. Covalent modifications of the peptides are within the scope of the present disclosure. Such modifications may be introduced into a peptide by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. The following examples of chemical derivatives are provided by way of illustration and not by way of limitation.

The design of peptide mimics which are resistant to degradation by proteolytic enzymes is known to those skilled in the art. See e.g., Sawyer, Structure-Based Drug Design, P. Verapandia, Ed., N.Y. (1997) ; U.S. Patent Nos. 5,552,534 and 5,550,251. Both peptide backbone and side chain modifications may be used in designing secondary structure mimicry. Possible modifications include substitution of D-amino acids, N ^α-Me-amino acids, C _α-Me-amino acids, and dehydroamino acids. To this date, a variety of secondary structure mimetics have been designed and incorporated in peptides or peptidomimetics.

Other modifications include substitution of a natural amino acid with an unnatural hydroxylated amino acid, substitution of the carboxy groups in acidic amino acids with nitrile derivatives, substitution of the hydroxyl groups in basic amino acids with alkyl groups, or substitution of methionine with methionine sulfoxide. In addition, an amino acid of a HER-2/Neu peptide or a fusion peptide of this disclosure can be replaced by the same amino acid but of the opposite chirality, i.e., a naturally-occurring L-amino acid may be replaced by its D-configuration.

I. Recombinant Proteins and Synthetic Peptides

Full length recombinant whole or variants thereof of SARS-CoV or SARS-CoV-2 virus and/or other coronavirous, or recombinant functional proteins, e.g., S protein, N protein, M protein, from SARS-CoV or SARS-CoV-2, and/or other coronavirous may be generated and purified using technologies known in the field. For example, full length SARS-CoV-2 can be generated based on “transformation-associated recombination” (TAR) in yeast (Thao et al. (2020) , “Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform, ” . bioRxiv, 2020.02.21.959817) . As an example, synthetic proteins (e.g., defined as >35 amino acids) and peptides from the SARS-CoV or SARS-CoV-2 recombinant binding domain (RBD) may be synthesized in the Peptide Core at Los Alamos National Laboratory. Positive control rabbit serum (polyclonal, against SARS/SARS-CoV-2 Coronavirus spike protein subunit 1) is Invitrogen PA5-81795. See e.g., Schein et al., 2021. “Synthetic proteins for COVID-19 diagnostics. ” Peptides. 143: 170583. doi: 10.1016/j. peptides. 2021.170583.

In some embodiments, provided herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical) of a reference protein having a length of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or longer than 500 amino acids. In another example, any protein that includes a stretch of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 (contiguous) amino acids that are 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%identical to any of the sequences described herein can be utilized in accordance with the disclosure. In some embodiments, a polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided herein or referenced herein. In another example, any protein that includes a stretch of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids that are greater than 40%, 50, 60%, 70%, 80%, 90%, 95%, or 100%identical to any of the sequences described herein, wherein the protein has a stretch of 5, 10, 15, 20, 25, or 30 amino acids that are less than 95%, 90%, 85%, 80%, 75%, 70%, 65%to 60%identical to any of the sequences described herein can be utilized in accordance with the disclosure.

IV. FUSING T CELL AND/OR B CELL EPITOPE WITH A HETEROLOGOUS POLYPEPTIDE

In one aspect of this disclosure, a peptide corresponding to a SARS-CoV or SARS-CoV-2 promiscuous T cell epitope or B cell epitope is attached to a heterologous polypeptide via a covalent bond to form a fusion peptide, such that the ability of the SARS-CoV or SARS-CoV-2 epitope to induce a T cell or B cell response is enhanced. Usually, this covalent bond is a peptide bond and the SARS-CoV and/or SARS-CoV-2 epitope and the heterologous polypeptide form a new polypeptide. This peptide bond may be a direct peptide bond between the SARS-CoV and/or SARS-CoV-2 epitope and the heterologous polypeptide, or it may be an indirect peptide bond provided by way of a peptide linker between the SARS-CoV or SARS-CoV-2 epitope and the heterologous polypeptide. As an example, the SARS-CoV and/or SARS-CoV-2 epitope may be linked to a detecting molecule such as a GFP, RFP, dsRed, or the like. As another example, the SARS-CoV and/or SARS-CoV-2 epitope may be linked to an affinity tag such as a six histidine (6His) on the N-terminal or the C-terminal, or the like.

Other covalent bonds are also suitable for the purpose of fusing the SARS-CoV or SARS-CoV-2 peptide with the heterologous polypeptide. For instance, a functional group (such as a non- terminal amine group, a non-terminal carboxylic acid group, a hydroxyl group, and a sulfhydryl group) of one peptide may easily react with a functional group of the other peptide and establish a covalent bond, other than a peptide bond, that conjugates the two peptides. A covalent connection between a peptide of a SARS-CoV or SARS-CoV-2 epitope and a heterologous polypeptide can also be provided by way of a linker molecule with suitable functional group (s) . Such a linker molecule can be a peptide linker or a non-peptide linker. A linker may be derivatized to expose or to attach additional reactive functional groups prior to conjugation. The derivatization may involve attachment of any of a number of molecules such as those available from Pierce Chemical Company, Rockford, Illinois.

V. FUNCTIONAL ASSAYS

Adaptive immune response is mediated by the T lymphocytes and B lymphocytes. T lymphocytes express T-cell receptors (TCR) and detect short peptides produced by a proteolytic machinery and presented by major histocompatibility complex (MHC) molecules at the cell surface. (Bercovici et al., 2000) . Through antigen processing, T lymphocytes are able to detect foreign peptides synthesized by infected cells. T cells which express CD8 molecules have the capacity to lyse directly the target cells. A subset of CD4 ⁺ T lymphocytes is specialized in regulating the immune response via cytokine secretion and activation of the antigen-presenting cells (APC) . Chromium release assays and limiting-dilution analyses have been commonly used to measure specific T-cell responses. Additionally, various assays are available for immune monitoring of specific T-cell responses. These various assays are schematically divided into functional assays, which measure the secretion of a particular cytokine (ELISPOT and intracellular cytokines) ; assays which assess the specificity of the T cells irrespective of their functionality and which are based on structural features of the TCR (tetramers and immunoscope) ; and assays aimed at detecting T-cell precursors by amplifying cells that proliferate in response to antigenic stimulation.

A SARS-CoV or SARS-CoV-2 epitope of this disclosure (or a fusion peptide comprising a SARS-CoV or SARS-CoV-2 epitope and a heterologous polypeptide) is useful for its capability to induce a T cell immune response specific to a SARS-CoV or SARS-CoV-2 protein, when the epitope is presented by an APC that may have one of a HLA-A, HLA-B, and/or HLA-DR allele. Various functional assays can be used to confirm the ability of a SARS-CoV or SARS-CoV-2 epitope to induce such a SARS-CoV or SARS-CoV-2 specific T cell immune response in a promiscuous manner with regard to antigen presenting cells of different HLA alleles, including proliferation assay and flow cytometry assays detecting the binding between a T cell receptor and a peptide epitope or the production of cytokines by T cells.

As an illustration, the function assay ELISPOT may be used for this purpose. The ELISPOT (enzyme-linked immunospot) technique detects T cells that secrete a given cytokine (e.g., gamma interferon [IFN-γ] ) in response to an antigenic stimulation (e.g., an antigen presented by SARS-CoV or SARS-CoV-2) . Briefly, T cells are cultured with antigen-presenting cells in wells which have been coated with anti-IFN-γ antibodies. The secreted IFN-γ is captured by the coated antibody and then revealed with a second antibody coupled to a chromogenic substrate. Thus, locally secreted cytokine molecules form spots, with each spot corresponding to one IFN-γ-secreting cell. The number of spots allows one to determine the frequency of IFN-γ-secreting cells specific for a given antigen in the analyzed sample. The ELISPOT assay has also been described for the detection of tumor necrosis factor alpha, interleukin-4 (IL-4) , IL-5, IL-6, IL-10, IL-12, granulocyte-macrophage colony-stimulating factor, and granzyme B-secreting lymphocytes.

As another illustration, the structural assay tetramers may be used for this purpose. T cells recognize short peptides presented by MHC molecules through their clonotypic TCR. Tetramers of MHC class I-peptide complexes have been used in cytometry to enumerate, characterize, and purify peptide-specific CD8 cells. The heavy and light chains of the MHC are produced in Escherichia coli, solubilized in urea, and refolded in vitro in the presence of high concentrations of the antigenic peptide. The refolded complexes are purified by gel filtration, and a single biotin is added at the C-terminal end of the heavy chain using the bacterial BirA enzyme. Incubation with fluorescent streptavidin yields tetramers which can be used like any clonotypic antibody. Tetramers of MHC class II molecules may also be produced and used to analyze CD4 ⁺ T-cell responses.

B lymphocytes recognize intact proteins and produce immunoglobulins (Ig) . (Chaplin, 2010) . B cell response is activated in two pathways: T cell-dependent and T cell-independent. In a T-cell dependent manner, antigens that activate T cells as well as B cells establish Ig responses in which T cells provide ‘help’ for the B cells to mature. T cell-independent B cell activation occurs without the assistance of T cell co-stimulatory proteins. In the absence of co-stimulators, monomeric antigens are unable to activate B cells. Polymeric antigens with a repeating structure, in contrast, are able to activate B cells, probably because they can crosslink and cluster Ig molecules on the B cell surface. T cell-independent antigens include bacterial lipopolysaccharide (LPS) , certain other polymeric polysaccharides, and certain polymeric proteins.

Several techniques have been used for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR) -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. (Jim Boonyaratanakornkit and Justin Taylor, 2019) . The ELISPOT assay is particularly suitable for the purpose of detecting B cell epitope response to SARS-CoV and/or SARS-CoV-2 infection in relation to the disclosure described herein.

Another example for confirming B cell immune response specific to a SARS-CoV or SARS-CoV-2 protein is flow cytometry-based analysis of antigen-specific B cells. This technique is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen may be set at a ratio ≤1 biotin to 1 antigen. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression.

VI. METHOD FOR ELICITING AN IMMUNE RESPONSE IN A SUBJECT

The present disclosure further provides a method for eliciting an immune response in a subject in need thereof such as at risk of exposure to SARS-CoV or SARS-CoV-2 infection. According to the CDC, the term exposure refers to contact with infectious agents (bacteria or virus, e.g., SARS-CoV or SARS-CoV-2) in a manner that promotes transmission and increases the likihood of disease. For instance, a subject who has close contact with someone who has COVID-19 is considered at risk of exposure to the disease. The term close contact refers to within 6 feet of someone for a cumulative total of 15 minites or more over a 24-hour period. Subjects who have underlying medical conditions (e.g., immune-comprimised due to treatment of immunosuppressants, under chemotherapy, prior heart and cardio diseases, prior pulmonary diseases, cancer, diabetes, obesity, etc. ) , age (e.g., 65 or over) , geneteic predisposition, and/or pregnant or recently pregnant. The subject may be at risk for severe illness when contracted with COVID-19 such that the subject may need hospitalization, intensive care, a ventilator to help them breathe or they may even die. Elicitation an immune response in a subject may provide prophylactic and therapeutic applications. The method includes the following steps: first, lymphocytes including at least a T cell epitope, and/or at least a B cell epitope, and/or optionally an antigen-presenting cell are obtained from a patient. Suitable samples that yield such lymphocytes include blood, and lymph nodes or lymphatic fluids. Second, at least a T cell epitope, and/or at least a B cell epitope, and/or optionally an antigen-presenting cell are exposed to a SARS-CoV or SARS-CoV-2 peptide (or a fusion peptide comprising the SARS-CoV or SARS-CoV-2 peptide and a heterologous peptide) of this disclosure under conditions that would allow, e.g., proper presentation of a T cell epitope by the antigen-presenting cell to the T cell. Third, signs of a T cell response and/or B cell response is measured in vitro by means well known in the art such as ELISPOT, ELISA, proliferation assay, or flow cytometry. When a T cell response and/or B cell response is detected by any of these methods, it can be concluded that there exists a T cell and/or a B cell immune response specific to a SARS-CoV or SARS-CoV-2 protein in the patient.

A. Vaccines

In certain preferred embodiments of the present disclosure, vaccines are provided. The vaccines will generally comprise a peptide comprising one or more T cell and/or B cell epitopes derived from the S protein, N protein, or full length or a fragment thereof, of a SARS-CoV and/or SARS-CoV-2, such as those discussed above, in combination with an immunostimulant. An immunostimulant may be any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen. Examples of immunostimulants include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes (into which the compound is incorporated; see, e.g., U.S. Patent No. 4,235,877) . Vaccine preparation is generally described in, for example, Powell &Newman, eds., Vaccine Design (the subunit and adjuvant approach) (1995) . Pharmaceutical compositions and vaccines within the scope of the present disclosure may also contain other compounds, which may be biologically active or inactive. For example, one or more T cell and/or B cell epitopes derived from immunogenic portions of other coronavirus antigens (e.g., MERS-CoV, HCoV-HKU1, HCoV-229E, HCoV-OC43, and HCoV- NL63) may be present, either incorporated into a fusion polypeptide or as a separate compound, within the composition or vaccine. Accordingly, a pharmaceutical vaccine formulation may comprise the petide comprising the one or more T cell and/or B cell epitopes derived from the S protein, N protein, or full length or a fragment thereof, of a SARS-CoV and/or SARS-CoV-2, one or more adjuvnts, pharmaceutically-acceptable carriers or other ingredients including immunological adjuvants routinely provided in vaccine formulation. Suitable adjuvants are described in detail below.

In some embodiments, the peptide comprising the one or more T cell and/or B cell epitopes derived from the S protein, N protein, or full length or a fragment thereof, of a SARS-CoV and/or SARS-CoV-2 is biologically produced such as using an expression cassette in a host cell. Short to medium length peptides, for example peptides that do no require specific folding, can be chemically synthesized. Large scale production of chemically synthesized peptides may be used for manufacturing large quantities of peptide vaccines. See e.g., Bray, B.L., 2003. Large-scale manufacture of peptide therapeutics by chemical synthesis. Nature Reviews Drug Discovery, 2 (7) , pp. 587-593.

In some embodiments, the peptide comprising the one or more T cell and/or B cell epitopes derived from the S protein, N protein, or full length or a fragment thereof, of a SARS-CoV and/or SARS-CoV-2 is a cationic peptide. As described herein, a “cationic peptide” refers to a peptide that is positively charged at a pH in the range of 5.0 to 8.0. The net charge on the peptide or peptide cocktails is calculated by assigning a +1 charge for each lysine (K) , arginine (R) or histidine (H) , a -1 charge for each aspartic acid (D) or glutamic acid (E) and a charge of 0 for the other amino acid within the sequence. The charge contributions from the N-terminal amine (+1) and C-terminal carboxylate (-1) end groups of each peptide effectively cancel each other when unsubstituted. The charges are summed for each peptide and expressed as the net average charge. A suitable peptide has a net average positive charge of +1. Preferably, the peptide has a net positive charge in the range that is larger than +2.

In some embodiments, the peptide comprising the one or more T cell and/or B cell epitopes derived from the S protein, N protein, or full length or a fragment thereof, of a SARS-CoV and/or SARS-CoV-2 is an anionic peptide. As described herein, an “anionic molecule” refers to a molecule that is negatively charged at a pH in the range of 5.0-8.0. The net negative charge on the oligomer or polymer is calculated by assigning a -1 charge for each phosphodiester or phosphorothioate group in the oligomer.

Illustrative vaccines may contain polynucleotides encoding one or more of the polypeptides (e.g., T cell and/or B cell epitopes derived from one or more immunogenic portions of a SARS-CoV, SARS-CoV-2, and/or other coronaviruses) as described above, such that the polypeptide is generated in situ. As noted above, the polynucleotides may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15: 143-198 (1998) , and references cited therein. Appropriate nucleic acid expression systems contain the necessary polynucleotides sequences for expression in the patient (such as a suitable promoter and terminating signal) . Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope. In one embodiment, the polynucleotides may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus) , which may involve the use of a non-pathogenic (defective) , replication competent virus. Suitable systems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86: 317-321 (1989) ; Flexner et al., Ann. N.Y. Acad. Sci. 569: 86-103 (1989) ; Flexner et al., Vaccine 8: 17-21 (1990) ; U.S. Patent Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Patent No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6: 616-627 (1988) ; Rosenfeld et al., Science 252: 431-434 (1991) ; Kolls et al., Proc. Natl. Acad. Sci. USA 91: 215-219 (1994) ; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90: 11498-11502 (1993) ; Guzman et al., Circulation 88: 2838-2848 (1993) ; and Guzman et al., Cir. Res. 73: 1202-1207 (1993) . Techniques for incorporating polynucleotides into such expression systems are well known to those of ordinary skill in the art. The polynucleotides may also be “naked, ” as described, for example, in Ulmer et al., Science 259: 1745-1749 (1993) and reviewed by Cohen, Science 259: 1691-1692 (1993) . The uptake of naked polynucleotides may be increased by coating the polynucleotides onto biodegradable beads, which are efficiently transported into the cells. It will be apparent that a vaccine may comprise both a polynucleotide and a polypeptide component. Such vaccines may provide for an enhanced immune response.

In some embodiments, the polynucleotides is formulated in lipid nanoparticles.. In some embodiments, the lipid nanoparticle is a mucus penetrating lipid nanoparticle. In some embodiments, the lipid nanoparticle is a solid lipid nanoparticle. Formulations for polynucleotides in lipid nanoparticles are discussed in U.S. Patent No: 10272150, which is incorporated herein in its entirety.

It will be apparent that a vaccine may contain pharmaceutically acceptable salts of the polynucleotides and polypeptides provided herein. Such salts may be prepared from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts) .

In some embodiments the peptide vaccines and/or the polynucleotide vaccines of the disclosure are superior to conventional vaccines by a factor of at least 2 fold, 5 fold, 10 fold, 20 fold, 40 fold, 50 fold, 100 fold, 500 fold or 1,000 fold.

While any suitable carrier known to those of ordinary skill in the art may be employed in the vaccine compositions of this disclosure, the type of carrier will vary depending on the mode of administration. Compositions of the present disclosure may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactate polyglycolate) may also be employed as carriers for the pharmaceutical compositions of this disclosure. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252. One may also employ a carrier comprising the particulate-protein complexes described in U.S. Patent No. 5,928,647, which are capable of inducing a class I-restricted cytotoxic T lymphocyte responses in a host.

Such compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline) , carbohydrates (e.g., glucose, mannose, sucrose or dextrans) , mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) , solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, compositions of the present disclosure may be formulated as a lyophilizate. Compounds may also be encapsulated within liposomes using well known technology.

B. Adjuvants

Any of a variety of immunostimulants may be employed in the vaccines of this disclosure. For example, an adjuvant or an immune potentiator may be included. An adjuvant may act as a co-signal to prime T-cells and/or B-cells and/or NK cells as to the existence of an infection. Adjuvants useful in the present disclosure may include, but are not limited to, natural or synthetic. They may be organic or inorganic. In some embodiments, adjuvants useful in the present disclosure include adjuvants for vaccines (e.g., influenza vaccines) as shown in Table 48. Adjuvants for DNA nucleic acid vaccines (DNA) have been disclosed in, for example, Kobiyama, et al Vaccines, 2013, 1 (3) , 278-292, the contents of which are incorporated herein by reference in their entirety.

Table 48. Adjuvants.

Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium species or Mycobacterium derived proteins. For example, delipidated, deglycolipidated M. vaccae ( “pVac” ) can be used. Suitable adjuvants are commercially available as, for example, Freund’s Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI) ; Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ) ; AS-2 and derivatives thereof (SmithKline Beecham, Philadelphia, PA) ; CWS, TDM, Leif, aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

Selection of appropriate adjuvants will be evident to one of ordinary skill in the art. Specific adjuvants may include, without limitation, cationic liposome-DNA complex JVRS-100, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, aluminum potassium sulfate adjuvant, alhydrogel, ISCOM (s) ^TM, Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, CpG DNA Vaccine Adjuvant, Cholera toxin, Cholera toxin B subunit, Liposomes, Saponin Vaccine Adjuvant, DDA Adjuvant, Squalene-based Adjuvants, Etx B subunit Adjuvant, IL-12 Vaccine Adjuvant, LTK63 Vaccine Mutant Adjuvant, TiterMax Gold Adjuvant, Ribi Vaccine Adjuvant, Corynebacterium-derived P40 Vaccine Adjuvant, MPL ^TM Adjuvant, AS04, AS02, Lipopolysaccharide Vaccine Adjuvant, Muramyl Dipeptide Adjuvant, CRL1005, Killed Corynebacterium parvum Vaccine Adjuvant, Montanide ISA 51, Bordetella pertussis component Vaccine Adjuvant, Cationic Liposomal Vaccine Adjuvant, Adamantylamide Dipeptide Vaccine Adjuvant, Arlacel A, VSA-3 Adjuvant, Aluminum vaccine adjuvant, Polygen Vaccine Adjuvant, Adjumer ^TM, Algal Glucan, Bay R1005,

Stearyl Tyrosine, Specol, Algammulin,

Calcium Phosphate Gel, CTA1-DD gene fusion protein, DOC/Alum Complex, Gamma Inulin, Gerbu Adjuvant, GM-CSF, GMDP, Recombinant hIFN-gammallnterferon-g, Interleukin-1β, Interleukin-2, Interleukin-7, Sclavo peptide, Rehydragel LV, Rehydragel HPA, Loxoribine, MF59, MTP-PE Liposomes, Murametide, Murapalmitine, D-Murapalmitine, NAGO, Non-Ionic Surfactant Vesicles, PMMA, Protein Cochleates, QS-21, SPT (Antigen Formulation) , nanoemulsion vaccine adjuvant, AS03, Quil-A vaccine adjuvant, LTR192G Vaccine Adjuvant, E. coli heat-labile toxin, LT, amorphous aluminum hydroxyphosphate sulfate adjuvant, Calcium phosphate vaccine adjuvant, Montanide Incomplete Seppic Adjuvant, Imiquimod, Resiquimod, AF03, Flagellin, Poly (I: C) ,

Abisco-100 vaccine adjuvant, Albumin-heparin microparticles vaccine adjuvant, AS-2 vaccine adjuvant, B7-2 vaccine adjuvant, DHEA vaccine adjuvant, Immunoliposomes Containing Antibodies to Costimulatory Molecules, Sendai Proteoliposomes, Sendai-containing Lipid Matrices, Threonyl muramyl dipeptide (TMDP) , Ty Particles vaccine adjuvant, Bupivacaine vaccine adjuvant, DL-PGL (Polyester poly (DL-lactide-co-glycolide) ) vaccine adjuvant, IL-15 vaccine adjuvant, LTK72 vaccine adjuvant, MPL-SE vaccine adjuvant, non-toxic mutant E112K of Cholera Toxin mCT-E112K, and/or Matrix-S.

Other adjuvants which may be co-administered with the polypeptides of the disclosure include, but are not limited to interferons, TNF-alpha, TNF-beta, chemokines such as CCL21, eotaxin, HMGB1, SA100-8alpha, GCSF, GMCSF, granulysin, lactoferrin, ovalbumin, CD-40L, CD28 agonists, PD-1, soluble PD1, L1 or L2, or interleukins such as IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-21, IL-23, IL-15, IL-17, and IL-18.

Within the vaccines provided herein, the adjuvant composition is preferably designed to induce an immune response predominantly of the Th1 type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a patient will support an immune response that includes Th1-and Th2-type responses. Within a preferred embodiment, in which a response is predominantly Th1-type, the level of Th1-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann &Coffman, Ann. Rev. Immunol. 7: 145-173 (1989) .

Preferred adjuvants for use in eliciting a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) , together with an aluminum salt. MPL adjuvants are available from Corixa Corporation (Seattle, WA; see US Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094) . CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Th1 response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Patent Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273: 352 (1996) . Another preferred adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, MA) ; Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins . Other preferred formulations include more than one saponin in the adjuvant combinations of the present disclosure, for example combinations of at least two of the following group comprising QS21, QS7, Quil A, β-escin, or digitonin.

Alternatively the saponin formulations may be combined with vaccine vehicles composed of chitosan or other polycationic polymers, polylactide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc. The saponins may also be formulated in the presence of cholesterol to form particulate structures such as liposomes or ISCOMs. Furthermore, the saponins may be formulated together with a polyoxyethylene ether or ester, in either a non-particulate solution or suspension, or in a particulate structure such as a paucilamelar liposome or ISCOM. The saponins may also be formulated with excipients such as Carbopol ^R to increase viscosity, or may be formulated in a dry powder form with a powder excipient such as lactose.

In one embodiment, the adjuvant system includes the combination of a monophosphoryl lipid A and a saponin derivative, such as the combination of QS21 and

adjuvant, as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion and tocopherol. Another particularly preferred adjuvant formulation employing QS21,

adjuvant and tocopherol in an oil-in-water emulsion is described in WO 95/17210.

Another enhanced adjuvant system involves the combination of a CpG-containing oligonucleotide and a saponin derivative particularly the combination of CpG and QS21 as disclosed in WO 00/09159. Preferably the formulation additionally comprises an oil in water emulsion and tocopherol.

Other adjuvants include Montanide ISA 720 (Seppic, France) , SAF-1 (Chiron, California, United States) , ISCOMS (CSL) , MF-59 (Chiron) , the SBAS series of adjuvants (e.g., SBAS-2, AS2’, AS2, ” SBAS-4, or SBAS6, available from SmithKline Beecham, Rixensart, Belgium) , Detox (Corixa, Hamilton, MT) , RC-529 (Corixa, Hamilton, MT) and other aminoalkyl glucosaminide 4-phosphates (AGPs) , such as those described in pending U.S. Patent Application Serial Nos. 08/853,826 and 09/074, 720, the disclosures of which are incorporated herein by reference in their entireties, and polyoxyethylene ether adjuvants such as those described in WO 99/52549A1.

Other adjuvants include adjuvant molecules of the general formula (I) : HO (CH ₂CH ₂O) _n-A-R, wherein, n is 1-50, A is a bond or –C (O) -, R is C _1-50 alkyl or Phenyl C _1-50 alkyl.

One embodiment of the present disclosure consists of a vaccine formulation comprising a polyoxyethylene ether of general formula (I) , wherein n is between 1 and 50, preferably 4-24, most preferably 9; the R component is C _1-50, preferably C ₄-C ₂₀ alkyl and most preferably C ₁₂ alkyl, and A is a bond. The concentration of the polyoxyethylene ethers should be in the range 0.1-20%, preferably from 0.1-10%, and most preferably in the range 0.1-1%. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12 ^th edition: entry 7717) . These adjuvant molecules are described in WO 99/52549.

The polyoxyethylene ether according to the general formula (I) above may, if desired, be combined with another adjuvant. For example, a preferred adjuvant combination is preferably with CpG as described in the pending UK patent application GB 9820956.2.

Any vaccine provided herein may be prepared using well known methods that result in a combination of antigen, immune response enhancer and a suitable carrier or excipient. The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule, sponge or gel (composed of polysaccharides, for example) that effects a slow release of compound following administration) . Such formulations may generally be prepared using well known technology (see, e.g., Coombes et al., Vaccine 14: 1429-1438 (1996) ) and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. In some embodiments, the vaccine is formulated for induction of systemic or localized mucosal immunity through immunogen entrapment and co-administration with microparticles.

Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. Such carriers include microparticles of poly (lactide-co-glycolide) , polyacrylate, latex, starch, cellulose, dextran and the like. Other delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see, e.g., U.S. Patent No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638) . The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

Vaccines and pharmaceutical compositions may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve sterility of the formulation until use. In general, formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, a vaccine or pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.

VII. KITS

The disclosure provides a variety of kits for conveniently and/or effectively carrying out methods of the present disclosure. Typically kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject (s) . The disclosure provides kits for eliciting an immune response in a subject according to the method of the present disclosure. The kits typically include a container that contains a pharmaceutical composition having an effective amount of SARS-CoV-derived or SARS-CoV-2-derived T cell epitope polypeptides or a function fragment thereof, and/or B cell epitope polypeptides or a function fragment thereof) , and/or the polypeptides optionally further comprising at least one heterologous polypeptide sequence. The kit may comprise nucleic acid sequences encoding the SARS-CoV-derived or SARS-CoV-2-derived T cell and/or B cell polypeptides, and/or the expression cassettes for expressing the T cell and/or B cell polypeptides, and/or the vectors for expressing the expression cassettes. The kit may optionally comprise an additional container containing a therapeutic agent against SARS-CoV or variants thereof, SARS-CoV-2 or variants thereof, and/or other coronavirus or variants (e.g., MERS-CoV, HCoV-HKU1, HCoV-229E, HCoV-OC43, and HCoV-NL63) .

In some embodiments, the present disclosure provides kits comprising the SARS-CoV-and/or SARS-CoV-2-derived T cell and/or B cell epitopes (including any polypeptides, proteins or function fragments thereof) of the disclosure. The T cell and/or B cell epitopes may be derived from one or more functional proteins (e.g., S protein, N protein, or M protein) or full length SARS-CoV or SARS-CoV-2. In some embodiments, the kit further comprises polypeptides comprising one or more T cell and/or B cell epitopes derived from one or more of a SARS-CoV and/or SARS-CoV-2 variants. In some embodiments, the kit further comprises polypeptides comprising one or more T cell and/or B cell epitopes derived from one or more coronaviruses or variants thereof (e.g., MERS-CoV, HCoV-HKU1, HCoV-229E, HCoV-OC43, and HCoV-NL63) . The kit may further comprise packaging and instructions and/or a delivery agent to form a formulation composition. The delivery agent may comprise a saline, a buffered solution, a lipidoid or any delivery agent disclosed herein.

The kits can be for protein or polypeptide production, comprising a polynucleotide comprising a translatable region of one or more T cell and/or B cell epitopes derived from a functional protein (e.g., S protein, N protein, or M protein) or full length SARS-CoV or SARS-CoV-2. In some embodiments, the kit further comprises a polynucleotide comprising a translatable region of one or more T cell and/or B cell epitopes derived from one or more coronaviruses (e.g., MERS-CoV, HCoV-HKU1, HCoV-229E, HCoV-OC43, and HCoV-NL63) . The kit may further comprise packaging and instructions and/or a delivery agent to form a formulation composition. The delivery agent may comprise a saline, a buffered solution, a lipidoid or any delivery agent disclosed herein.

In some embodiments, the kit further comprises devices for administering the polypeptides or a protein or polypeptide product descried herein. For instance, the kit may comprise syringes, needles, and instructions on how to dispense the pharmaceutical composition, including description of the type of patients who may be treated (e.g., subjects who are at risk of having COVID-19 due to individual health condition, pre-existing condition of any kind, compromised immune system, living condition, age, or occupation; subjects who has close contact with someone who has COVID-19) .

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results.

EXAMPLE 1 –PRELIMINARY IDENTIFICATION OF POTENTIAL VACCINE TARGETS FOR THE COVID-19 CORONAVIRUS (SARS-COV-2) BASED ON SARS-COV IMMUNOLOGICAL STUDIES

Introduction

In this study, we sought to gain insights for vaccine design against SARS-CoV-2 by considering the high genetic similarity between SARS-CoV-2 and SARS-CoV, which caused the outbreak in 2003, and leveraging existing immunological studies of SARS-CoV. By screening the experimentally-determined SARS-CoV-derived B cell and T cell epitopes in the immunogenic structural proteins of SARS-CoV, we identified a set of B cell and T cell epitopes derived from the spike (S) and nucleocapsid (N) proteins that map identically to SARS-CoV-2 proteins. As no mutation has been observed in these identified epitopes among the 120 available SARS-CoV-2 sequences (as of 21 February 2020) , immune targeting of these epitopes may potentially offer protection against this novel virus. For the T cell epitopes, we performed a population coverage analysis of the associated MHC alleles and proposed a set of epitopes that is estimated to provide broad coverage globally, as well as in China. Our findings provide a screened set of epitopes that can help guide experimental efforts towards the development of vaccines against SARS-CoV-2.

Worldwide collaborative efforts from scientists are working on this disease and SARS-CoV-2 to develop effective interventions for controlling and preventing it [6–9] .

Coronaviruses are positive-sense single-stranded RNA viruses belonging to the family Coronaviridae. These viruses mostly infect animals, including birds and mammals. In humans, they generally cause mild respiratory infections, such as those observed in the common cold. However, some recent human coronavirus infections have resulted in lethal endemics, which include the SARS (Severe Acute Respiratory Syndrome) and MERS (Middle East Respiratory Syndrome) endemics. Both of these are caused by zoonotic coronaviruses that belong to the genus Betacoronavirus within Coronaviridae. SARS-CoV originated from Southern China and caused an endemic in 2003. A total of 8098 cases of SARS were reported globally, including 774 associated deaths, and an estimated case-fatality rate of 14%–15% [10] . The first case of MERS occurred in Saudi Arabia in 2012. Since then, a total of 2, 494 cases of infection have been reported, including 858 associated deaths, and an estimated high case-fatality rate of 34.4% [11] . While no case of SARS-CoV infection has been reported since 2004, MERS-CoV has been around since 2012 and has caused multiple sporadic outbreaks in different countries.

Like SARS-CoV and MERS-CoV, the recent SARS-CoV-2 belongs to the Betacoronavirus genus [12] . It has a genome size of ～30 kilobases which, like other coronaviruses, encodes for multiple structural and non-structural proteins. The structural proteins include the spike (S) protein, the envelope (E) protein, the membrane (M) protein, and the nucleocapsid (N) protein. With SARS-CoV-2 being discovered very recently, there is currently a lack of immunological information available about the virus (e.g., information about immunogenic epitopes eliciting antibody or T cell responses) . Preliminary studies suggest that SARS-CoV-2 is quite similar to SARS-CoV based on the full-length genome phylogenetic analysis [9, 12] , and the putatively similar cell entry mechanism and human cell receptor usage [9, 13, 14] . Due to this apparent similarity between the two viruses, previous research that has provided an understanding of protective immune responses against SARS-CoV may potentially be leveraged to aid vaccine development for SARS-CoV-2.

Various reports related to SARS-CoV suggest a protective role of both humoral and cell-mediated immune responses. For the former case, antibody responses generated against the S protein, the most exposed protein of SARS-CoV, have been shown to protect from infection in mouse models [15–17] . In addition, multiple studies have shown that antibodies generated against the N protein of SARS-CoV, a highly immunogenic and abundantly expressed protein during infection [18] , were particularly prevalent in SARS-CoV-infected patients [19, 20] . While being effective, the antibody response was found to be short-lived in convalescent SARS-CoV patients [21] . In contrast, T cell responses have been shown to provide long-term protection [21–23] , even up to 11 years post-infection [24] , and thus have also attracted interest for a prospective vaccine against SARS-CoV [reviewed in [25] ] . Among all SARS-CoV proteins, T cell responses against the structural proteins have been found to be the most immunogenic in peripheral blood mononuclear cells of convalescent SARS-CoV patients as compared to the non-structural proteins [26] . Further, of the structural proteins, T cell responses against the S and N proteins have been reported to be the most dominant and long-lasting [27] .

Here, by analyzing available experimentally-determined SARS-CoV-derived B cell epitopes (both linear and discontinuous) and T cell epitopes, we identify and report those that are completely identical and comprise no mutation in the available SARS-CoV-2 sequences (as of 21 February 2020) . These epitopes have the potential, therefore, to elicit a cross-reactive/effective response against SARS-CoV-2. We focused particularly on the epitopes in the S and N structural proteins due to their dominant and long-lasting immune response previously reported against SARS-CoV. For the identified T cell epitopes, we additionally incorporated the information about the associated MHC alleles to provide a list of epitopes that seek to maximize population coverage globally, as well as in China. Our presented results can potentially narrow down the search for potent targets for an effective vaccine against SARS-CoV-2, and help guide experimental studies focused on vaccine development.

Materials and Methods

Acquisition and Processing of Sequence Data. A total of 120 whole genome sequences of SARS-CoV-2 were downloaded on 21 February 2020 from the GISAID database (gisaid. org/CoV2020/) (Table 6) . We excluded sequences that likely had spurious mutations resulting from sequencing errors, as indicated in the comment field of the GISAID data. These nucleotide sequences were aligned to the GenBank reference sequence (accession ID: NC_045512.2) and then translated into amino acid residues according to the coding sequence positions provided along the reference sequence for SARS-CoV-2 proteins (orf1a, orf1b, S, ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N, and ORF10) . These sequences were aligned separately for each protein using the MAFFT multiple sequence alignment program [28] . Reference protein sequences for SARS-CoV and MERS-CoV were obtained following the same procedure from GenBank using the accession IDs NC_004718.3 and NC_019843.3, respectively.

Table 6. Whole genome sequences of SARS-CoV-2 downloaded from theGISAID database.

Acquisition and Filtering of Epitope Data. SARS-CoV-derived B cell and T cell epitopes were searched on the NIAID Virus Pathogen Database and Analysis Resource (ViPR) (www. viprbrc. org/; accessed 21 February 2020) [29] by querying for the virus species name: “Severe acute respiratory syndrome-related coronavirus” from “human” hosts. We limited our search to include only the experimentally-determined epitopes that were associated with at least one positive assay: (i) Positive B cell assays (e.g., enzyme-linked immunosorbent assay (ELISA) -based qualitative binding) for B cell epitopes; and (ii) either positive T cell assays (such as enzyme-linked immune absorbent spot (ELISPOT) or intracellular cytokine staining (ICS) IFN-γ release) , or positive major histocompatibility complex (MHC) binding assays for T cell epitopes. Strictly speaking, the latter set of epitopes, determined using positive MHC binding assays, are antigens which are candidate epitopes, since a T cell response has not been confirmed experimentally. However, for brevity and to be consistent with the terminology used in the ViPR database, we will not make this qualification, and will simply refer to them as epitopes in this study. The number of B cell and T cell epitopes obtained from the database following the above procedure is listed in Table 1.

Table 1. Filtering criteria and corresponding number of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) -derived epitopes obtained from the Virus Pathogen Database and Analysis Resource (ViPR) database.

Population-Coverage-Based T Cell Epitope Selection. Population coverages for sets of T cell epitopes were computed using the tool provided by the Immune Epitope Database (IEDB) (tools. iedb. org/population/; accessed 21 February 2020) [30] . This tool uses the distribution of MHC alleles (with at least 4-digit resolution, e.g., A*02: 01) within a defined population (obtained from allelefrequencies. net/) to estimate the population coverage for a set of T cell epitopes. The estimated population coverage represents the percentage of individuals within the population that are likely to elicit an immune response to at least one T cell epitope from the set. To identify the set of epitopes associated with MHC alleles that would maximize the population coverage, we adopted a greedy approach: (i) We first identified the MHC allele with the highest individual population coverage and initialized the set with their associated epitopes, then (ii) we progressively added epitopes associated with other MHC alleles that resulted in the largest increase of the accumulated population coverage. We stopped when no increase in the accumulated population coverage was observed by adding epitopes associated with any of the remaining MHC alleles.

Constructing the Phylogenetic Tree. We used the publicly available software PASTA v1.6.4 [31] to construct a maximum-likelihood phylogenetic tree of each structural protein using the unique set of sequences in the available data of SARS-CoV, MERS-CoV, and SARS-CoV-2. We additionally included the Zaria Bat coronavirus strain (accession ID: HQ166910.1) to serve as an outgroup. The appropriate parameters for tree estimation are automatically selected in the software based on the provided sequence data. For visualizing the constructed phylogenetic trees, we used the publicly available software Dendroscope v3.6.3 [32] . Each constructed tree was rooted with the outgroup Zaria Bat coronavirus strain, and circular phylogram layout was used

Data and Code Availability. All sequence and immunological data, and all scripts (written in R) for reproducing the results are available online [33] .

Results

Structural Proteins of SARS-CoV-2 Are Genetically Similar to SARS-CoV, but Not to MERS-CoV. SARS-CoV-2 has been observed to be close to SARS-CoV-much more so than MERS-CoV-based on full-length genome phylogenetic analysis [9, 12] . We checked whether this is also true at the level of the individual structural proteins (S, E, M, and N) . A straightforward reference-sequence-based comparison indeed confirmed this, showing that the M, N, and E proteins of SARS-CoV-2 and SARS-CoV have over 90%genetic similarity, while that of the S protein was notably reduced (but still high) (FIG. 1A) . The similarity between SARS-CoV-2 and MERS-CoV, on the other hand, was substantially lower for all proteins (FIG. 1A) ; a feature that was also evident from the corresponding phylogenetic trees (FIG. 1B) . We note that while the former analysis (FIG. 1A) was based on the reference sequence of each coronavirus, it is indeed a good representative of the virus population, since few amino acid mutations have been observed in the corresponding sequence data (FIG. 3) . It is also noteworthy that while MERS-CoV is the more recent coronavirus to have infected humans, and is comparatively more recurrent (causing outbreaks in 2012, 2015, and 2018) (who. int/emergencies/mers-cov/en/) , SARS-CoV-2 is closer to SARS-CoV, which has not been observed since 2004.

Given the close genetic similarity between the structural proteins of SARS-CoV and SARS-CoV-2, we attempted to leverage immunological studies of the structural proteins of SARS-CoV to potentially aid vaccine development for SARS-CoV-2. We focused specifically on the S and N proteins as these are known to induce potent and long-lived immune responses in SARS-CoV [15–17, 19, 20, 25, 27] . We used the available SARS-CoV-derived experimentally-determined epitope data (see Materials and Methods) and searched to identify T cell and B cell epitopes that were identical-and hence potentially cross-reactive-across SARS-CoV and SARS-CoV-2. We first report the analysis for T cell epitopes, which have been shown to provide a long-lasting immune response against SARS-CoV [27] , followed by a discussion of B cell epitopes.

Mapping the SARS-CoV-Derived T Cell Epitopes that ARE identical in SARS-CoV-2, and Determining those with Greatest Estimated Population Coverage. The SARS-CoV-derived T cell epitopes used in this study were experimentally-determined from two different types of assays [29] : (i) Positive T cell assays, which tested for a T cell response against epitopes, and (ii) positive MHC binding assays, which tested for epitope-MHC binding. We aligned these T cell epitopes across the SARS-CoV-2 protein sequences. Among the 115 T cell epitopes that were determined by positive T cell assays (Table 1) , we found that 27 epitope-sequences were identical within SARS-CoV-2 proteins and comprised no mutation in the available SARS-CoV-2 sequences (as of 21 February 2020) (Table 2) . Interestingly, all of these were present in either the N (16) or S (11) protein. MHC binding assays were performed for 19 of these 27 epitopes, and these were reported to be associated with only five distinct MHC alleles (at 4-digit resolution) : HLA-A*02: 01, HLA-B*40: 01, HLA-DRA*01: 01, HLA-DRB1*07: 01, and HLA-DRB1*04: 01. Consequently, the accumulated population coverage of these epitopes (see Materials and Methods for details) is estimated to not be high for the global population (59.76%) , and was quite low for China (32.36%) . For the remaining 8 epitopes, since the associated MHC alleles are unknown, they could not be used in the population coverage computation. Additional MHC binding tests to identify the MHC alleles that bind to these 8 epitopes may reveal additional distinct alleles, beyond the five determined so far, that may help to improve population coverage.

To further expand the search and identify potentially effective T cell targets covering a higher percentage of the population, we next additionally considered the set of T cell epitopes that have been experimentally-determined from positive MHC binding assays (Table 1) , but, unlike the previous epitope set, their ability to induce a T cell response against SARS-CoV was not experimentally determined. Nonetheless, they also present promising candidates for inducing a response against SARS-CoV-2. For the expanded set of epitopes, all of which have at least one positive MHC binding assay, we found that 229 epitope-sequences have an identical match in SARS-CoV-2 proteins and have associated MHC allele information available (listed in Table 7) . Of these 229 epitopes, ～82%were MHC Class I restricted epitopes (Table 8) . Importantly, 102 of the 229 epitopes were derived from either the S (66) or N (36) protein. Mapping all 66 S-derived epitopes onto the resolved crystal structure of the SARS-CoV S protein (FIG. 4) revealed that 3 of these (GYQPYRVVVL, QPYRVVVLSF, and PYRVVVLSF) were located entirely in the SARS-CoV receptor-binding motif (uniprot. org/uniprot/P59594) , known to be important for virus cell entry [34] .

Table 7. List of all SARS-CoV-derived T cell epitopes determined using positive MHC binding assays (with associated MHC allele information available at 4-digit resolution) and found to be identical in SARS-CoV-2.

Table 8. Distribution of all SARS-CoV-derived T cell epitopes obtained using positive MHC binding assays (with associated MHC allele information available at 4-digit resolution) and that are identical in SARS-CoV-2.

Protein	MHC Allele Class I	MHC Allele Class II	Total
S
	40	26	66
orf1b	45	0	45
N	32	4	36
orf1a	29	0	29
E	1	0	1
M	19	3	22
ORF7a	5	1	6
ORF7b	15	8	23
ORF6	1	0	1
Total	187	42	229

Similar to previous studies on HIV and HCV [35–38] , we estimated population coverages for various combinations of MHC alleles associated with these 102 epitopes. Our aim was to determine sets of epitopes associated with MHC alleles with maximum population coverage, potentially aiding the development of vaccines against SARS-CoV-2. For selection, we adopted a greedy computational approach (see Materials and Methods) , which identified a set of T cell epitopes estimated to maximize global population coverage. This set comprised of multiple T cell epitopes associated with 20 distinct MHC alleles and was estimated to provide an accumulated population coverage of 96.29% (Table 3) . Interestingly, the majority of the T cell epitopes for which a positive immune response has been determined using T cell assays (Table 2) were presented by the globally most-prevalent MHC allele (shown in bold text in Table 3) . Moreover, the functionally important epitopes located in the SARS-CoV receptor binding motif were associated with the second and third most-prevalent MHC alleles (underlined in Table 3) . Thus, while the ordering of T cell epitopes in Table 3 is based on the estimated global population coverage of the associated MHC alleles, it is also a natural order in which these epitopes should be tested experimentally for determining their potential to induce a positive immune response against SARS-CoV-2. We also computed the population coverage of this specific set of epitopes in China, the country most affected by the COVID-19 outbreak, which was estimated to be slightly lower (88.11%) , as certain MHC alleles (e.g., HLA-A*02: 01) associated with some of these epitopes are less frequent in the Chinese population (Table 3) . Repeating the same greedy approach but focusing on the Chinese population, instead of a global population, the maximum population coverage was estimated to be 92.76% (Table 9) .

Table 2. SARS-CoV-derived T cell epitopes obtained using positive T cell assays that are identical in SARS-CoV-2 (27 epitopes in total) .

¹NA: Not available.

Table 9. Set of SARS-CoV-derived S and N protein T cell epitopes (obtained using positive MHC binding assays) that are identical in SARS-CoV-2 and that maximize estimated population coverage in China (86 distinct epitopes) .

Due to the promiscuous nature of binding between peptides and MHC alleles, multiple S and N peptides were reported to bind to individual MHC alleles. Thus, while we list all the S and N epitopes that bind to each MHC allele (Table 3) , the estimated maximum population coverage may be achieved by selecting at least one epitope for each listed MHC allele. Likewise, many individual S and N epitopes were found to be presented by multiple alleles and thereby estimated to have varying global population coverage (listed in Table 10) .

Table 3. Set of the SARS-CoV-derived spike (S) and nucleocapsid (N) protein T cell epitopes (obtained from positive MHC binding assays) that are identical in SARS-CoV-2 and that maximize estimated population coverage globally (87 distinct epitopes) .

¹ Multiple SARS-CoV-derived epitopes that were determined using MHC binding assays are shown for each allele. Epitopes that were also tested for positive T cell response (listed also in Table 2) are shown in bold text. Epitopes that lie within the SARS-CoV receptor-binding motif are underlined. ² Epitopes are ordered according to the estimated global accumulated population coverage.

Table 10. Estimated global and Chinese population coverages for the individual SARS-CoV-derived S or N protein T cell epitopes (obtained using positive MHC binding assays) that are identical in SARS-CoV-2.

Mapping the SARS-CoV-Derived B cell Epitopes that Are Identical in SARS-CoV-2 . Similar to T cell epitopes, we used in our study the SARS-CoV-derived B cell epitopes that have been experimentally-determined from positive B cell assays [29] . These epitopes were classified as: (i) Linear B cell epitopes (antigenic peptides) , and (ii) discontinuous B cell epitopes (conformational epitopes with resolved structural determinants) .

We aligned the 298 linear B cell epitopes (Table 1) across the SARS-CoV-2 proteins and found that 49 epitope-sequences, all derived from structural proteins, have an identical match and comprised no mutation in the available SARS-CoV-2 protein sequences (as of 21 February 2020) . Interestingly, a large number (45) of these were derived from either the S (23) or N (22) protein (Table 4) , while the remaining (4) were from the M protein (Table 11) .

Table 4. SARS-CoV-derived linear B cell epitopes from S (23; 20 of which are located in subunit S2) and N (22) proteins that are identical in SARS-CoV-2 (45 epitopes in total) .

Table 11. SARS-CoV-derived linear B cell epitopes, excluding those in S and N proteins, that are identical in SARS-CoV-2.

Protein	IEDB ID	Epitope
M	21996	GRCDIKDLPKEITVATSR
M	29127	ITVATSRT
M	48052	PKEITVATSRTLSYYKL
M	66409	TSRTLSYYKLGASQRV

On the other hand, all 6 SARS-CoV-derived discontinuous B cell epitopes obtained from the ViPR database (Table 5) were derived from the S protein. Based on the pairwise alignment between the SARS-CoV and SARS-CoV-2 reference sequences (FIG. 5) , we found that none of these mapped identically to the SARS-CoV-2 S protein, in contrast to the linear epitopes. For 3 of these discontinuous B cell epitopes (corresponding to antibodies S230, m396, and 80R [39–41] ) , there was a partial mapping, with at least one site having an identical residue at the corresponding site in the SARS-CoV-2 S protein (Table 5) .

Table 5. SARS-CoV-derived discontinuous B cell epitopes (and associated known antibodies [39–41] ) that have at least one site with an identical amino acid to the corresponding site in SARS-CoV-2.

Mapping the residues of the linear and discontinuous B cell epitopes onto the available structure of the SARS-CoV S protein revealed their distinct association with the two functional subunits of the S protein [42] : S1, important for interaction with the host cell receptor, and S2, involved in fusion of the cellular and virus membranes (FIG. 2A) . Specifically, 20 of the 23 linear epitopes (Table 4) mapped to S2 (FIG. 2B) . Thus, the antibodies targeting the identified linear epitopes in the S2 subunit might cross-react and neutralize both SARS-CoV and SARS-CoV-2, as suggested in a very recent study [43] . While S2 is comparatively less exposed than S1, it may be accessible to antibodies during the complex conformational changes involved in viral entry of coronaviruses [44–46] ; though this remains to be more clearly understood. In contrast, the 3 discontinuous B cell epitopes (Table 5) mapped onto the more exposed S1 subunit (FIG. 2C, left panel) , which contains the receptor-binding motif of the SARS-CoV S protein [34] . We observed that very few residues of the 3 discontinuous epitopes were identical within SARS-CoV and SARS-CoV-2 (Figure 2c, right panel) . These differences suggest that the SARS-CoV-specific antibodies S230, m396, and 80R known to bind to these epitopes in SARS-CoV might not be able to bind to the same regions in SARS-CoV-2 S protein. Interestingly, while this paper was under review, this has been confirmed experimentally [47] . Further studies are currently under way to identify other SARS-CoV antibodies that may bind to discontinuous epitopes of the SARS-CoV-2 S protein [48] .

Discussion

The quest for a vaccine against the novel SARS-CoV-2 is recognized as an urgent problem. Effective vaccination could indeed play a significant role in curbing the spread of the virus, and help to eliminate it from the human population. However, scientific efforts to address this challenge are only just beginning. Much remains to be learnt about the virus, its biological properties, epidemiology, etc. At this early stage, there is also a lack of information about specific immune responses against SARS-CoV-2, which presents a challenge for vaccine development.

This study has sought to assist with the initial phase of vaccine development by providing recommendations of epitopes that may potentially be considered for incorporation in vaccine designs. Despite having limited understanding of how the human immune system responds naturally to SARS-CoV-2, these epitopes are motivated by responses they have recorded in SARS-CoV (or, for the case of T cell epitopes, to at least confer MHC binding) , and the fact that they map identically to SARS-CoV-2, based on the available sequence data (as of 21 February 2020) . This important observation should not be taken for granted. Despite the apparent similarity between SARS-CoV and SARS-CoV-2, there is still considerable genetic variation between the two, and it is not obvious a-prior if epitopes that elicit an immune response against SARS-CoV are likely to be effective against SARS-CoV-2. We found that only 23%and 16%of known SARS-CoV T cell and B cell epitopes map identically to SARS-CoV-2, respectively, and with no mutation having been observed in these epitopes among the available SARS-CoV-2 sequences (as of 21 February 2020) . This provides a strong indication of their potential for eliciting a robust T cell or antibody response in SARS-CoV-2.

EXAMPLE 2 –OPTIMIZED PEPTIDE POOLS FOR ASSESSING REGION-SPECIFIC SARS-COV2 CD8+ T CELL RESPONSES AND USES THEREOF

Introduction

Global efforts to combat COVID-19 have led to the rapid development of multiple vaccines. These vaccines have been shown to induce a robust neutralizing antibody response and provide protection against severe disease and hospitalization (Hall et al. 2021; Mor et al. 2021) . As the virus continues to circulate worldwide, virus variants have emerged in several regions, concerns about their potential to escape vaccine-induced antibody responses. Preliminary results suggest that most current vaccines remain effective against emerging virus variants (Abdool Karim and de Oliveira 2021; Abu-Raddad et al. 2021; Collier et al. 2021; Emary et al. 2021; Liu et al. 2021; Planas et al. 2021) . However, specific variants such as Beta (B. 1.351) , Gamma (P. 1) and Delta (B1.617.2) , that first emerged in South Africa, Brazil, and India respectively, are currently under investigation due to the observed reduction in neutralizing antibody titres against them in sera of vaccinated individuals (Abdool Karim and de Oliveira 2021; Liu et al. 2021; Madhi et al. 2021; Planas et al. 2021; Wall et al. 2021; Zhou et al. 2021) .

In addition to eliciting neutralizing antibodies, SARS-CoV-2 vaccines in use or development also stimulate T cell responses. There is increasing evidence of the role of T cells in protection from severe disease in SARS-CoV-2 infected patients (Altmann and Boyton 2020; Chen and John Wherry 2020; Liao et al. 2020; Mazzoni et al. 2020; Reynolds et al. 2020; Rydyznski Moderbacher et al. 2020; Wyllie et al. 2020; Bergamaschi et al. 2021; Bertoletti et al. 2021; Cohen et al. 2021) , and of their robustness to mutations associated with SARS-CoV-2 variants (Quadeer et al. 2021; Tarke et al. 2021; Woldemeskel et al. 2021) . However, in contrast to neutralizing antibody responses (Piccoli et al. 2020; Pinto et al. 2020; Fedry et al. 2021) , specific T cell responses have not been characterized in detail for any COVID-19 vaccine thus far.

For most currently administered SARS-CoV-2 vaccines, T cell responses have been coarsely measured using immune assays that stimulate blood samples of vaccinated individuals using overlapping peptide pools (Anderson et al. 2020; Folegatti et al. 2020; Keech et al. 2020; Logunov et al. 2020; Ramasamy et al. 2020; Sahin et al. 2020; Zhu et al. 2020; Ella et al. 2021; Klasse et al. 2021; Sadoff et al. 2021) . Use of these pools, however, may underestimate T cell responses due to peptide competition, where immunogenic peptides compete with a large number of irrelevant peptides in the pool that are not recognized by T cells (Pala et al. 1988; Sahin et al. 2021) . In contrast, assays based on optimized peptide pools would be more efficient at estimating T cell responses as these comprise of a selected set of most relevant peptides against which a T cell response is expected to be stimulated. Such pools also enable identifying precise T cell epitopes in the context of the associated human leukocyte antigen (HLA) alleles presenting them.

Designing optimized peptides pools for assessing T cell responses is challenging due to the diversity of these responses. This is because T cells recognize peptides restricted by an individual’s HLA alleles which are highly diverse across the global population (albeit with some commonalities in a given region) . Consequently, the peptides restricted by these HLA alleles are also different, and hence T cell responses are expected to differ between geographical regions, even for the same vaccine. Moreover, current vaccines employ different SARS-CoV-2 antigens (e.g., based on the spike (S) protein only or employing the whole inactivated virion) , and these are expected to elicit distinct T cell responses. To our knowledge, no tool or platform is currently available that provides peptide pools for measuring SARS-CoV-2-specific T cell responses in a particular region where population is immunized by a specific COVID-19 vaccine. Here, we fill this important gap by developing a software platform, SARS2TPools, that provides optimized peptide pools for assessing region-specific vaccine-induced SARS-CoV-2 T cell responses. These pools are designed by exploiting information of prevalent HLA alleles in a population, the experimentally-determined and in-silico-predicted SARS-CoV-2 T cell epitopes associated with these alleles, and the antigen employed in the vaccine. The optimized pools provided by SARS2TPools, in addition to characterizing the vaccine-induced T cell responses in detail, can be useful for designing T cell based diagnostics, monitoring durability of T cell response, and any change in T cell responses due to emerging SARS-CoV-2 variants.

Methods and Materials

Data Collection. We downloaded experimentally-determined HLA class I and class II restricted SARS-CoV-2 T cell epitope data (CD8 ⁺ and CD4 ⁺, respectively) from the immune epitope database (IEDB) (Vita et al. 2019) on March 10, 2021. We included all epitopes that were reported in positive T cell assays with associated HLA information available. The data consisted of 768 and 445 unique class I and class II epitope-HLA pairs, respectively. Majority of the HLA class I restricted epitopes (474/768) were nine residues long, which is the canonical length of epitopes restricted by HLA class I alleles. The epitope data was found to be biased towards a handful of HLA alleles, with only 10 HLA class I alleles (HLA-A*02: 01, HLA-A*03: 01, HLA-A*11: 01, HLA-A*24: 02, HLA-A*29: 02, HLA-A*68: 01, HLA-B*07: 02, HLA-B*35: 01, HLA-B*51: 01, HLA-B*57: 01) having 20 or more nine-residue-long epitopes. Collectively, the epitopes restricted by these 10 HLA alleles corresponded to ～62% (295/474) of nine-residue-long epitopes in the data. In the case of HLA class II restricted epitopes, all the available epitopes were 15 resides long, and only 3 HLA alleles had more than 20 epitopes in the data.

In Silico Prediction Methods. Performance of several in silico epitope prediction methods were benchmarked against the set of experimentally-determined SARS-CoV-2 epitopes associated with the 10 HLA class I alleles having the most data. The considered methods included the current state-of-the-art methods such as MHCflurry (O’ Donnell et al. 2020) , NetMHCpan4.1 (Reynisson et al. 2020) , HLAthena (Sarkizova et al. 2020) , NetMHCpan4.0 (Jurtz et al. 2017) , NetMHC4.0 (Andreatta and Nielsen 2016) , along with other common prediction methods that have been employed for predicting SARS-CoV-2 epitopes (Sohail et al. 2021) such as NetMHCpan3.0 (Nielsen and Andreatta 2016) , SMM (Peters and Sette 2005) , SMMPMBEC (Kim et al. 2009) , and IEDB consensus (Moutaftsi et al. 2006) . We considered the eluted ligand and binding affinity predictions (denoted by suffix BA and EL respectively) of NetMHCpan4.1 and NetMHCpan4.0 as separate methods, as was done for the latter method in (Sarkizova et al. 2020) and (Paul et al. 2020) . Similarly, we considered the binding affinity and the presentation score predictions of MHCflurry as two separate methods, referred to as MHCflurry2.0BA and MHCflurry2.0P. In cases where a method required an input other than the protein sequence, HLA allele, and length of the predicted peptides, we used the default parameter settings for that method.

Union Approach. In this work, we have proposed a union approach based on combining the top-ranked predictions of MHCflurry2.0P and NetMHCpan4.1BA to obtain a set of peptides restricted by a given HLA. This approach was motivated by performance comparison analysis of the 12 in silico epitope prediction methods listed above. Briefly, we ranked peptides in ascending order of their predicted score using each method and compared the histograms of ranks of experimentally-determined SARS-CoV-2 CD8 ⁺ T cell epitopes associated with the 10 HLA class I alleles with the most data. We found that these histograms were bi-modal for all 12 methods. That is, while the top predictions of each method contained a large number of experimentally-determined epitopes, a good number of epitopes were also ranked quite low by each method (FIG. 10) . Exploring the relationships among the set of top 20 ranked peptides per HLA allele predicted by these methods revealed that the predictions of MHCflurry2.0P were most distinct from those of other methods (FIGS. 11A-D) . Consistent results were obtained when this set was constructed by pooling the top 10 to top 25 ranked peptides restricted by each HLA allele (FIGS. 11A-D) . Predictions of MHCflurry2.0P also contained a large number of experimentally-determined SARS-CoV-2 epitopes that were not present in the set of top-ranked peptides predicted by any other method (FIG. 7A) . Given the uniqueness of the predictions of MHCflurry2.0P, we asked if a strategy that combines the predictions of MHCflurry2.0P with any of the other 11 methods would work better than any individual method. The union strategy combines the top x predictions of any two methods and provides a set of peptides whose size can vary between x and 2x depending on the number of common peptides predicted by each method. We fixed one of the methods as MHCflurry2.0P and predicted 11 peptide pools by combining predictions of MHCflurry2.0P with those of the other 11 methods. Our analysis showed that the pool predicted by the union approach always had a higher hit-rate (the fraction of experimentally-determined SARS-CoV-2 epitopes present in the set of top-ranked peptides) than those predicted by the individual methods (FIG. 12) . While comparison among the various union approaches did not readily reveal a clear winner, the unions of MHCflurry2.0P with the in silico methods NetMHC4.0, NetMHCpan4.0BA, NetMHCpan4.1BA, and NetMHCpan4.1EL ranked among the top (FIG. 13, Table 13) .

The union method implemented in SARS2TPools combines the predictions of MHCflurry2.0P with NetMHCpan4.1BA. SARS2TPools provides optimized peptide pools by supplementing experimentally-determined epitopes with small or large sized group of in silico predicted epitopes corresponding respectively to top 10 and top 20 predictions of each method being combined. We used the default thresholds of MHCflurry2.0P and NetMHCpan4.1BA to assess whether or not a peptide is predicted to be an epitope. However, the platform also provides a relaxed threshold which can be particularly useful for specific proteins with very limited number of predicted epitopes.

Statistical Analysis. Statistical analyses were performed using the R language (version 3.6) on the RStudio server (version 1.3) . The software platform was developed using the open source R Shiny (version 1.5) development framework.

Results

SARS2TPools provides optimized peptide pools for assessing T cell responses leveraging experimentally-determined SARS-CoV-2 epitope data. The designed CD8+ T cell peptide pools also include in silico predictions obtained using a computational approach optimized for predicting SARS-CoV-2 CD8 ⁺ T cell epitopes. This approach is based on benchmarking predictions of state- of-the-art in silico methods against the ample experimentally-determined SARS-CoV-2 CD8+epitope data that is now available (Methods) .

Global HLA class I diversity and summary of experimentally-determined SARS-CoV-2 CD8+ T cell epitope data. Each individual possesses three major HLA class I alleles, HLA-A, HLA-B, and HLA-C, which are among the most polymorphic loci of the human genome (Jin et al. 2018) . In fact, more than 6, 500 alleles for each of these three loci have been identified so far (Robinson et al. 2015) . In order to design a peptide pool for assessing T cell response in a specific geographical region, information of the set of most common HLA alleles in that population is required. Based on extensive population studies, the allele frequency net database (AFND) has curated a list of ten most common HLA alleles per locus for 11 distinct geographical regions encompassing the global population (Gonzalez-Galarza et al. 2019) . These sets of HLA alleles have a population coverage of 96%or more in the respective regions. By combining alleles prevalent in all regions, we obtained a total of 136 distinct HLA class I alleles (FIG. 6A, Table 12) . Ideally, a peptide pool for assessing T cell responses in a specific region should include a set of experimentally-determined epitopes against all HLA alleles prevalent in that region. However, the available experimental data of SARS-CoV-2 CD8 ⁺ T cell epitopes with associated HLA class I information is limited (see Methods) . Comparing HLA class I alleles for which experimental data is available with the compiled list of 30 most prevalent HLA alleles per region suggested that a peptide pool comprising of only experimentally-determined epitopes does not cover more than half of the alleles prevalent in majority of the regions (FIG. 6B) . This is because epitopes associated with several of the prevalent HLA alleles in different regions have not been experimentally determined yet. Further analysis showed that even at the level of individual HLA alleles, there is large disparity in the number of experimentally reported epitopes, particularly for proteins other than S (FIG. 6C) . These data limitations are addressed in the developed platform SARS2TPools by supplementing experimentally-determined SARS-CoV-2 epitope data with in silico predictions to design peptide pools optimized for assessing vaccine-induced T cell responses in a specific region.

Table 12. List of 136 HLA alleles that are ranked in the top 10 most-prevalent HLA alleles in at least one of the 13 geographical regions defined by AFND.

In silico prediction of CD8+ T cell epitopes leveraging SARS-CoV-2 experimental epitope data. We have developed an in silico strategy optimized to predict CD8 ⁺ T cell epitopes by leveraging experimentally-determined SARS-CoV-2 immunological data. Briefly, we used the available information of experimentally-determined SARS-CoV-2 immunological data. Briefly, we used the available information of experimentally-determined SARS-CoV-2 CD8 ⁺ T cell epitopes as ground-truth data for comparing the sets of top-ranked peptides predicted by 12 in silico HLA class I epitope prediction methods (Methods) . Using the number of experimentally-determined epitopes present in the set of top ranked peptides as a metric to quantify the ability of a method to predict SARS-CoV-2 epitopes, we found that the NetMHCpan family of methods outperformed others (FIG. 7A) . Importantly, this comparison revealed the uniqueness of MHCflurry2.0P (O’Donnell et al. 2020) predictions; i.e., it predicted the most number of SARS-CoV-2 epitopes that were not predicted by any other method (FIG. 7A) . Our analysis showed that the optimum SARS-CoV-2 epitope prediction strategy was one that combined the predictions of MHCflurry2.0P and NetMHCpan4.1BA (Reynisson et al. 2020) (see Methods for details) . This union strategy performed better than either of the individual methods (FIG. 7B) and was thus used to obtain a set of in silico predicted epitopes to supplement the experimentally-determined epitopes in the optimized peptide pools provided by SARS2TPools. We note that a similar approach to predict CD4 ⁺ T cell epitopes could not be pursued at present due to the scarcity of experimentally-determined SARS-CoV-2 CD4 ⁺ epitopes having the information of cognate HLA allele (Methods) .

Optimized peptide pools from SARSTPools –Software platform. The platform SARS2TPools integrates experimentally-determined SARS-CoV-2 epitope data, in silico predictions, and information of prevalent HLA alleles across regions to provide optimized peptide pools for assessing vaccine-induced SARS-CoV-2 T cell responses (FIG. 8A) . It enables users to obtain region-specific, host-specific, and protein-specific optimized peptide pools through a simple and user-friendly interface (FIG. 8B) . Peptide pools optimized for each of the 11 regions, by taking into account the information of HLA alleles prevalent in these regions (FIG. 6A) , can be obtained by selecting the ‘Region-specific’ tab on SARS2TPools interface (FIG. 8B) . This optimization is important due to the heterogeneity of prevalent HLA alleles among regions that may result in presentation of different epitopes and consequently different T cell responses (FIG. 6A) . Region-specific pools can be useful to contrast T cell responses induced by the same vaccine in different geographical regions, and to understand the role of population heterogeneity in mediating different disease outcomes.

Host-specific (or cohort-specific) peptide pools, for a range of peptide lengths (8–11 residues) , optimized for HLA haplotypes of the host (or cohort) can be obtained by selecting the ‘Host-specific’ tab on SARS2TPools. This optimization can be useful for cases when host (or cohort) HLA typing has been performed.

SARS2TPools also provides the flexibility to select protein-specific pools derived from the entire SARS-CoV-2 proteome or any number of specific proteins for both region-specific and host-specific options. Moreover, the user can also select peptides belonging to a specific domain of a protein (e.g., receptor binding domain of the spike protein) by specifying a range of amino- acid positions. Protein-specific peptide pools are important for assessing T cell responses induced by vaccine comprising of different antigens, e.g., S only, S with other proteins, or whole-virion.

The CD8 ⁺ T cell peptide pools provided by SARS2TPools comprise of both experimentally-determined and in silico predicted epitopes, while those for measuring CD4 ⁺ T cell responses comprise only of experimentally-determined epitopes. In the former case, the platform indicates whether a peptide is an experimentally-determined epitope, predicted epitope, or both. It also provides users the flexibility to select peptide pools consisting exclusively of experimentally-determined CD8 ⁺ T cell epitopes. SARS2TPools ranks peptides within an optimized pool based on HLA promiscuity, which can be used as an additional prioritization criterion among peptides. In summary, SARS2TPools allows users to obtain pools for assessing T cell responses by flexibly selecting various optimization criteria.

Region-specific CD8+ T cell pools from the whole SARS-CoV-2 proteome. As an illustrative example, we used SARS2TPools to obtain region-specific optimized pools, comprising of peptides derived from the whole SARS-CoV-2 proteome, for measuring CD8 ⁺ T cell responses for each of the 11 regions defined by AFND. These pools were designed to include around 20 peptides corresponding to each HLA allele prevalent in a region as follows: (i) If an HLA allele prevalent in a region had 20 or more associated experimentally-determined SARS-CoV-2 epitopes, we selected 20 of these based on response frequency (proportion of responding donors (Quadeer et al. 2020) ) ; (ii) if an HLA allele had less than 20 associated experimentally-determined epitopes, we complemented them with in silico predictions based on the union approach; and (iii) if an HLA allele had no associated experimentally-determined epitopes, all peptides associated with it were predicted based on the union approach.

We found that the number of peptides in each of the region-specific optimized pools was roughly similar, with the fraction of experimentally-determined epitopes in these pools varying between ～35%for Oceania to ～78%for Europe (FIG. 9A) . The HLA alleles covered by each optimized pool can be grouped into 3 classes based on whether their associated peptides in the pool are all (i) experimentally-determined, (ii) in silico predicted, or (iii) a mix of both (FIG. 9B) . Importantly, in silico predictions help to fill the gap for alleles for which limited or no experimentally-determined epitopes are available at present. Comparing the 11 region-specific optimized pools, we found that these pools are largely distinct from each other (FIG. 9C) . These differences in region-specific pools underscore further the importance of designing optimized peptide pools for assessing T cell responses in different regions.

Listing of all region-specific optimized pools. In addition to providing pools for the whole SARS-CoV-2 proteome, SARS2TPools provides optimized peptide pools for assessing CD8 ⁺ T cell responses against any of the 30 individual proteins of SARS-CoV-2 for each of the 11 regions. Thus, a total of 31 x 11 = 341 region-specific optimized peptide pools are provided by the platform. We list all the 3, 860 peptides belonging to any of the 341 pools in Table 15, while the peptides belonging to each specific pools are indicated Table 16 (whole proteome) and Tables 17-47 (individual proteins) .

Table 15. List of all peptide sequences (with their IDs) for CD8 T cell pools.

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
1	STECSNLLLQY	51	AIVSTIQRK	101	VYSSANNCTF
2	FADDLNQLTGY	52	YMSALNHTK	102	VYRGTTTYKL
3	VTVKNGSIHLY	53	RIAGHHLGR	103	AYILFTRFF
4	SSANNCTFEY	54	QLRARSVSPK	104	EYHDVRVVL
5	FSAVGNICY	55	SVYAWNRKR	105	IFFITGNTL
6	VVDYGARFY	56	TLADAGFIK	106	DVFYKENSY
7	YKIEELFYSY	57	GVYYHKNNK	107	NTVKSVGKF
8	SSEAFLIGCNY	58	AIVSTIQRKYK	108	DTFCAGSTF
9	LADAGFIKQY	59	ALDPLSETK	109	FTISVTTEI
10	TDEMIAQY	60	RASANLAATK	110	YVNTFSSTF
11	SSPDDQIGYYR	61	IQITISSFK	111	STAALGVLM
12	AGDSGFAAY	62	RMYIFFASFY	112	DSAEVAVKM
13	TSEDMLNPNY	63	TSFGPLVRK	113	DTIANYAKPF
14	YTELEPPCRF	64	KTFPPTEPK	114	DVVAIDYKHY
15	AAISDYDYY	65	VTNNTFTLK	115	ETIQITISSF
16	FTCASEYTGNY	66	KLFDRYFKY	116	EVARDLSLQF
17	LGDVRETMSY	67	ATVVIGTSK	117	EVAVKMFDAY
18	TIEVNSFSGY	68	FAVSKGFFK	118	EVGHTDLMAAY
19	TITQMNLKY	69	AISDYDYYR	119	NSTNVTIATY
20	TSSGDATTAY	70	VVSTGYHFR	120	SVPWDTIANY
21	WLDMVDTSL	71	YIATNGPLK	121	TVKNGSIHLY
22	FLYENAFLP	72	KVAGFAKFLK	122	ELIRQGTDY
23	FLPGVYSV	73	QTVKPGNFNK	123	EVTPSGTWLTY
24	YLITPVHV	74	KSAAEASKK	124	ETKCTLKSF
25	YLTNDVSFLA	75	AGFSLWVYK	125	WTFGAGAAL
26	AQFAPSASA	76	VVNARLRAK	126	TLKEILVTY
27	KLDDKDPNF	77	ASMPTTIAK	127	YIFFASFYY
28	KLNDLCFTNV	78	STFNVPMEK	128	HSYFTSDYY
29	FLAFVVFL	79	GTHWFVTQR	129	LEAPFLYLY
30	YLGTGPEAGL	80	SASKIITLK	130	SFYYVWKSY
31	NTASWFTAL	81	KTIQPRVEK	131	AGLEAPFLY
32	VLQLPQGTTL	82	SAFAMMFVK	132	VGGNYNYLY
33	ALWEIQQV	83	GVYFASTEK	133	KFCLEASFNY
34	SLIYSTAAL	84	TISLAGSYK	134	WFVTQRNFY
35	TLMNVLTLV	85	QYIKWPWYIW	135	VLKGVKLHY
36	KLKDCVMYA	86	LYDKLVSSF	136	GAAAYYVGY
37	WLLWPVTLA	87	SYATHSDKF	137	KVGGNYNYLY
38	TVYSHLLLV	88	VYDPLQPELDSF	138	SFKEELDKY
39	VLSEARQHL	89	YYVGYLQPRTF	139	SWMESEFRVY
40	GLEAPFLYL	90	IYLYLTFYL	140	LVAEWFLAY

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
41	FVVPGLPGT	91	YYKKDNSYF	141	VFAQVKQIY
42	FVENPDILRV	92	LYLYALVYF	142	CVADYSVLY
43	YQDVNCTEV	93	IYQTSNFRV	143	WTAGAAAYY
44	FLAHIQWMV	94	MFTPLVPFW	144	FVFKNIDGY
45	VVFLHVTYV	95	SFLPGVYSV	145	VASQSIIAY
46	ASFDNFKFV	96	AYILFTRF	146	RIKASMPTT
47	VMYMGTLSY	97	AYVDNSSLTI	147	RLFARTRSM
48	QVVNVVTTK	98	AYVNTFSSTF	148	KASMPTTIA
49	VVTTKIALK	99	KMFDAYVNTF	149	MSALNHTKK
50	KSAGFPFNK	100	GNYNYLYRLF	150	TTIAKNTVK
151	YSRYRIGNYK	201	LTAVVIPTK	251	VLAAECTIF
152	RNRFLYIIK	202	ESKPSVEQR	252	KLVSSFLEM
153	GTRNPANNA	203	DTVIEVQGYK	253	VVYRAFDIY
154	AGLPYGANK	204	SSSDNIALL	254	ITILDGISQY
155	RVYSTGSNV	205	FVLAAVYRI	255	LVKQGDDYVY
156	VTYVPAQEK	206	VPHISRQRL	256	QLYLGGMSYY
157	RYRIGNYKL	207	RARSVSPKL	257	VLTESNKKF
158	ASRELKVTF	208	FPLKLRGTA	258	SQRVAGDSGF
159	KVFRSSVLH	209	LPKEITVAT	259	LVQMAPISAM
160	ATSRTLSYYK	210	LPFAMGIIAM	260	FVVEVVDKY
161	QLTPTWRVY	211	NPIQLSSYSL	261	YLKLTDNVY
162	VTPSGTWLTY	212	RPLLESEL	262	KQFDTYNLW
163	LAYYFMRFR	213	KPFERDISTEI	263	LLNKHIDAY
164	GAMDTTSYR	214	LPNNTASWF	264	KIEELFYSY
165	WVLNNDYYR	215	KPVETSNSFDVL	265	VVQQLPETY
166	WFFSNYLKR	216	RPQGLPNNTA	266	QRNAPRITF
167	GSVAYESLR	217	VPGLPGTIL	267	LVSDIDITF
168	KSNLKPFER	218	QPYRVVVLSF	268	VVVNAANVY
169	YNYLYRLFR	219	VPLHGTIL	269	VPFWITIAY
170	QTNSPRRAR	220	RIRGGDGKM	270	TPSKLIEY
171	KFLPFQQFGR	221	APHGVVFLHV	271	QIPFAMQMAY
172	RFASVYAWNR	222	QPGQTFSVL	272	TPSGTWLTY
173	LSYFIASFR	223	LEIPRRNVATL	273	SPDDQIGYY
174	RLFRKSNLK	224	APHGVVFL	274	DVLLPLTQY
175	STGSNVFQTR	225	SPIFLIVAA	275	VAAGLEAPF
176	KLMGHFAWW	226	SAMVRMYIF	276	LPAADLDDF
177	YVMHANYIF	227	TPKYKFVRI	277	LPLTQYNRY
178	KLINIIIWF	228	TLKKRWQLA	278	SIIQFPNTY
179	KVAGFAKFL	229	HLRIAGHHL	279	DASGKPVPY
180	AMYTPHTVL	230	SLYVNKHAF	280	FAPSASAFF
181	SLDNVLSTF	231	HLKDGTCGL	281	TNVLEGSVAY
182	GVVFLHVTY	232	TFKVSIWNL	282	FAMQMAYRF
183	STNVTIATY	233	LTIKKPNEL	283	LGAENSVAY
184	HVTFFIYNK	234	NLKTLLSL	284	NATRFASVY
185	MASLVLARK	235	MLRIMASL	285	LPPLLTDEM
186	FTIGTVTLK	236	FRLFARTRSM	286	IPFAMQMAY
187	AVILRGHLR	237	HPLADNKFAL	287	SEIIGYKAI
188	HVSGTNGTK	238	INITRFQTL	288	FGEYSHVVAF
189	AAISDYDYYR	239	KIYSKHTPI	289	YENFNQHEV
190	FVVSTGYHFR	240	ANRNRFLYI	290	LEMELTPVV
191	YAISAKNRAR	241	NITRFQTL	291	VEVQPQLEM
192	QIAPGQTGK	242	MFDAYVNTF	292	SELLTPLGI
193	NSASFSTFK	243	LLADKFPVL	293	FELDERIDKVL
194	LVIGAVILR	244	LIIMRTFKV	294	LEFGATSAAL
195	FASVYAWNR	245	VPQEHYVRI	295	MELTPVVQTI
196	SVLNDILSR	246	SSAKSASVY	296	SEDAQGMDNL
197	NASVVNIQK	247	VVAIDYKHY	297	FDEDDSEPVL
198	GTITVEELK	248	RLYYDSMSY	298	FEYVSQPFLM
199	FVIRGDEVR	249	YLFDESGEF	299	SEPVLKGVKL
200	DSGFAAYSR	250	EIKESVQTF	300	WEPEFYEAM
301	LEYHDVRVVL	351	YLPYPDPSRI	401	YAKPFLNKV
302	GETLPTEVL	352	IPYNSVTSSIVI	402	VRQALLKTV
303	TEVVGDIIL	353	LPFGWLIV	403	ERHSLSHFV
304	FERDISTEI	354	APYIVGDVV	404	SAKNRARTV
305	AEVQIDRL	355	VPMEKLKTL	405	MYKGLPWNV

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
306	NESLIDLQEL	356	EGYLNSTNV	406	YQPYRVVVL
307	FEPSTQYEY	357	EAKKVKPTV	407	YRYNLPTMC
308	IEVNSFSGY	358	MPTTIAKNTV	408	LHKPIVWHV
309	VELGTEVNEF	359	MPYFFTLL	409	VKNGSIHLY
310	QELIRQGTDY	360	YPQVNGLTSI	410	LRPDTRYV
311	AELAKNVSL	361	LPYGANKDGI	411	VGYQPYRVV
312	SELVIGAVI	362	LPYPDPSRI	412	VRIQPGQTF
313	YERHSLSHF	363	LPLVSSQCV	413	WRNTNPIQL
314	AEVAVKMF	364	FAYTKRNVI	414	VRNLQHRLY
315	AEWFLAYILF	365	EVFAQVKQI	415	SRVLGLKTL
316	EESSAKSASVY	366	NPLLYDANY	416	LRVEAFEYY
317	SEYKGPITDVFY	367	FLPFFSNVTW	417	VRETMSYLF
318	SEYTGNYQCGHY	368	IHADQLTPTW	418	FKNLREFVF
319	TETDLTKGPHEF	369	LPPAYTNSF	419	NVIPTITQM
320	VENPDILRVY	370	IAIPTNFTI	420	ARAGEAANF
321	VENPHLMGWD	371	FPQSAPHGV	421	WKYPQVNGL
322	YSDVENPHLMGW	372	AVHFISNSW	422	FWRNTNPIQL
323	AAGLEAPFLYLY	373	NSIAIPTNF	423	FRSSVLHST
324	ADAGFIKQY	374	KMKDLSPRW	424	LRGTAVMSL
325	YENQKLIANQF	375	ATIPIQASL	425	GRVDGQVDL
326	YEQYIKWPW	376	VAMPNLYKM	426	TANPKTPKY
327	VENPDILRV	377	RSVASQSII	427	KKQQTVTLL
328	EEVVENPTI	378	VARDLSLQF	428	MKDLSPRWY
329	EEVGHTDLMAAY	379	STVFPPTSF	429	YRSLPGVF
330	VENMTPRDL	380	AKSHNIALIW	430	SRYWEPEF
331	REGVFVSNGTHW	381	ITFDNLKTL	431	RNRFLYIIKL
332	EEIAIILASF	382	LTAFGLVAEW	432	MYASAVVLL
333	EEAIRHVRAW	383	NKATYKPNTW	433	TRTQLPPAY
334	QEILGTVSW	384	SAKSASVYY	434	YADVFHLYL
335	SEFSSLPSY	385	ISTKHFYW	435	FLYLYALVY
336	MEVTPSGTW	386	RTTNGDFLHF	436	KFADDLNQL
337	AEAELAKNV	387	KSAGFPFNKW	437	YFDKAGQKTY
338	AEVQIDRLI	388	LTNDNTSRYW	438	YYKKDNSY
339	AEIRASANL	389	CATVHTANKW	439	YDYLVSTQEF
340	KEIDRLNEV	390	GVAPGTAVLRQW	440	QSAPHGVVF
341	GEVFNATRF	391	GVFVSNGTHW	441	LRIMASLVL
342	QELGKYEQY	392	VRSIFSRTL	442	YFTSDYYQL
343	KQEILGTVSW	393	YRGTTTYKL	443	VRIIMRLWL
344	QEYADVFHLY	394	YASAVVLLI	444	LVKPSFYVY
345	AEHVNNSY	395	GFMGRIRSV	445	FYYVWKSY
346	IAAVITREV	396	YVYSRVKNL	446	ARLYYDSMSY
347	DAVNLLTNM	397	AHAEETRKL	447	IYKTPPIKDF
348	LPGVYSVI	398	LRKHFSMMI	448	YFIKGLNNL
349	LPRVF SAV	399	CRSKNPLLY	449	TRFASVYAW
350	DAMRNAGIV	400	NSFSGYLKL	450	FYLITPVHV
451	IYDEPTTTT	501	KMQRMLLEK	551	MTYRRLISM
452	FLLPSLATV	502	AVAKHDFFK	552	FTSDYYQLY
453	FLLNKEMYL	503	HVVGPNVNK	553	MVMCGGSLY
454	TMADLVYAL	504	TMLFTMLRK	554	EVNSFSGYL
455	YLQPRTFLL	505	MTSCCSCLK	555	EVVGDIILK
456	YLNSTNVTI	506	AQCFKMFYK	556	SFYEDFLEY
457	HLVDFQVTI	507	HLYLQYIRK	557	VVYRGTTTY
458	VLNDILSRL	508	RQFHQKLLK	558	YILFTRFFY
459	ALWEIQQVV	509	TTIKPVTYK	559	FAIGLALYY
460	SVVSKVVKV	510	GVAMPNLYK	560	SMMGFKMNY
461	ALSKGVHFV	511	SSTCMMCYK	561	GVYSVIYLY
462	NLIDSYFVV	512	GTLSYEQFK	562	ATSRTLSYY
463	KIADYNYKL	513	QTMLFTMLR	563	ASHMYCSFY
464	LLYDANYFL	514	QTFFKLVNK	564	KMNYQVNGY
465	FVNEFYAYL	515	NYMPYFFTL	565	AVKTQFNYY
466	YLYALVYFL	516	IYNDKVAGF	566	ALCEKALKY
467	ILFTRFFYV	517	VYMPASWVM	567	MMSAPPAQY
468	FLNRFTTTL	518	YFVVKRHTF	568	RISNCVADY
469	YLNTLTLAV	519	VYIGDPAQL	569	GTFTCASEY
470	YLTNDVSFL	520	VYDPLQPEL	570	VYYPDKVFR

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
471	FLPRVFSAV	521	QYIKWPWYI	571	CSLSHRFYR
472	KLNIKLLGV	522	YFIASFRLF	572	HFYSKWYIR
473	FIAGLIAIV	523	YYTSNPTTF	573	AYYFMRFRR
474	FLNGSCGSV	524	YYQLYSTQL	574	AVHECFVKR
475	KLSYGIATV	525	TYKPNTWCI	575	KAIDGGVTR
476	VLAWLYAAV	526	VYFLQSINF	576	RVVRSIFSR
477	RTIKVFTTV	527	WSMATYYLF	577	NSLLTPFAR
478	VLWAHGFEL	528	SYYSLLMPI	578	RVKNLNSSR
479	LMIERFVSL	529	TYACWHHSI	579	NFYGPFVDR
480	SMWALIISV	530	SYFIASFRL	580	VTHSKGLYR
481	YTMADLVYA	531	DYQGKPLEF	581	ALHFLLFFR
482	FVAAIFYLI	532	NYNYLYRLF	582	CMMCYKRNR
483	SLPGVFCGV	533	FFASFYYVW	583	MSKFPLKLR
484	RIMTWLDMV	534	TYASALWEI	584	RTIKGTHHW
485	LQLGFSTGV	535	LYSPIFLIV	585	KAYNVTQAF
486	AVIKTLQPV	536	RYLALYNKY	586	KLLHKPIVW
487	KVDGVVQQL	537	EYADVFHLY	587	RMYIFFASF
488	FVDGVPFVV	538	MYIFFASFY	588	KFYGGWHNM
489	YMPYFFTLL	539	QYNRYLALY	589	KTPKYKFVR
490	YLDAYNMMI	540	YYPSARIVY	590	LFALLQRYR
491	MLDMYSVML	541	ETISLAGSY	591	RAMPNMLRI
492	GLMWLSYFI	542	DTYNLWNTF	592	KLAKKFDTF
493	NLSDRVVFV	543	DVTDVTQLY	593	KSYELQTPF
494	FLARGIVFM	544	ETKAIVSTI	594	RTNVYLAVF
495	AMDEFIERY	545	EIVDTVSAL	595	NVFAFPFTI
496	TLKSFTVEK	546	EAIRHVRAW	596	KVYPIILRL
497	HLMGWDYPK	547	EIAIILASF	597	VMFTPLVPF
498	LLFFRALPK	548	VVIPDYNTY	598	DYGDAVVYR
499	KLFAAETLK	549	DAQSFLNGF	599	DFDTWFSQR
500	RLISMMGFK	550	DVRETMSYL	600	DFYDFAVSK
601	VYADSFVIR	651	KPREQIDGY	701	MKIILFLAL
602	MTQMYKQAR	652	KPRQKRTAT	702	FAVDAAKAY
603	NYAKPFLNK	653	LPSLATVAY	703	FLHFLPRVF
604	IASFRLFAR	654	YLRKHFSMM	704	QLYLGGMSY
605	NTVIWDYKR	655	YLKLRSDVL	705	LMNVLTLVY
606	YAFASEAAR	656	FVKHKHAFL	706	YLVQQESPF
607	TVIEVQGYK	657	CLLNRYFRL	707	VQMAPISAM
608	TTDPSFLGR	658	DLFMRIFTI	708	ILMTARTVY
609	FSSEIIGYK	659	YFMRFRRAF	709	MISAGFSLW
610	MSAFAMMFV	660	FLKTNCCRF	710	NMVYMPASW
611	NATNVVIKV	661	DAPAHISTI	711	LPFFSNVTW
612	STSAFVETV	662	TQMNLKYAI	712	MSMTYGQQF
613	NTFSSTFNV	663	SQLGGLHLL	713	FISNSWLMW
614	SVAALTNNV	664	GEYSHVVAF	714	NMMVTNNTF
615	QSFLNGFAV	665	MMISAGFSL	715	HADQLTPTW
616	ETFKLSYGI	666	TQWSLFFFL	716	NVLEGSVAY
617	YTACSHAAV	667	REHEHEIAW	717	HMLDMYSVM
618	FSASTSAFV	668	RLVDPQIQL	718	FMGRIRSVY
619	HTIDGSSGV	669	NQMCLSTLM	719	VPWDTIANY
620	HVISTSHKL	670	RQWLPTGTL	720	DEWSMATYY
621	FSYFAVHFI	671	MQTMLFTML	721	VEHVTFFIY
622	DAQSFLNRV	672	FQFCNDPFL	722	DEISMATNY
623	NTQEVFAQV	673	RELHLSWEV	723	FELEDFIPM
624	TTFDSEYCR	674	MQVESDDYI	724	HEFCSQHTM
625	LSTFISAAR	675	YELQTPFEI	725	LEWLAMAVM
626	FLAYILFTR	676	RQLLFVVEV	726	NETLVTMPL
627	RVYANLGER	677	RSLKVPATV	727	LEIKDTEKY
628	FPRGQGVPI	678	KQIYKTPPI	728	SEVGPEHSL
629	IPRRNVATL	679	KQLIKVTLV	729	TEETFKLSY
630	SPRRARSVA	680	MQLFFSYFA	730	EEFEPSTQY
631	KPNELSRVL	681	SQNAVASKI	731	SEFDRDAAM
632	SPRWYFYYL	682	TQYNRYLAL	732	TELEPPCRF
633	SPYNSQNAV	683	NRFLYIIKL	733	MPYFFTLLL
634	IPVAYRKVL	684	DQFKHLIPL	734	LPFNDGVYF
635	RPDTRYVLM	685	EHYVRITGL	735	NPHLMGWDY

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
636	KPCIKVATV	686	TRFQTLLAL	736	YPNASFDNF
637	MARKTLNSL	687	DKVFRSSVL	737	LPGVYSVIY
638	HPTQAPTHL	688	YRLFRKSNL	738	CPDGVKHVY
639	IPIGAGICA	689	SHVVAFNTL	739	VPFVVSTGY
640	LPQNAVVKI	690	EHFIETISL	740	KRWQLALSK
641	MPNMLRIMA	691	VRFPNITNL	741	RRLISMMGF
642	IPIQASLPF	692	FRNARNGVL	742	GRWVLNNDY
643	KPVETSNSF	693	TRFFYVLGL	743	KRVDWTIEY
644	IPTNFTISV	694	HHMELPTGV	744	ARFYFYTSK
645	TPAFDKSAF	695	NNLNRGMVL	745	ARYMRSLKV
646	FPPTSFGPL	696	SHFVNLDNL	746	NRFNVAITR
647	MIAQYTSAL	697	QHEETIYNL	747	MRIMTWLDM
648	NPAWRKAVF	698	QHMVVKAAL	748	GRIRSVYPV
649	TPKGPKVKY	699	FRYMNSQGL	749	SRYRIGNYK
650	QPRTFLLKY	700	SHFAIGLAL	750	ARTRSMWSF
751	SRLSFKELL	801	GEQKSILSP	851	DAYNMMISA
752	RRVWTLMNV	802	TERLKLFAA	852	LPFKLTCAT
753	LPVNVAFEL	803	REAACCHLA	853	SPSGVYQCA
754	FPFTIYSLL	804	LQAAVGELL	854	KSHNIALIW
755	SAPPAQYEL	805	FEYVSQPFL	855	LSDLQDLKW
756	MPASWVMRI	806	FQVTIAEIL	856	RSFIEDLLF
757	FPDLNGDVV	807	AEIVDTVSA	857	VSFLAHIQW
758	YPSLETIQI	808	LEPEYFNSV	858	MACLVGLMW
759	FGADPIHSL	809	MEKLKTLVA	859	KAYKIEELF
760	VADAVIKTL	810	APFLYLYAL	860	LAAVYRINW
761	EAVGTNLPL	811	KPTVVVNAA	861	LAGTITSGW
762	YPLECIKDL	812	VENPHLMGW	862	VMPLSAPTL
763	FSSTFNVPM	813	SEKQVEQKI	863	SAPHGVVFL
764	QPTESIVRF	814	SEMHPALRL	864	GVAPGTAVL
765	FVSLAIDAY	815	SEDMLNPNY	865	HANEYRLYL
766	YPGQGLNGY	816	CPIHFYSKW	866	FLPGVYSVI
767	YANRNRFLY	817	TAFGLVAEW	867	VSPTKLNDL
768	FAYANRNRF	818	TASDTYACW	868	NVPLHGTIL
769	LAKDTTEAF	819	TPGDSSSGW	869	QLPAPRTLL
770	HHSIGFDYV	820	GETLGVLVP	870	TAPHGHVMV
771	MHAASGNLL	821	REAVGTNLP	871	IGPERTCCL
772	YHTTDPSFL	822	QEAYEQAVA	872	SANNCTFEY
773	LHSTQDLFL	823	SEYDYVIFT	873	FVLTSHTVM
774	THHWLLLTI	824	EELFYSYAT	874	IAMSAFAMM
775	VRDPQTLEI	825	NEYRLYLDA	875	VATSRTLSY
776	KHITSKETL	826	SEAGVCVST	876	FAQDGNAAI
777	THTGTGQAI	827	YLITPVHVM	877	FSNSGSDVL
778	THLSVDTKF	828	YSSANNCTF	878	YSTAALGVL
779	VHFVCNLLL	829	FASEAARVV	879	FVSDADSTL
780	IHFYSKWYI	830	MELPTGVHA	880	ISTSHKLVL
781	YKVYYGNAL	831	FENKTTLPV	881	FCYMHHMEL
782	LRSDVLLPL	832	MPLSAPTLV	882	VAKSHNIAL
783	NRALTGIAV	833	CPAEIVDTV	883	RTAPHGHVM
784	AEWFLAYIL	834	SPFELEDFI	884	TFDNLKTLL
785	HEGKTFYVL	835	MPTIFFAGI	885	RFDNPVLPF
786	GEAANFCAL	836	FPLCANGQV	886	ILDITPCSF
787	AEAAVKPLL	837	SAFYILPSI	887	KYDFTEERL
788	LENVAFNVV	838	LAWLYAAVI	888	LFDMSKFPL
789	TEVPVAIHA	839	MAYITGGVV	889	YGDFSHSQL
790	NESGLKTIL	840	VEYCPIFFI	890	YFTEQPIDL
791	HEVLLAPLL	841	CQYLNTLTL	891	VYDDGARRV
792	AECTIFKDA	842	TQFNYYKKV	892	VTDVTQLYL
793	REFLTRNPA	843	IQLSSYSLF	893	YVDNSSLTI
794	SEFRVYS SA	844	LAAVNSVPW	894	YSDVENPHL
795	YENAFLPFA	845	MPILTLTRA	895	VVDSYYSLL
796	IELKFNPPA	846	LPFAMGIIA	896	YIDIGNYTV
797	MEIDFLELA	847	MPVCVETKA	897	MADQAMTQM
798	GECPNFVFP	848	FPFNKWGKA	898	ANDPVGFTL
799	VELKHFFFA	849	FPREGVFVS	899	VSDIDITFL
800	LEFGATSAA	850	CPFGEVFNA	900	ISDEVARDL

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
901	ISDEFSSNV	951	GQVDLFRNA	1001	HSSGVTREL
902	VAFNTLLFL	952	AMRPNFTIK	1002	VTRELMREL
903	YYRYNLPTM	953	EILPVSMTK	1003	QSASKIITL
904	YYHTTDPSF	954	TVYDDGARR	1004	VGYLQPRTF
905	FYLTNDVSF	955	TIDYTEISF	1005	LSDRVVFVL
906	LYYQNNVFM	956	YTRYVDNNF	1006	EAANFCALI
907	AADPAMHAA	957	YYRSLPGVF	1007	MGYINVFAF
908	LSDGLLLAL	958	TVNVLAWLY	1008	KVDGVDVEL
909	ITDAVDCAL	959	KLIEYTDFA	1009	FGDDTVIEV
910	VSDIDYVPL	960	VLLSVLQQL	1010	LLLDRLNQL
911	VAYFNMVYM	961	PYNMRVIHF	1011	LLLEWLAMA
912	SFSASTSAF	962	TTIQTIVEV	1012	KIFVDGVPF
913	ISAMVRMYI	963	LATNNLVVM	1013	MLVYCFLGY
914	KTLLSLREV	964	VLVPHVGEI	1014	MPLKAPKEI
915	ISAGFSLWV	965	SQSIIAYTM	1015	DGYFKIYSK
916	YTNSFTRGV	966	NTVCTVCGM	1016	DTLKNLSDR
917	HAASGNLLL	967	EAFEKMVSL	1017	VMAYITGGV
918	AVASKILGL	968	YFYTSKTTV	1018	EAVMYMGTL
919	ALNNIINNA	969	LLDDFVEII	1019	FGDSVEEVL
920	KLWAQCVQL	970	HLDGEVITF	1020	VYSTGSNVF
921	SLSHRFYRL	971	HFYWFFSNY	1021	VVNVVTTKI
922	TLIGDCATV	972	FTVLCLTPV	1022	ELTPVVQTI
923	QMAPISAMV	973	KSVNITFEL	1023	ELPDEFVVV
924	VLSDRELHL	974	TTAAKLMVV	1024	YFPLQSYGF
925	YLATALLTL	975	LSDDAVVCF	1025	AIKITEHSW
926	MLAKALRKV	976	LPNDDTLRV	1026	ALDQAISMW
927	HSIGFDYVY	977	YTVELGTEV	1027	SLIDFYLCF
928	NYSGVVTTV	978	LEYHDVRVV	1028	RVESSSKLW
929	YQKVGMQKY	979	IADKYVRNL	1029	YKKPASREL
930	TVAYFNMVY	980	THVQLSLPV	1030	LMDGSIIQF
931	ALNTLVKQL	981	LLLALHFLL	1031	LAVPYNMRV
932	YFKYWDQTY	982	KLLEQWNLV	1032	SGFAAYSRY
933	VFLGIITTV	983	YITGGVVQL	1033	QEYADVFHL
934	VTWFHAIHV	984	NVLTLVYKV	1034	FAFACPDGV
935	KVQIGEYTF	985	ALCTFLLNK	1035	QVVDMSMTY
936	FLTENLLLY	986	RVDGQVDLF	1036	FTNVYADSF
937	S SDNIALLV	987	YGIATVREV	1037	KTIGPDMFL
938	RSVSPKLFI	988	LLFNKVTLA	1038	NRGMVLGSL
939	IVDTVSALV	989	ERSEKSYEL	1039	YVFCTVNAL
940	KYTQLCQYL	990	HHWLLLTIL	1040	TLKNTVCTV
941	TSDLATNNL	991	YFNSVCRLM	1041	NLWNTFTRL
942	TVASLINTL	992	VSNGTHWFV	1042	SALWEIQQV
943	VSDADSTLI	993	FVSNGTHWF	1043	KNFKSVLYY
944	VLYENQKLI	994	ISNSWLMWL	1044	EAAVKPLLV
945	NYLKRRVVF	995	DTVIEVQGY	1045	ILFALLQRY
946	EAMYTPHTV	996	FLAFLLFLV	1046	YLCFLAFLL
947	NQKLIANQF	997	KLNVGDYFV	1047	LANECAQVL
948	FLAFVVFLL	998	AYANSVFNI	1048	LPQLEQPYV
949	LLLDDFVEI	999	YYVGYLQPR	1049	SWVMRIMTW
950	ALLADKFPV	1000	DALFAYTKR	1050	RTATKAYNV
1051	KVFTTVDNI	1101	MSDVKCTSV	1151	FEYYHTTDP
1052	KKFLPFQQF	1102	DEFIERYKL	1152	YVWKSYVHV
1053	FHQKLLKSI	1103	ISMDNSPNL	1153	LASHMYCSF
1054	NHTSPDVDL	1104	NPNYEDLLI	1154	SVNPYVCNA
1055	AHIQWMVMF	1105	SPNLAWPLI	1155	DFNLVAMKY
1056	TTTIKPVTY	1106	LIISVTSNY	1156	RLRAKHYVY
1057	KYKYFSGAM	1107	MSYEDQDAL	1157	STTTNIVTR
1058	QFAPSASAF	1108	SSLPSYAAF	1158	DIQLLKSAY
1059	YTNDKACPL	1109	ASANLAATK	1159	KENSYTTTI
1060	VPHHVVATV	1110	VQQESPFVM	1160	VLSFCAFAV
1061	KWDLTAFGL	1111	IVSTIQRKY	1161	FLALCADSI
1062	FVTVYSHLL	1112	ETMSYLFQH	1162	ETICAPLTV
1063	LRLGSPLSL	1113	FEEAALCTF	1163	FLFVAAIFY
1064	VINGDRWFL	1114	YLASGGQPI	1164	RYFRLTLGV
1065	DILSRLDKV	1115	MSNLGMPSY	1165	AIDAYPLTK

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
1066	YECDIPIGA	1116	MVDTSLSGF	1166	SAGFPFNKW
1067	KSHKPPISF	1117	RAFGEYSHV	1167	IAIAMACLV
1068	TVEEAKTVL	1118	TQAPTHLSV	1168	ETTADIVVF
1069	AVKPLLVPH	1119	AQLPAPRTL	1169	HFISNSWLM
1070	ATLPKGIMM	1120	SAFFGMSRI	1170	HSWNADLYK
1071	HTDLMAAYV	1121	FLGRYMSAL	1171	AIMQLFFSY
1072	VPYCYDTNV	1122	CAMRPNFTI	1172	QHEVLLAPL
1073	NRNRFLYII	1123	AIMTRCLAV	1173	MYDPKTKNV
1074	VLYYQNNVF	1124	YYKLGASQR	1174	LSPRWYFYY
1075	KTQFNYYKK	1125	TVVIGTSKF	1175	KVSIWNLDY
1076	REETGLLMP	1126	LVAVPTGYV	1176	CSFGGVSVI
1077	QVDVVNFNL	1127	YSGVVTTVM	1177	FSTFEEAAL
1078	VMYASAVVL	1128	ILSPLYAFA	1178	EETGLLMPL
1079	AAYVDNSSL	1129	MSLSEQLRK	1179	MTNRQFHQK
1080	HFAIGLALY	1130	FEIKLAKKF	1180	SLRPDTRYV
1081	SLVKPSFYV	1131	ILHCANFNV	1181	SYLFQHANL
1082	LQLPQGTTL	1132	IGAGICASY	1182	DYKHYTPSF
1083	KLVNKFLAL	1133	LRPDTRYVL	1183	ESGLKTILR
1084	NVAFNVVNK	1134	FSKQLQQSM	1184	LSFKELLVY
1085	SRYWEPEFY	1135	AANTVIWDY	1185	NAANVYLKH
1086	TSNPTTFHL	1136	TPLIQPIGA	1186	AFPFTIYSL
1087	FYAYLRKHF	1137	NYDLSVVNA	1187	KATEETFKL
1088	QLFFSYFAV	1138	YKTPPIKDF	1188	TSMKYFVKI
1089	AMSAFAMMF	1139	AWPLIVTAL	1189	IINNTVYTK
1090	WLPTGTLLV	1140	QSINFVRII	1190	SFKWDLTAF
1091	ETAQNSVRV	1141	KHAFHTPAF	1191	MFVKHKHAF
1092	TYFTQSRNL	1142	LLMPLKAPK	1192	KLIFLWLLW
1093	FVVSTGYHF	1143	TVKPGNFNK	1193	WVYKQFDTY
1094	SQLMCQPIL	1144	YKGPITDVF	1194	LALGGSVAI
1095	GSIHLYFDK	1145	MFLARGIVF	1195	FAAYSRYRI
1096	LQTPFEIKL	1146	ALAYYNTTK	1196	FVSEETGTL
1097	TPCNGVEGF	1147	GTYEGNSPF	1197	STKHFYWFF
1098	AHGFELTSM	1148	LIDFYLCFL	1198	INFVRIIMR
1099	GTSKFYGGW	1149	TLDSKTQSL	1199	TTIVYLTIV
1100	WMESEFRVY	1150	KWDLIISDM	1200	DTYPSLETI
1201	KDLPKEITV	1251	GLPWNVVRI	1301	LLIIMRTFK
1202	TTKGGRFVL	1252	NLLKDCPAV	1302	GQQFGPTYL
1203	LVLSVNPYV	1253	DYLVSTQEF	1303	FVMMSAPPA
1204	NVLAWLYAA	1254	DSKEGFFTY	1304	QWSLFFFLY
1205	WEIQQVVDA	1255	KVNSTLEQY	1305	FVNLKQLPF
1206	ASAFFGMSR	1256	NELSPVALR	1306	SLPSYAAFA
1207	IANQFNSAI	1257	APISAMVRM	1307	FTINCQEPK
1208	WLMWLIINL	1258	FACPDGVKH	1308	DVFHLYLQY
1209	LEIPRRNVA	1259	FYWFFSNYL	1309	YFVKIGPER
1210	ILTSLLVLV	1260	IAATRGATV	1310	HVGEIPVAY
1211	TIVEVQPQL	1261	IAQYTSALL	1311	VAELEGIQY
1212	ELYHYQECV	1262	NSFTRGVYY	1312	TEQPIDLVP
1213	IIIGGAKLK	1263	FNATRFASV	1313	LPKGIMMNV
1214	VFAFPFTIY	1264	IMASLVLAR	1314	YTPSKLIEY
1215	VTANVNALL	1265	WQLALSKGV	1315	LLLTILTSL
1216	ALLTKSSEY	1266	VLSTFISAA	1316	NRQFHQKLL
1217	LTDEMIAQY	1267	KAIVSTIQR	1317	RLQSLQTYV
1218	LLALHRSYL	1268	LYQPPQTSI	1318	KHAFLCLFL
1219	LLNKEMYLK	1269	STDTCFANK	1319	AAAYYVGYL
1220	SILSPLYAF	1270	RYKLEGYAF	1320	MGIIAMSAF
1221	VVNAANVYL	1271	TTITVNVLA	1321	AIASEFS SL
1222	GFDYVYNPF	1272	MLQSCYNFL	1322	FKHLIPLMY
1223	IFTIGTVTL	1273	FLKKDAPYI	1323	EAFLIGCNY
1224	RVVFNGVSF	1274	DIAANTVIW	1324	AHSCNVNRF
1225	QQWGFTGNL	1275	RVWTLMNVL	1325	KHADFDTWF
1226	REVGFVVPG	1276	MVYMPASWV	1326	EPKLGSLVV
1227	SAQTGIAVL	1277	LTNMFTPLI	1327	LPFGWLIVG
1228	HSLSHFVNL	1278	QAWQPGVAM	1328	IAQVDVVNF
1229	RVFSAVGNI	1279	ASLPFGWLI	1329	RFRRAFGEY
1230	LYLDAYNMM	1280	MSYYCKSHK	1330	VYANGGKGF

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
1231	SASVYYSQL	1281	QTFSVLACY	1331	EVQELYSPI
1232	NLHSSRLSF	1282	VSFCYMHHM	1332	VMVELVAEL
1233	SYEDQDALF	1283	AFDIYNDKV	1333	YPIILRLGS
1234	FELTSMKYF	1284	FPLNSIIKT	1334	KATNNAMQV
1235	VLGSLAATV	1285	VHFISNSWL	1335	DISGINASV
1236	NVLSTFISA	1286	ALLQRYRYK	1336	RTIAFGGCV
1237	STEKSNIIR	1287	LESELVIGA	1337	EVITFDNLK
1238	GIITTVAAF	1288	VSIINNTVY	1338	HSMQNCVLK
1239	IELSLIDFY	1289	RELKVTFFP	1339	HFDGQQGEV
1240	FTDGVCLFW	1290	DGARRVWTL	1340	VTVYSHLLL
1241	KATYKPNTW	1291	LTNDNTSRY	1341	TLGVLVPHV
1242	FRKSNLKPF	1292	LYENAFLPF	1342	DTANPKTPK
1243	VLSGHNLAK	1293	YPANSIVCR	1343	VEVEKGVLP
1244	QLIKVTLVF	1294	EVNEFACVV	1344	AARVVRSIF
1245	RVCGVSAAR	1295	EVFNATRFA	1345	SSRVPDLLV
1246	STVLSFCAF	1296	TLKATEETF	1346	REQIDGYVM
1247	MATNYDLSV	1297	VLNEKCSAY	1347	TSVDCTMYI
1248	KLVLSVNPY	1298	RDLPQGFSA	1348	RHHANEYRL
1249	TLVTMPLGY	1299	LLFLMSFTV	1349	YIVDSVTVK
1250	VQLHNDILL	1300	LVYAADPAM	1350	TILDGISQY
1351	TLAVPYNMR	1401	RNYVFTGYR	1451	IEDLLFNKV
1352	RQEEVQELY	1402	AVDAAKAYK	1452	IAKNTVKSV
1353	HEETIYNLL	1403	ITHDVSSAI	1453	CPACHNSEV
1354	GYLPQNAVV	1404	NTWCIRCLW	1454	YSLFDMSKF
1355	VGTNLPLQL	1405	FQTRAGCLI	1455	GLNDNLLEI
1356	LYLGGMSYY	1406	AIFYLITPV	1456	DKAYKIEEL
1357	TLNDLNETL	1407	YLEGSVRVV	1457	WPWYIWLGF
1358	EAARYMRSL	1408	SIIAYTMSL	1458	SLEDKAFQL
1359	STYASQGLV	1409	FQPTNGVGY	1459	KISEMHPAL
1360	FEHIVYGDF	1410	TDYKHWPQI	1460	DEFVVVTVK
1361	TAQNSVRVL	1411	HDIGNPKAI	1461	ASAVVLLIL
1362	FVFPLNSII	1412	LLLQILFAL	1462	LLTNMFTPL
1363	MKFLVFLGI	1413	YFVLTSHTV	1463	KWYIRVGAR
1364	KAPKEIIFL	1414	SVIYLYLTF	1464	TSNSFDVLK
1365	SYSGQSTQL	1415	VMHANYIFW	1465	LQIPFAMQM
1366	ATVHTANKW	1416	MESEFRVYS	1466	VYSVIYLYL
1367	MFYKGVITH	1417	YTEISFMLW	1467	SLLSVLLSM
1368	IIFWF SLEL	1418	RLSFKELLV	1468	GVRRSFYVY
1369	AQVLSEMVM	1419	SVFNICQAV	1469	TTIVNGVRR
1370	FYGGWHNML	1420	VFVSNGTHW	1470	RQVVNVVTT
1371	CVPLNIIPL	1421	DSIIIGGAK	1471	KIQEGVVDY
1372	NDLCFTNVY	1422	GEVITFDNL	1472	MYTPHTVLQ
1373	YTDFATSAC	1423	DAMMFTSDL	1473	RLYLDAYNM
1374	DEFTPFDVV	1424	SAFVNLKQL	1474	LKKRWQLAL
1375	FFGMSRIGM	1425	DQAISMWAL	1475	ATVAYFNMV
1376	ETLGVLVPH	1426	VAVKMFDAY	1476	ALVYFLQSI
1377	YWFFSNYLK	1427	RVCTNYMPY	1477	DEMIAQYTS
1378	ITVATSRTL	1428	KTSVDCTMY	1478	ATAEAELAK
1379	WPVTLACFV	1429	EKALKYLPI	1479	SEYTGNYQC
1380	LPSYAAFAT	1430	GHFAWWTAF	1480	TVYTKVDGV
1381	MVTNNTFTL	1431	SIIKTIQPR	1481	VVDKYFDCY
1382	ELLHAPATV	1432	YYFMRFRRA	1482	DVRVVLDFI
1383	KHDFFKFRI	1433	TSHKLVLSV	1483	TTLPVNVAF
1384	VYNPFMIDV	1434	FIASFRLFA	1484	LPLQLGFST
1385	KSFDLGDEL	1435	AYLRKHFSM	1485	KNFTTAPAI
1386	ILIVTTIVY	1436	LYYPSARIV	1486	NAAISDYDY
1387	RLWLCWKCR	1437	DLQDLKWAR	1487	DLPQGFSAL
1388	CVMYASAVV	1438	CTDDNALAY	1488	NSQGLLPPK
1389	KLQFTSLEI	1439	TPRDLGACI	1489	LTNIFGTVY
1390	HTDFSSEII	1440	NEEIAIILA	1490	GPFVDRQTA
1391	KQGDDYVYL	1441	TLAILTALR	1491	SALNHTKKW
1392	LTYTGAIKL	1442	RTILGSALL	1492	VGDSAEVAV
1393	ETSWQTGDF	1443	VPHVGEIPV	1493	TTFTYASAL
1394	DAVTAYNGY	1444	NYTVSCLPF	1494	QSSYIVDSV
1395	QEIQLQAAV	1445	FLCWHTNCY	1495	AVDCALDPL

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
1396	NALDQAISM	1446	EVPANSTVL	1496	LVASIKNFK
1397	GHMLDMYSV	1447	QVVSDIDYV	1497	TTFHLDGEV
1398	RYMNSQGLL	1448	SVSPKLFIR	1498	DHVDILGPL
1399	LLDKRTTCF	1449	VETVKGLDY	1499	QLCQYLNTL
1400	LAHIQWMVM	1450	TESNKKFLP	1500	KLFIRQEEV
1501	GVYDYLVST	1551	TLGVYDYLV	1601	SFNPETNIL
1502	GERVRQALL	1552	TTVDNINLH	1602	FLRDGWEIV
1503	IDYVPLKSA	1553	STDVVYRAF	1603	YMPASWVMR
1504	NFCALILAY	1554	LTILTSLLV	1604	KVKYLYFIK
1505	YHFRELGVV	1555	QLMCQPILL	1605	LTFYLTNDV
1506	HKPPISFPL	1556	ARNGVLITE	1606	FALLQRYRY
1507	SFRLFARTR	1557	KHTDFSSEI	1607	WLTNIFGTV
1508	ILGTVSWNL	1558	AEYHNESGL	1608	LAAECTIFK
1509	SSNVANYQK	1559	LEQYVFCTV	1609	WHNMLKTVY
1510	TPVCINGLM	1560	KPVPEVKIL	1610	VSQPFLMDL
1511	FYYVWKSYV	1561	AKPPPGDQF	1611	GLFKDCSKV
1512	IVAGGIVAI	1562	MMFVKHKHA	1612	NELSRVLGL
1513	KLMPVCVET	1563	AMPNMLRIM	1613	GADLKSFDL
1514	AFATAQEAY	1564	IQYIDIGNY	1614	AMQTMLFTM
1515	WFSQRGGSY	1565	STCMMCYKR	1615	TPVVQTIEV
1516	TFEYVSQPF	1566	MVSLLSVLL	1616	NYYKKDNSY
1517	MLRIMASLV	1567	SFLAHIQWM	1617	LVSTQEFRY
1518	YRARAGEAA	1568	GTTTLNGLW	1618	CASEYTGNY
1519	YPSARIVYT	1569	ALLAVFQSA	1619	GHSMQNCVL
1520	LVYDNKLKA	1570	TEVPANSTV	1620	LSVCLGSLI
1521	AAVDALCEK	1571	DESGEFKLA	1621	TIAFGGCVF
1522	TLRVEAFEY	1572	GVFCGVDAV	1622	KVVKVTIDY
1523	FRVQPTESI	1573	FMRFRRAFG	1623	EQPYVFIKR
1524	YVLPNDDTL	1574	YKQFDTYNL	1624	LEILQKEKV
1525	LHDELTGHM	1575	IATNGPLKV	1625	EPVLKGVKL
1526	WVPRASANI	1576	HVVAFNTLL	1626	SIWNLDYII
1527	IVVTCLAYY	1577	RLANECAQV	1627	SRTLSYYKL
1528	TLLALHRSY	1578	YVVDDPCPI	1628	LQSCYNFLK
1529	TTPGSGVPV	1579	EEIAIILAS	1629	SVLLFLAFV
1530	VMCGGSLYV	1580	LAMAVMLLL	1630	KMVSLLSVL
1531	QMCLSTLMK	1581	RVVVLSFEL	1631	LNDFNLVAM
1532	NLKTLLSLR	1582	EFTPFDVVR	1632	NSPNLAWPL
1533	STQDLFLPF	1583	NQHEVLLAP	1633	VVNQNAQAL
1534	TLSEQLDFI	1584	CEFCGTENL	1634	VTIAEILLI
1535	FLEYHDVRV	1585	FLQSINFVR	1635	SEYCRHGTC
1536	KRFDNPVLP	1586	LQSINFVRI	1636	KYVRNLQHR
1537	REVLSDREL	1587	SVLYNSASF	1637	KMAFPSGKV
1538	QEKNFTTAP	1588	IANYAKPFL	1638	VVIGTSKFY
1539	IVDEPEEHV	1589	LPGCDGGSL	1639	QTYVTQQLI
1540	AQYTSALLA	1590	MRIFTIGTV	1640	DKFKVNSTL
1541	ETVKGLDYK	1591	VLLAPLLSA	1641	KLMVVIPDY
1542	AHVASCDAI	1592	LQGPPGTGK	1642	VYQLRARSV
1543	MGFKMNYQV	1593	LYLQYIRKL	1643	SGDGTTSPI
1544	QALLKTVQF	1594	DMFLGTCRR	1644	FELLHAPAT
1545	VPYNMRVIH	1595	MPSYCTGYR	1645	QELYSPIFL
1546	SHTVMPLSA	1596	FKMFYKGVI	1646	TFTYASALW
1547	EEAIRHVRA	1597	YYSLLMPIL	1647	TENLTKEGA
1548	VPNQPYPNA	1598	NLKQLPFFY	1648	TLVPQEHYV
1549	STDTGVEHV	1599	GFAAYSRYR	1649	HLMSFPQSA
1550	QAISMWALI	1600	DYGARFYFY	1650	AKYTQLCQY
1651	GELGDVRET	1701	LFLMSFTVL	1751	NIFGTVYEK
1652	VEAPLVGTP	1702	NYMLTYNKV	1752	ALPETTADI
1653	SVAIKITEH	1703	ADSIIIGGA	1753	FFLYENAFL
1654	RNAGIVGVL	1704	TQLGIEFLK	1754	KTLQPVSEL
1655	GYYRRATRR	1705	VGMQKYSTL	1755	SAQCFKMFY
1656	LWLLWPVTL	1706	LAPLLSAGI	1756	IMRTFKVSI
1657	GSFCTQLNR	1707	AAKKNNLPF	1757	HVVATVQEI
1658	GIATVREVL	1708	ISTIGVCSM	1758	KLTDNVYIK
1659	QECVRGTTV	1709	TFFKLVNKF	1759	DTVRTNVYL
1660	KKNNLPFKL	1710	GLLLALHFL	1760	AEETRKLMP

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
1661	RLDKVEAEV	1711	AQVAKSHNI	1761	SIIIGGAKL
1662	TTAYANSVF	1712	KVTLVFLFV	1762	FFFLYENAF
1663	TGDSCNNYM	1713	VRIKIVQML	1763	MADLVYALR
1664	QESPFVMMS	1714	ARVECFDKF	1764	YINVFAFPF
1665	GYKSVNITF	1715	SYIVDSVTV	1765	SEAKCWTET
1666	EYVSQPFLM	1716	YQVNGYPNM	1766	CTSCCFSER
1667	KGLPWNVVR	1717	TVLCLTPVY	1767	YVPLKSATC
1668	KEKVNINIV	1718	KWGKARLYY	1768	QQGEVPVSI
1669	AAFHQECSL	1719	LEGNFYGPF	1769	VHTANKWDL
1670	LAYILFTRF	1720	CPAVAKHDF	1770	KDTEKYCAL
1671	KFLKTNCCR	1721	RYRYKPHSL	1771	VSTSGRWVL
1672	GVKHVYQLR	1722	TLSYEQFKK	1772	FTALTQHGK
1673	EAALCTFLL	1723	AFEKMVSLL	1773	QASLNGVTL
1674	AQKFNGLTV	1724	SLVPGFNEK	1774	YFDKAGQKT
1675	ITLATCELY	1725	LVLVQSTQW	1775	LLSAGIFGA
1676	VPLKSATCI	1726	DTTEAFEKM	1776	YSLRLIDAM
1677	IPTITQMNL	1727	HEIAWYTER	1777	NKHAFHTPA
1678	AALQIPFAM	1728	DEVARDLSL	1778	MFTPLIQPI
1679	KLNEEIAII	1729	GEFKLASHM	1779	AVPYNMRVI
1680	ILGLPTQTV	1730	TPSDFVRAT	1780	DFVNEFYAY
1681	TTLKGVEAV	1731	GSLAATVRL	1781	SANLAATKM
1682	EEVQELYSP	1732	WMVMFTPLV	1782	FAFPFTIYS
1683	KPLLVPHHV	1733	SNYLKRRVV	1783	TQSRNLQEF
1684	FYVLGLAAI	1734	AYSNNSIAI	1784	GTITSGWTF
1685	MDNSPNLAW	1735	ANGQVFGLY	1785	AAVGELLLL
1686	EVGKPRPPL	1736	VYSFLPGVY	1786	LTSHTVMPL
1687	AAVINGDRW	1737	LNRYFRLTL	1787	LAAIMQLFF
1688	IAKKPTETI	1738	LSPVALRQM	1788	LSVLQQLRV
1689	KHWPQIAQF	1739	WEIVKFIST	1789	IFWRNTNPI
1690	LAILTALRL	1740	LIVNSVLLF	1790	DLYKLMGHF
1691	AFGGCVFSY	1741	VFLFVAAIF	1791	YSQLMCQPI
1692	ASFSTFKCY	1742	IEELFYSYA	1792	TIDGSSGVV
1693	LHCANFNVL	1743	LQTYVTQQL	1793	LWPVTLACF
1694	TRVLSNLNL	1744	IVNSVLLFL	1794	WLAMAVMLL
1695	ISAARQGFV	1745	LYIDINGNL	1795	FLNKVVSTT
1696	CEESSAKSA	1746	DLSPRWYFY	1796	VFVLWAHGF
1697	CGPKKSTNL	1747	NPKTPKYKF	1797	FTPLVPFWI
1698	SSTASALGK	1748	KPYIKWDLL	1798	HFVNLDNLR
1699	DHSSSSDNI	1749	DADSKIVQL	1799	ASKIITLKK
1700	AYANRNRFL	1750	RFKESPFEL	1800	IIAMSAFAM
1801	EAVKTQFNY	1851	SLAIDAYPL	1901	IAMACLVGL
1802	STASALGKL	1852	VVENPTIQK	1902	LAFVVFLLV
1803	RITGLYPTL	1853	GYAFEHIVY	1903	TSRTLSYYK
1804	DPFLGVYYH	1854	QQLIRAAEI	1904	TLNDFNLVA
1805	QKFNGLTVL	1855	VANYQKVGM	1905	YLKSPNFSK
1806	MNVAKYTQL	1856	KYTMADLVY	1906	ELKINAACR
1807	YYVWKSYVH	1857	YAADPAMHA	1907	QTVTLLPAA
1808	NLYDKLVSS	1858	YLAVFDKNL	1908	NEKCSAYTV
1809	ALDISASIV	1859	SPFVMMSAP	1909	VAFELWAKR
1810	KWADNNCYL	1860	ETKDVVECL	1910	SLSDGLLLA
1811	EQKSILSPL	1861	SSPDAVTAY	1911	LEQPTSEAV
1812	RKSAPLIEL	1862	IGYYRRATR	1912	KTILRKGGR
1813	IAIVMVTIM	1863	KSWMESEFR	1913	VAAIVFITL
1814	AANFCALIL	1864	IFFASFYYV	1914	QFTSLEIPR
1815	MVPHISRQR	1865	KVQHMVVKA	1915	GPKVYPIIL
1816	SEAVEAPLV	1866	TTLNDFNLV	1916	HYVRITGLY
1817	HWFVTQRNF	1867	IPKEEVKPF	1917	LPIDKCSRI
1818	IMQLFFSYF	1868	NYQHEETIY	1918	QIYKTPPIK
1819	IQPGQTFSV	1869	YIKWPWYIW	1919	EAARVVRSI
1820	IIMRLWLCW	1870	RAAEIRASA	1920	VLCNSQTSL
1821	SLPINVIVF	1871	YVLGLAAIM	1921	LTSMKYFVK
1822	KIITLKKRW	1872	GMSRIGMEV	1922	MLSDTLKNL
1823	SKVGGNYNY	1873	ASIKNFKSV	1923	LTNDVSFLA
1824	IQASLPFGW	1874	AFLPFAMGI	1924	VIYLYLTFY
1825	ITLKKRWQL	1875	GVDIAANTV	1925	CEIVGGQIV

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
1826	LMSFTVLCL	1876	KFKEGVEFL	1926	SLLMPILTL
1827	LAWPLIVTA	1877	NSYFTEQPI	1927	LLKSIAATR
1828	ETIQITISS	1878	FNPETNILL	1928	AYWVPRASA
1829	AVVCFNSTY	1879	TSLSGFKLK	1929	RNFYEPQII
1830	IERFVSLAI	1880	FSTFKCYGV	1930	HEHEIAWYT
1831	VTDTPKGPK	1881	INDMILSLL	1931	LVDSDLNDF
1832	LPIGINITR	1882	TEKYCALAP	1932	LLMPILTLT
1833	QAITVTPEA	1883	QINDMILSL	1933	YGQQFGPTY
1834	ILKPANNSL	1884	YTVEEAKTV	1934	HFLLFFRAL
1835	TVQFCDAMR	1885	KPFLNKVVS	1935	ALYNKYKYF
1836	QPILLLDQA	1886	KQGNFKNLR	1936	GDMVPHISR
1837	YSKHTPINL	1887	KPHSLSDGL	1937	QSTQWSLFF
1838	HMVVKAALL	1888	RLIDAMMFT	1938	LQKAAITIL
1839	KRAKVTSAM	1889	NTLTLAVPY	1939	VLDMCASLK
1840	IPYNSVTSS	1890	NENGTITDA	1940	KEGATTCGY
1841	ASCDAIMTR	1891	LTYNKVENM	1941	HSLSDGLLL
1842	TGSNVFQTR	1892	VAYRKVLLR	1942	WLSYFIASF
1843	VEQKIAEIP	1893	IEYTDFATS	1943	FLPFAMGII
1844	NIDYDCVSF	1894	CYFGLFCLL	1944	SMWSFNPET
1845	KSPNFSKLI	1895	NPPALQDAY	1945	GSVRVVTTF
1846	YYHKNNKSW	1896	SDRVVFVLW	1946	MLFTMLRKL
1847	TTCCSLSHR	1897	LTKHPNQEY	1947	YEAMYTPHT
1848	VSEETGTLI	1898	NCYDYCIPY	1948	ILLLDQALV
1849	STECSNLLL	1899	TVREVLSDR	1949	RIMASLVLA
1850	TVYEKLKPV	1900	QPITNCVKM	1950	QEGVVDYGA
1951	EKMVSLLSV	2001	NIDGYFKIY	2051	YIKWDLLKY
1952	ISTKHFYWF	2002	DLKWARFPK	2052	GTSTDVVYR
1953	SNVTWFHAI	2003	YFRLTLGVY	2053	LYIIKLIFL
1954	TSNQVAVLY	2004	AALTNNVAF	2054	EYPIIGDEL
1955	RNIKPVPEV	2005	ESSAKSASV	2055	SVCLGSLIY
1956	APLLSAGIF	2006	LLQLCTFTR	2056	NQTTTIQTI
1957	LVS SFLEMK	2007	LLTKSSEYK	2057	RIIPARARV
1958	FSNYLKRRV	2008	EEAALCTFL	2058	VVISSDVLV
1959	LTTAAKLMV	2009	CVDIPGIPK	2059	AYKIEELFY
1960	ISDYDYYRY	2010	EYFNSVCRL	2060	QIDRLITGR
1961	RLNEVAKNL	2011	FFSYFAVHF	2061	ESVQTFFKL
1962	GVYYPDKVF	2012	LPDEFVVVT	2062	MQMAYRFNG
1963	NVAFELWAK	2013	NYIAQVDVV	2063	ASVYAWNRK
1964	LSYGIATVR	2014	EANMDQESF	2064	LTALRLCAY
1965	VRATATIPI	2015	RPLLESELV	2065	MSFPQSAPH
1966	YLVSTQEFR	2016	NTYLEGSVR	2066	TIAEILLII
1967	NMLRIMASL	2017	DLKGKYVQI	2067	VVFDEISMA
1968	MMILSDDAV	2018	KLPDDFTGC	2068	VTFFIYNKI
1969	FLLVTLAIL	2019	QMAYRFNGI	2069	LATCELYHY
1970	GQGLNGYTV	2020	ASQGLVASI	2070	RLKLFDRYF
1971	GPKVKYLYF	2021	WHHSIGFDY	2071	TSTDVVYRA
1972	VLKLKVDTA	2022	SEETGTLIV	2072	QHGKEDLKF
1973	EVGFVVPGL	2023	KYAISAKNR	2073	HDELTGHML
1974	FIDTKRGVY	2024	LAVFDKNLY	2074	LPTGVHAGT
1975	AFQLTPIAV	2025	YDANYFLCW	2075	TYRRLISMM
1976	GTTTYKLNV	2026	FTTVDNINL	2076	CALAPNMMV
1977	EEMLDNRAT	2027	AQALNTLVK	2077	FLHVTYVPA
1978	LAFLLFLVL	2028	VEKGVLPQL	2078	YSRYRIGNY
1979	YLALYNKYK	2029	IAEIPKEEV	2079	IAPGQTGKI
1980	DLVYALRHF	2030	KALNLGETF	2080	CYKRNRATR
1981	TVSCLPFTI	2031	LPRVFSAVG	2081	FSSLPSYAA
1982	TPGSGVPVV	2032	STQWSLFFF	2082	KEILVTYNC
1983	DKAFQLTPI	2033	DNLKTLLSL	2083	LALSKGVHF
1984	REVRTIKVF	2034	KWDLLKYDF	2084	YEYGTEDDY
1985	LQHRLYECL	2035	LIDSYFVVK	2085	KLKPVLDWL
1986	EEHVQIHTI	2036	GDCATVHTA	2086	KIILFLALI
1987	ADQAMTQMY	2037	KSAQCFKMF	2087	AESHVDTDL
1988	AGIVGVLTL	2038	RKGGRTIAF	2088	FYEPQIITT
1989	SEISMDNSP	2039	SHEGKTFYV	2089	GYPNMFITR
1990	NLDYIINLI	2040	LLTILTSLL	2090	HSDKFTDGV

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
1991	ELFENKTTL	2041	QWMVMFTPL	2091	IRKSNHNFL
1992	VVPGLPGTI	2042	LRAKHYVYI	2092	LTLVYKVYY
1993	SLENVAFNV	2043	NFGAISSVL	2093	SVKGLQPSV
1994	KFTDGVCLF	2044	FAMMFVKHK	2094	DYIINLIIK
1995	AFLIGCNYL	2045	MRNAGIVGV	2095	VDILGPLSA
1996	LPETTADIV	2046	SALEPLVDL	2096	GPKKSTNLV
1997	KYLYFIKGL	2047	TVMFLARGI	2097	VLDWLEEKF
1998	TVMPLSAPT	2048	KPANNSLKI	2098	VFITLCFTL
1999	ITLCFTLKR	2049	ESNKKFLPF	2099	VVYCPRHVI
2000	ISQYSLRLI	2050	TALLTLQQI	2100	LVKNKCVNF
2101	YREAACCHL	2151	KVVSTTTNI	2201	NIIPLTTAA
2102	KPASRELKV	2152	MLLLQILFA	2202	EQFKKGVQI
2103	GTDLEGNFY	2153	NLKYAISAK	2203	AVITREVGF
2104	NLAKHCLHV	2154	LDYKAFKQI	2204	FWITIAYII
2105	GELLLLEWL	2155	LPAPRTLLT	2205	YAFEHIVYG
2106	DEFSSNVAN	2156	FVKRVDWTI	2206	DFQENWNTK
2107	TAAKLMVVI	2157	FPNTYLEGS	2207	ARTVAGVSI
2108	KARLYYDSM	2158	FVLALLSDL	2208	TPINLVRDL
2109	VFDEISMAT	2159	NRNYVFTGY	2209	IMRLWLCWK
2110	TCDGTTFTY	2160	YHPNCVNCL	2210	EISFMLWCK
2111	GHNLAKHCL	2161	YIRKLHDEL	2211	YAAVINGDR
2112	ITPVHVMSK	2162	LALYYPSAR	2212	GVVREFLTR
2113	PQNAVVKIY	2163	RLIIRENNR	2213	KRGDKSVYY
2114	RKAVFISPY	2164	YQIGGYTEK	2214	KHIDAYKTF
2115	SRIIPARAR	2165	NNAAIVLQL	2215	TAHSCNVNR
2116	PYPDPSRIL	2166	LAVVVCNSL	2216	FACVVADAV
2117	TIYSLLLCR	2167	VRQCSGVTF	2217	SSRSRNSSR
2118	VPATVSVSS	2168	SQLDEEQPM	2218	LATHGLAAV
2119	CSMTDIAKK	2169	GQQQQGQTV	2219	RWVLNNDYY
2120	VLHDIGNPK	2170	CTNYMPYFF	2220	LEKMADQAM
2121	RVVISSDVL	2171	GLFCLLNRY	2221	LIVTTIVYL
2122	FLYIIKLIF	2172	NLDSCKRVL	2222	SIDAFKLNI
2123	VESSSKLWA	2173	ATTAYANSV	2223	DIADTTDAV
2124	NLVAVPTGY	2174	LGDIAARDL	2224	SNFGAISSV
2125	LSAQTGIAV	2175	VEIIKSQDL	2225	YNMMISAGF
2126	MLKTVYSDV	2176	TEVNEFACV	2226	ALNLGETFV
2127	IPLTTAAKL	2177	VYRGTTTYK	2227	NLGERVRQA
2128	EQWNLVIGF	2178	NPTIQKDVL	2228	QLSLPVLQV
2129	MCDIRQLLF	2179	RHVRAWIGF	2229	ILFLALITL
2130	VQAGNVQLR	2180	RINWITGGI	2230	QPRVEKKKL
2131	GLQPSVGPK	2181	TLQCIMLVY	2231	VPVSIINNT
2132	QIGEYTFEK	2182	DIASTDTCF	2232	DFVKATCEF
2133	SSVELKHFF	2183	NVVIKVCEF	2233	LEKCDLQNY
2134	YNSASFSTF	2184	RDLICAQKF	2234	CLTPVYSFL
2135	NVVTTKIAL	2185	LIPLMYKGL	2235	SLNVAKSEF
2136	QIGGYTEKW	2186	CFANKHADF	2236	ADIVVFDEI
2137	TSQWLTNIF	2187	NSWLMWLII	2237	SNYQHEETI
2138	SLPFGWLIV	2188	WTNAGDYIL	2238	FSYVGCHNK
2139	KPSFYVYSR	2189	ALRANSAVK	2239	EPEFYEAMY
2140	TTNGDFLHF	2190	LFVTVYSHL	2240	FVSGNCDVV
2141	VVAFNTLLF	2191	ALLSDLQDL	2241	TPVYSFLPG
2142	ASLNGVTLI	2192	NYQKVGMQK	2242	WLIVGVALL
2143	WICLLQFAY	2193	DTKFKTEGL	2243	NVTQAFGRR
2144	GPEHSLAEY	2194	TEKSNIIRG	2244	KKLDGFMGR
2145	IFVDGVPFV	2195	GDELKINAA	2245	FEKMVSLLS
2146	VTSNYSGVV	2196	YNLWNTFTR	2246	GMPSYCTGY
2147	CVEEVTTTL	2197	TNNLVVMAY	2247	LEILDITPC
2148	IRQEEVQEL	2198	LLSDLQDLK	2248	VLKKCKSAF
2149	NFNKDFYDF	2199	WSFNPETNI	2249	QEGVLTAVV
2150	YPVASPNEC	2200	PYNSVTSSI	2250	LTLAVPYNM
2251	SEQLDFIDT	2301	DATPSDFVR	2351	LYKMQRMLL
2252	ELSLIDFYL	2302	NRYLALYNK	2352	ALYYPSARI
2253	LSKGRLIIR	2303	ASPNECNQM	2353	TSEAVEAPL
2254	GVKDCVVLH	2304	GRTILGSAL	2354	CIMSDRDLY
2255	AGILIVTTI	2305	VLQKAAITI	2355	AIVVTCLAY

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
2256	SEMVMCGGS	2306	WTLMNVLTL	2356	VQPQLEMEL
2257	WEVGKPRPP	2307	GTTLPKGFY	2357	RYVLMDGSI
2258	QITISSFKW	2308	CTTIVNGVR	2358	EWFLAYILF
2259	YTSALLAGT	2309	LHSYFTSDY	2359	VLITEGSVK
2260	FADDLNQLT	2310	SLSSTASAL	2360	TTCFSVAAL
2261	TFVTHSKGL	2311	ELEGIQYGR	2361	GANKDGIIW
2262	RAFDIYNDK	2312	LTSQWLTNI	2362	SSQGSEYDY
2263	EFLTRNPAW	2313	NVYLAVFDK	2363	MLWCKDGHV
2264	HLAKALNDF	2314	YSHLLLVAA	2364	EVVLKTGDL
2265	VVDADSKIV	2315	VVQEGVLTA	2365	SLRVCVDTV
2266	WKCRSKNPL	2316	PYRVVVLSF	2366	CFVLAAVYR
2267	SYGADLKSF	2317	YELKHGTFT	2367	VRGTTVLLK
2268	MADSNGTIT	2318	FVFLVLLPL	2368	VQHMVVKAA
2269	AENVTGLFK	2319	TVQEIQLQA	2369	IPARARVEC
2270	EEVGHTDLM	2320	LYRKCVKSR	2370	VLLRKNGNK
2271	MLIIFWFSL	2321	SAGFSLWVY	2371	FASFYYVWK
2272	CRMNSRNYI	2322	IIQFPNTYL	2372	GDFLHFLPR
2273	KQLSSNFGA	2323	EFSSNVANY	2373	SICSTMTNR
2274	LVGLMWLSY	2324	SKSLTENKY	2374	TGVEHVTFF
2275	NEFACVVAD	2325	AHFPREGVF	2375	YIIKLIFLW
2276	TLACFVLAA	2326	FLPFQQFGR	2376	FGAGAALQI
2277	SVQTFFKLV	2327	NSPRRARSV	2377	EAACCHLAK
2278	DEPTTTTSV	2328	KGFCKLHNW	2378	LPVLQVRDV
2279	TSCCFSERF	2329	SMQNCVLKL	2379	LSLQFKRPI
2280	VSTQEFRYM	2330	ELKKLLEQW	2380	NPTDQSSYI
2281	VIPDYNTYK	2331	VVTTFDSEY	2381	KGDYGDAVV
2282	TMCDIRQLL	2332	RKVQHMVVK	2382	CSQHTMLVK
2283	RGWIFGTTL	2333	RPNFTIKGS	2383	WADNNCYLA
2284	CQVHGNAHV	2334	NASFDNFKF	2384	QFAYANRNR
2285	KFVCDNIKF	2335	VTTIVYLTI	2385	LRANSAVKL
2286	QEFKPRSQM	2336	NSRNYIAQV	2386	ATNNLVVMA
2287	QPSVGPKQA	2337	RRIRGGDGK	2387	LFLPFFSNV
2288	IISDMYDPK	2338	IEVQGYKSV	2388	ETKFLTENL
2289	LALCADSII	2339	AVLDMCASL	2389	TYVTQQLIR
2290	YPDKVFRSS	2340	ADAQSFLNR	2390	RLNQLESKM
2291	NPDILRVYA	2341	FKLKDCVMY	2391	ASSSEAFLI
2292	FKEGSSVEL	2342	YYLGTGPEA	2392	VLSFELLHA
2293	VIGFLFLTW	2343	CAFAVDAAK	2393	SVAYSNNSI
2294	VTLAILTAL	2344	FTEQPIDLV	2394	SLREVRTIK
2295	MNVLTLVYK	2345	CRFVTDTPK	2395	ALILAYCNK
2296	TSAMQTMLF	2346	ITPCSFGGV	2396	HVMSKHTDF
2297	GIYQTSNFR	2347	VSLVKPSFY	2397	GGDAALALL
2298	LLALHFLLF	2348	ALGGSVAIK	2398	YQTQTNSPR
2299	FSHSQLGGL	2349	CPRHVICTS	2399	VIAWNSNNL
2300	GAWNIGEQK	2350	STGYHFREL	2400	YYRRATRRI
2401	VLKKLKKSL	2451	ARGIVFMCV	2501	HGFELTSMK
2402	IDHPNPKGF	2452	LNVPLHGTI	2502	MIDVQQWGF
2403	LSLREVRTI	2453	FYLCFLAFL	2503	CRKVQHMVV
2404	SVTSNYSGV	2454	NLVAMKYNY	2504	YFLQSINFV
2405	SSSSDNIAL	2455	VGFTLKNTV	2505	MMPTIFFAG
2406	FCSQHTMLV	2456	CTSVVLLSV	2506	NPYVCNAPG
2407	FLGIITTVA	2457	TSAFVETVK	2507	MHANYIFWR
2408	VVNPVMEPI	2458	VVHNQDVNL	2508	ESPFVMMSA
2409	GACIRRPFL	2459	LPVSMTKTS	2509	LMWLIINLV
2410	FLGYFCTCY	2460	TEHSWNADL	2510	GLWLDDVVY
2411	TITVNVLAW	2461	CISTKHFYW	2511	CYGVSPTKL
2412	MRSLKVPAT	2462	LLAKDTTEA	2512	YQCAMRPNF
2413	LKLFAAETL	2463	SLCLQLAVV	2513	HFVCNLLLL
2414	GVVTTVMFL	2464	IVNNWLKQL	2514	VQMLSDTLK
2415	TANKWDLII	2465	HVASCDAIM	2515	FFSNVTWFH
2416	LPDDFTGCV	2466	VTSAMQTML	2516	TFLLNKEMY
2417	KENDSKEGF	2467	TVLSFCAFA	2517	ARHINAQVA
2418	LPFFYYSDS	2468	NSVLLFLAF	2518	RIDKVLNEK
2419	KLDGFMGRI	2469	IYSLLLCRM	2519	SIVCRFDTR
2420	KACPLIAAV	2470	NVNRFNVAI	2520	SLLSKGRLI

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
2421	LRKVPTDNY	2471	ALAPNMMVT	2521	FMCVEYCPI
2422	TRPLLESEL	2472	FSLWVYKQF	2522	EYRLYLDAY
2423	ISMATNYDL	2473	NARDGCVPL	2523	SVTTEILPV
2424	TAGAAAYYV	2474	FLELAMDEF	2524	KTLNSLEDK
2425	LALYNKYKY	2475	ITSGWTFGA	2525	VFKNIDGYF
2426	LLQFAYANR	2476	ITVNVLAWL	2526	TGNYQCGHY
2427	RILGAGCFV	2477	VLLSMQGAV	2527	GTILTRPLL
2428	KQLPFFYYS	2478	LTLGVYDYL	2528	FAMGIIAMS
2429	ELTSMKYFV	2479	RTCCLCDRR	2529	VESCGNFKV
2430	SSRLSFKEL	2480	YPQVNGLTS	2530	VRTNVYLAV
2431	LDYIINLII	2481	RVDFCGKGY	2531	YEDQDALFA
2432	TGPEAGLPY	2482	TTLPKGFYA	2532	TNDKACPLI
2433	GDSEVVLKK	2483	FVENPDILR	2533	NFKDQVILL
2434	YVRNLQHRL	2484	TETAHSCNV	2534	HPLADNKFA
2435	SREETGLLM	2485	LVTMPLGYV	2535	DYVYNPFMI
2436	MLAHAEETR	2486	FSTGVNLVA	2536	FLMSFTVLC
2437	KEGFFTYIC	2487	GCDGGSLYV	2537	ELTGHMLDM
2438	QQESPFVMM	2488	RENNRVVIS	2538	KHYTPSFKK
2439	KITEHSWNA	2489	QPYRVVVLS	2539	FHPLADNKF
2440	TPFEIKLAK	2490	TVVVNAANV	2540	DYDYYRYNL
2441	DPSFLGRYM	2491	VLLFLAFVV	2541	ELYSPIFLI
2442	LLFLAFVVF	2492	VYEKLKPVL	2542	RNLQEFKPR
2443	ITREVGFVV	2493	FPNITNLCP	2543	DIGNPKAIK
2444	GLCVDIPGI	2494	DGYVMHANY	2544	VAKHDFFKF
2445	TQDLFLPFF	2495	QILFALLQR	2545	LVDPQIQLA
2446	FVTHSKGLY	2496	KSDGTGTIY	2546	CWHTNCYDY
2447	WVMRIMTWL	2497	SSRGTSPAR	2547	DRYPANSIV
2448	RFLYIIKLI	2498	NVAKSEFDR	2548	RQRLTKYTM
2449	FNVLFSTVF	2499	FTSLEIPRR	2549	LFTRFFYVL
2450	SEAARVVRS	2500	CLEASFNYL	2550	NFTIKGSFL
2551	NRVCGVSAA	2601	VAGFAKFLK	2651	AENSVAYSN
2552	VFQTRAGCL	2602	TYLEGSVRV	2652	FLPFFSNVT
2553	NVAITRAKV	2603	ASEAARVVR	2653	HPALRLVDP
2554	IDFYLCFLA	2604	ITGGIAIAM	2654	FNEKTHVQL
2555	AYIICISTK	2605	DLDDFSKQL	2655	LVKQLSSNF
2556	YVQIPTTCA	2606	PFFSNVTWF	2656	FLKRGDKSV
2557	GFIQQKLAL	2607	NVYADSFVI	2657	NLNESLIDL
2558	KSILSPLYA	2608	NQPYPNASF	2658	QLHNDILLA
2559	FCLLNRYFR	2609	HISRQRLTK	2659	GYLQPRTFL
2560	LALHFLLFF	2610	RRATCFSTA	2660	NASSSEAFL
2561	CLAVHECFV	2611	FVVFLLVTL	2661	FITESKPSV
2562	AMAVMLLLL	2612	LAMDEFIER	2662	RELGVVHNQ
2563	ALKYLPIDK	2613	KPFERDIST	2663	TFTRSTNSR
2564	DELKINAAC	2614	TPLVPFWIT	2664	MFITREEAI
2565	GVVQLTSQW	2615	ILAYCNKTV	2665	DAVRDPQTL
2566	DDTLRVEAF	2616	YQCGHYKHI	2666	ATATIPIQA
2567	RFTTTLNDF	2617	FAWWTAFVT	2667	SDRELHLSW
2568	RNPANNAAI	2618	IWLGFIAGL	2668	EWSMATYYL
2569	SWNADLYKL	2619	GGNYNYLYR	2669	VGPKVYPII
2570	TASWFTALT	2620	FISTCACEI	2670	ETGLLMPLK
2571	LSWEVGKPR	2621	FFSNYLKRR	2671	IINLIIKNL
2572	SSGDATTAY	2622	EVVLKKLKK	2672	SINFVRIIM
2573	LFWNCNVDR	2623	QLGGLHLLI	2673	RPPLNRNYV
2574	RKMAFPSGK	2624	LKVGGSCVL	2674	DGISQYSLR
2575	RLTLGVYDY	2625	RANNTKGSL	2675	TFISDEVAR
2576	LIVAAIVFI	2626	KYCALAPNM	2676	RRAFGEYSH
2577	DVEGCHATR	2627	SLVVRCSFY	2677	TTEILPVSM
2578	VPLNIIPLT	2628	VRMYIFFAS	2678	RQGFVDSDV
2579	HFLPRVFSA	2629	HVICTSEDM	2679	MLVKQGDDY
2580	WPQIAQFAP	2630	RKHFSMMIL	2680	MPNLYKMQR
2581	MGHFAWWTA	2631	KESVQTFFK	2681	HHANEYRLY
2582	VQSTQWSLF	2632	SPLSLNMAR	2682	VAPGTAVLR
2583	RLCAYCCNI	2633	PQADVEWKF	2683	FELWAKRNI
2584	DVNCTEVPV	2634	VQQLPETYF	2684	SRIKASMPT
2585	LTVFFDGRV	2635	TEILPVSMT	2685	GEIKDATPS

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
2586	KLHDELTGH	2636	NFVFPLNSI	2686	DKRAKVTSA
2587	NTLQCIMLV	2637	YSYATHSDK	2687	TNSFTRGVY
2588	VDDPCPIHF	2638	MLNPNYEDL	2688	KWPWYIWLG
2589	IIWFLLLSV	2639	AFGEYSHVV	2689	TCFSTQFAF
2590	WESGVKDCV	2640	FATSACVLA	2690	YLGKPREQI
2591	AELEGIQYG	2641	MAYCWRCTS	2691	SEDNQTTTI
2592	TPKDHIGTR	2642	GFELTSMKY	2692	LRARSVSPK
2593	LATVAYFNM	2643	QKKQQTVTL	2693	CEFQFCNDP
2594	MRFRRAFGE	2644	NKYKYFSGA	2694	IGFLFLTWI
2595	LTRNPAWRK	2645	FAQVKQIYK	2695	KSPIQYIDI
2596	KEIKESVQT	2646	HFAWWTAFV	2696	YGDSATLPK
2597	LFARTRSMW	2647	NRKRISNCV	2697	AFVETVKGL
2598	LRKQIRSAA	2648	SSQAWQPGV	2698	TPTWRVYST
2599	MPLGYVTHG	2649	SQCVNLTTR	2699	CGYLPQNAV
2600	MELTPVVQT	2650	RFQNHNPQK	2700	EAPLVGTPV
2701	KAALLADKF	2751	ALQDAYYRA	2801	GSSGVVNPV
2702	LRKLDNDAL	2752	FAAETLKAT	2802	KRSFIEDLL
2703	VSALVYDNK	2753	TLQPVSELL	2803	AYRKVLLRK
2704	ATNYDLSVV	2754	LACFVLAAV	2804	CEKALKYLP
2705	RTLLTKGTL	2755	WNVKDFMSL	2805	VAKNLNESL
2706	YPKCDRAMP	2756	LLEWLAMAV	2806	FLALITLAT
2707	NYVFTGYRV	2757	IFGTVYEKL	2807	AVFQSASKI
2708	VDTVRTNVY	2758	YDDGARRVW	2808	KVEGCMVQV
2709	NGDSEVVLK	2759	QPTSEAVEA	2809	CVDTVRTNV
2710	YLYLTFYLT	2760	WAKRNIKPV	2810	HAFLCLFLL
2711	IVNNATNVV	2761	IRASANLAA	2811	EDQDALFAY
2712	SAPLIELCV	2762	SERFQNHNP	2812	QIVESCGNF
2713	NVGPKVYPI	2763	VSKVVKVTI	2813	HSQLGGLHL
2714	TGYKKPASR	2764	TVCTVCGMW	2814	GALDISASI
2715	SVGPKQASL	2765	SSKCVCSVI	2815	LYDANYFLC
2716	SQDLSVVSK	2766	FEKGDYGDA	2816	AAMQRKLEK
2717	SLINTLNDL	2767	MVRMYIFFA	2817	VFIKRSDAR
2718	NLREMLAHA	2768	VYANLGERV	2818	GLALYYPSA
2719	QYGSFCTQL	2769	FCDLKGKYV	2819	LQPRTFLLK
2720	EMLDNRATL	2770	VSIWNLDYI	2820	NGMNGRTIL
2721	CPLIAAVIT	2771	FLIVAAIVF	2821	GRFVLALLS
2722	FMSLSEQLR	2772	NYLYRLFRK	2822	KMFDAYVNT
2723	TIFKDASGK	2773	FVDRQTAQA	2823	ATRGATVVI
2724	DALCEKALK	2774	TAFVTNVNA	2824	FGGCVFSYV
2725	APHGHVMVE	2775	KLKTLVATA	2825	VRDVLVRGF
2726	LPKGFYAEG	2776	NNCYLATAL	2826	AGTDTTITV
2727	LKQLIKVTL	2777	QQTVTLLPA	2827	KGTHHWLLL
2728	TQALPQRQK	2778	TNNVAFQTV	2828	VDWTIEYPI
2729	DFLEYHDVR	2779	IAYTMSLGA	2829	AMACLVGLM
2730	ISEMHPALR	2780	KEMYLKLRS	2830	GVTLIGEAV
2731	LRVESSSKL	2781	SWMESEFRV	2831	AQEAYEQAV
2732	DHIGTRNPA	2782	AKHDFFKFR	2832	GDCLGDIAA
2733	VTTVMFLAR	2783	CKDGHVETF	2833	GSKSPIQYI
2734	TWLTYTGAI	2784	KEGQINDMI	2834	SGLKTILRK
2735	VMFLARGIV	2785	MVLGSLAAT	2835	QLTPIAVQM
2736	TPFDVVRQC	2786	LLSVCLGSL	2836	DMSKFPLKL
2737	ILASFSAST	2787	SCDQLREPM	2837	VYDNKLKAH
2738	LGFSTGVNL	2788	VAGGIVAIV	2838	VRVLQKAAI
2739	FVCNLLLLF	2789	QWLPTGTLL	2839	PFAMQMAYR
2740	TNILLNVPL	2790	GRLQSLQTY	2840	SAYENFNQH
2741	RDAAMQRKL	2791	SAVVLLILM	2841	NLQSNHDLY
2742	NRARTVAGV	2792	SEKSYELQT	2842	FYSKWYIRV
2743	QESFGGASC	2793	AMKYNYEPL	2843	GLVEVEKGV
2744	YDKLQFTSL	2794	TLKNLSDRV	2844	NIVTRCLNR
2745	YYKKVDGVV	2795	DFSSEIIGY	2845	CEEMLDNRA
2746	LTQDHVDIL	2796	DPAQLPAPR	2846	AAITILDGI
2747	YICGFIQQK	2797	NKDFYDFAV	2847	LLSVLQQLR
2748	AQTGSSKCV	2798	YWEPEFYEA	2848	GVLTESNKK
2749	MAPISAMVR	2799	QLNRALTGI	2849	DTTITVNVL
2750	NPANNAAIV	2800	LLKDCPAVA	2850	FLFLTWICL

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
2851	MPTTIAKNT	2901	TICAPLTVF	2951	CIRCLWSTK
2852	NIVNVSLVK	2902	SATLPKGIM	2952	NTLLFLMSF
2853	ISPYNSQNA	2903	VVTCLAYYF	2953	LIRKSNHNF
2854	GYQPYRVVV	2904	VFHLYLQYI	2954	IVNGVRRSF
2855	YMRSLKVPA	2905	DEDDSEPVL	2955	NVSLVKPSF
2856	VATVQEIQL	2906	KKADETQAL	2956	LLEDEFTPF
2857	ICAPLTVFF	2907	APGQTGKIA	2957	VFMSEAKCW
2858	NQFNSAIGK	2908	HRLYECLYR	2958	IVESCGNFK
2859	NLLLLFVTV	2909	FPKSDGTGT	2959	KPTETICAP
2860	NFVRIIMRL	2910	TETDLTKGP	2960	FYEDFLEYH
2861	KPLEFGATS	2911	PPISFPLCA	2961	TTEELPDEF
2862	MQNCVLKLK	2912	LTENKYSQL	2962	KQIRSAAKK
2863	FFKLVNKFL	2913	RSGETLGVL	2963	DMILSLLSK
2864	LVQAGNVQL	2914	QPYVVDDPC	2964	MLLLLCCCL
2865	FTEERLKLF	2915	EKTHVQLSL	2965	NLYKMQRML
2866	VFTGYRVTK	2916	QKLLKSIAA	2966	GPLVRKIFV
2867	CFSTASDTY	2917	SSRSSSRSR	2967	SFGGASCCL
2868	VLPQLEQPY	2918	QAAVGELLL	2968	CHNKCAYWV
2869	MAFPSGKVE	2919	VTDFNAIAT	2969	NSVFNICQA
2870	HTANKWDLI	2920	KSREETGLL	2970	VANGDSEVV
2871	LANTCTERL	2921	RDLSLQFKR	2971	QLLFVVEVV
2872	DVNLHSSRL	2922	EHSWNADLY	2972	ALRLVDPQI
2873	KKLLEQWNL	2923	EAPFLYLYA	2973	ITEGSVKGL
2874	LELAMDEFI	2924	DAFKLNIKL	2974	GFFTYICGF
2875	TDTPKGPKV	2925	TYKLNVGDY	2975	CSHAAVDAL
2876	SGAMDTTSY	2926	VGARKSAPL	2976	DKRTTCFSV
2877	FWNCNVDRY	2927	KRRVVFNGV	2977	SGKPVPYCY
2878	AVANGDSEV	2928	TSVVLLSVL	2978	FCGPDGYPL
2879	DVVYCPRHV	2929	VFQSASKII	2979	SVEEVLSEA
2880	SDVENPHLM	2930	CANGQVFGL	2980	GIMMNVAKY
2881	SEEVVENPT	2931	KAGQKTYER	2981	EELPDEFVV
2882	AQEKNFTTA	2932	HLLIGLAKR	2982	GYVMHANYI
2883	YKLNVGDYF	2933	EIDFLELAM	2983	HLSVDTKFK
2884	YNGSPSGVY	2934	FYEAMYTPH	2984	STIGVCSMT
2885	AMMFVKHKH	2935	ITIAYIICI	2985	VLTLVYKVY
2886	NRPQIGVVR	2936	STGVNLVAV	2986	LLVPHHVVA
2887	CDQLREPML	2937	RALTAESHV	2987	DYDCVSFCY
2888	SEHDYQIGG	2938	AVVKIYCPA	2988	SALVYDNKL
2889	ITVEELKKL	2939	YYNTTKGGR	2989	HVETFYPKL
2890	FKELLVYAA	2940	TACTDDNAL	2990	NRYFRLTLG
2891	IPKDMTYRR	2941	YVLMDGSII	2991	YHNESGLKT
2892	DIVKTDGTL	2942	QVDLFRNAR	2992	YRINWITGG
2893	TLLSLREVR	2943	VECFDKFKV	2993	FMRIFTIGT
2894	NWYDFGDFI	2944	LPWNVVRIK	2994	NLVIGFLFL
2895	LVIGFLFLT	2945	ECAQVLSEM	2995	NIKPVPEVK
2896	RDAPAHIST	2946	HPNQEYADV	2996	AMMFTSDLA
2897	LGYFCTCYF	2947	DFYLCFLAF	2997	VAIVVTCLA
2898	LLLCRMNSR	2948	TIFFAGILI	2998	KVITGLHPT
2899	FNYLKSPNF	2949	VTLIGEAVK	2999	YTVSCLPFT
2900	TREAVGTNL	2950	ALTCFSTQF	3000	SKQRRPQGL
3001	EVTPSGTWL	3051	NWITGGIAI	3101	LSMQGAVDI
3002	FFYYSDSPC	3052	RVVFVLWAH	3102	HQKLLKSIA
3003	VYAWNRKRI	3053	TALRANSAV	3103	LTWICLLQF
3004	LEPLVDLPI	3054	HLLLVAAGL	3104	PHSLSDGLL
3005	GTPVCINGL	3055	YLGGMSYYC	3105	YGNALDQAI
3006	LLKSAYENF	3056	FPSGKVEGC	3106	ASKKPRQKR
3007	RQMSCAAGT	3057	IPCTCGKQA	3107	LYECLYRNR
3008	ENAFLPFAM	3058	ARLRAKHYV	3108	EGFNCYFPL
3009	SLRCGACIR	3059	TKLATTEEL	3109	ERLKLFDRY
3010	DYTEISFML	3060	CVCSVIDLL	3110	SSGVVNPVM
3011	KVTSAMQTM	3061	LRTTNGDFL	3111	LQDLKWARF
3012	RKHTTCCSL	3062	NINIVGDFK	3112	RNSTPGSSR
3013	EVAKNLNES	3063	LKLRGTAVM	3113	SHMYCSFYP
3014	VQELYSPIF	3064	MMFTSDLAT	3114	CSLCLQLAV
3015	LLFLVLIML	3065	AAVYRINWI	3115	SEDKRAKVT

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
3016	LPQGTTLPK	3066	YAISAKNRA	3116	PPTSFGPLV
3017	TEVLTEEVV	3067	FGGASCCLY	3117	KGLDYKAFK
3018	IWVATEGAL	3068	FITLCFTLK	3118	TQKGAEAAV
3019	LLQILFALL	3069	LEGYAFEHI	3119	IERYKLEGY
3020	LLVAAGLEA	3070	IIFLEGETL	3120	ILRTTNGDF
3021	YNVTQAFGR	3071	MSFTVLCLT	3121	AVTANVNAL
3022	ILANTCTER	3072	RAKVGILCI	3122	GFCDLKGKY
3023	LECIKDLLA	3073	GSVGFNIDY	3123	GDYGDAVVY
3024	LVRGFGDSV	3074	ILLIIMRTF	3124	DAIMTRCLA
3025	VLTEEVVLK	3075	FDTRVLSNL	3125	LSLPVLQVR
3026	LKFPRGQGV	3076	LKLRSDVLL	3126	GDAALALLL
3027	DGVKHVYQL	3077	IILRLGSPL	3127	TRNPANNAA
3028	LPGTILRTT	3078	EGYAFEHIV	3128	GVGGKPCIK
3029	TIQPRVEKK	3079	SDRDLYDKL	3129	LPPKNSIDA
3030	FSNVTWFHA	3080	QYGRSGETL	3130	LVQSTQWSL
3031	QASSRSSSR	3081	LEGSVRVVT	3131	YEGNSPFHP
3032	RRSFYVYAN	3082	MDTTSYREA	3132	WDYKRDAP
3033	QLRVIGHSM	3083	YRVTKNSKV	3133	IVVFDEISM
3034	EMHPALRLV	3084	GASCCLYCR	3134	SPVALRQMS
3035	LSSYSLFDM	3085	LDISASIVA	3135	QQQGQTVT
3036	DKCSRIIPA	3086	CESHGKQVV	3136	LNNIINNAR
3037	APRITFGGP	3087	LNDNLLEIL	3137	QEPKLGSLV
3038	TSTLQGCSL	3088	FGPLVRKIF	3138	VPLHGTILT
3039	NLLLQYGSF	3089	IQWMVMFTP	3139	FLTRNPAWR
3040	TRMENAVGR	3090	KEPCSSGTY	3140	KGVAPGTAV
3041	PPQTSITSA	3091	ARSVSPKLF	3141	QLRVESSSK
3042	YRFNGIGVT	3092	VIDLLLDDF	3142	QEHYVRITG
3043	LLLLFVTVY	3093	PHGHVMVEL	3143	YMHHMELPT
3044	APLVGTPVC	3094	YPDPSRILG	3144	SEGLNDNLL
3045	FGLFCLLNR	3095	TQLSTDTGV	3145	AQSFLNGFA
3046	DAAKAYKDY	3096	LIRQGTDYK	3146	VCVSTSGRW
3047	AAKAYKDYL	3097	NSYECDIPI	3147	RTVYDDGAR
3048	NPIQLSSYS	3098	VGLMWLSYF	3148	SSKLWAQCV
3049	CDHCGETSW	3099	CQEPKLGSL	3149	TFNGECPNF
3050	NGVGYQPYR	3100	NSKVQIGEY	3150	VEGCMVQVT
3151	KVTKGKAK	3201	PVNVAFELW	3251	GSLVVRCSF
3152	YKLMGHFAW	3202	KLKKSLNVA	3252	QAGNVQLRV
3153	DAALALLLL	3203	LELQDHNET	3253	GKSHFAIGL
3154	NSVTSSIVI	3204	KHKHAFLCL	3254	ACPDGVKHV
3155	LVFLFVAAI	3205	PFTIYSLLL	3255	NKDGIIWVA
3156	VPTDNYITT	3206	QTSITSAVL	3256	CAQVLSEMV
3157	DTGVEHVTF	3207	KFDTFNGEC	3257	ALLSTDGNK
3158	AAFATAQEA	3208	CWRCTSCCF	3258	VLLILMTAR
3159	TPSKLIEYT	3209	SVLYYQNNV	3259	DEAGSKSPI
3160	NVTDFNAIA	3210	LMKTIGPDM	3260	GPLSAQTGI
3161	LAVFQSASK	3211	LPTQTVDSS	3261	FLLFLVLIM
3162	CERSEAGVC	3212	DTSLSGFKL	3262	KCSRIIPAR
3163	IAEILLIIM	3213	FDKSAFVNL	3263	WYIRVGARK
3164	LSVVNARLR	3214	KGPKVKYLY	3264	FKLNEEIAI
3165	LEDKAFQLT	3215	YKFVRIQPG	3265	VFLLVTLAI
3166	LLLLEWLAM	3216	TETICAPLT	3266	GLHLLIGLA
3167	KYVQIPTTC	3217	LVPHHVVAT	3267	LYHYQECVR
3168	LITLATCEL	3218	YFNMVYMPA	3268	DAYKTFPPT
3169	KNIDGYFKI	3219	IGDPAQLPA	3269	FVRIIMRLW
3170	RKSNHNFLV	3220	YTEKWESGV	3270	MVVKAALLA
3171	FGWLIVGVA	3221	GMVLGSLAA	3271	FLWLLWPVT
3172	SKIVQLSEI	3222	RSEDKRAKV	3272	ILRGHLRIA
3173	SQGSEYDYV	3223	NAQALNTLV	3273	YEDLLIRKS
3174	KRVLNVVCK	3224	YNKYKYFSG	3274	VVRIKIVQM
3175	TEAFEKMVS	3225	TPCGTGTST	3275	ELFYSYATH
3176	LGSPLSLNM	3226	CAAGTTQTA	3276	DVVRQCSGV
3177	KQVVSDIDY	3227	VYTACSHAA	3277	DRRATCFST
3178	ITVTPEANM	3228	IFFAGILIV	3278	NSQNAVASK
3179	DKFTDGVCL	3229	VPANSTVLS	3279	DTNVLEGSV
3180	GIVFMCVEY	3230	LRLIDAMMF	3280	DVKCTSVVL

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
3181	IIKNLSKSL	3231	HTNCYDYCI	3281	IVQMLSDTL
3182	HVQLSLPVL	3232	QPGVAMPNL	3282	PFMIDVQQW
3183	TEELPDEFV	3233	VAYSNNSIA	3283	NGVRRSFYV
3184	YVFTGYRVT	3234	RSGARSKQR	3284	MQGAVDINK
3185	AIVFITLCF	3235	ILRKGGRTI	3285	GPEQTQGNF
3186	GRSGETLGV	3236	CELYHYQEC	3286	NLIIKNLSK
3187	LTGHMLDMY	3237	IQPIGALDI	3287	SFYVYSRVK
3188	TLIVNSVLL	3238	RVECFDKFK	3288	LLIGLAKRF
3189	VILRGHLRI	3239	LADNKFALT	3289	SHAAVDALC
3190	SSAINRPQI	3240	SNIIRGWIF	3290	IKDFGGFNF
3191	RGTAVMSLK	3241	APRTLLTKG	3291	SLNMARKTL
3192	VLQVRDVLV	3242	ASIVAGGIV	3292	IKGTHHWLL
3193	KSTNLVKNK	3243	VVTTVMFLA	3293	VRITGLYPT
3194	SKEGFFTYI	3244	FLKEQHCQK	3294	NKSWMESEF
3195	VGEIPVAYR	3245	GVCVSTSGR	3295	RAGKASCTL
3196	AQPCSDKAY	3246	ESELVIGAV	3296	LGIITTVAA
3197	GTGTIYTEL	3247	NPKAIKCVP	3297	YANSVFNIC
3198	ELVAELEGI	3248	ELKFNPPAL	3298	AYCCNIVNV
3199	TLCFTLKRK	3249	GHHLGRCDI	3299	VPINTNSSP
3200	PLMYKGLPW	3250	YFCTCYFGL	3300	HKDKSAQCF
3301	FYYLGTGPE	3351	QRVAGDSGF	3401	CAKEIKESV
3302	KCDRAMPNM	3352	YSPIFLIVA	3402	IIPLTTAAK
3303	LLEQWNLVI	3353	VPRASANIG	3403	LLSKGRLII
3304	SFGPLVRKI	3354	ISSVLNDIL	3404	ECPNFVFPL
3305	TGLYPTLNI	3355	GLTVLPPLL	3405	GDYILANTC
3306	TVCGMWKGY	3356	IVQLSEISM	3406	SGWTAGAAA
3307	LLQNGMNGR	3357	TLETAQNSV	3407	LVYCFLGYF
3308	TEIYQAGST	3358	LFLTWICLL	3408	QRNFYEPQI
3309	FQQFGRDIA	3359	FLIGCNYLG	3409	VRAWIGFDV
3310	SQAWQPGVA	3360	SQWLTNIFG	3410	FPVLHDIGN
3311	LITGRLQSL	3361	LCDRRATCF	3411	CTLSEQLDF
3312	LMPILTLTR	3362	LLWPVTLAC	3412	QENWNTKHS
3313	SIKNFKSVL	3363	ICISTKHFY	3413	ICYTPSKLI
3314	VELFENKTT	3364	DFATSACVL	3414	TEGLCVDIP
3315	DPNFKDQVI	3365	QAAGTDTTI	3415	TDFVNEFYA
3316	RPQGLPNNT	3366	QSRNLQEFK	3416	QMEIDFLEL
3317	VTLKQGEIK	3367	CNDPFLGVY	3417	NSTLEQYVF
3318	LTPFARCCW	3368	GSSKCVCSV	3418	RPFLCCKCC
3319	RIRSVYPVA	3369	DDYFNKKDW	3419	QECSLQSCT
3320	MDSTVKNYF	3370	FITREEAIR	3420	DQAMTQMYK
3321	QEFRYMNSQ	3371	MCVEYCPIF	3421	AISAKNRAR
3322	IRENNRVVI	3372	LPTEVLTEE	3422	PANSTVLSF
3323	VGVALLAVF	3373	QRQKKQQTV	3423	KEQHCQKAS
3324	CLAYYFMRF	3374	DLTKPYIKW	3424	QVNGYPNMF
3325	YLKRRVVFN	3375	AISMWALII	3425	NKHADFDTW
3326	YCALAPNMM	3376	KSAPLIELC	3426	CYNGSPSGV
3327	IFLWLLWPV	3377	SETKCTLKS	3427	CNLGGAVCR
3328	CLFLLPSLA	3378	TGTLIVNSV	3428	ITRAKVGIL
3329	TESIVRFPN	3379	MFHLVDFQV	3429	YDFAVSKGF
3330	CTQHQPYVV	3380	SSGVTRELM	3430	CAYWVPRAS
3331	DGWEIVKFI	3381	VEGFNCYFP	3431	EGNSPFHPL
3332	IVGVALLAV	3382	EQYIKWPWY	3432	DEVRQIAPG
3333	VEWKFYDAQ	3383	LNEEIAIIL	3433	KAFQLTPIA
3334	LIGCNYLGK	3384	NRVVISSDV	3434	NEFYAYLRK
3335	TLQGCSLCL	3385	KLALGGSVA	3435	GPLKVGGSC
3336	APAHISTIG	3386	MRTFKVSIW	3436	LQFTSLEIP
3337	TEISFMLWC	3387	KGFYAEGSR	3437	AGNGGDAAL
3338	REFVFKNID	3388	IIRENNRVV	3438	FKLSYGIAT
3339	SGVYQCAMR	3389	LGTVSWNLR	3439	NGQVFGLYK
3340	KQQTVTLLP	3390	NHNFLVQAG	3440	NTDFSRVSA
3341	LFKDCSKVI	3391	QSCTQHQPY	3441	VPVVDSYYS
3342	RQKRTATKA	3392	SDFVRATAT	3442	RMNSRNYIA
3343	CSTMTNRQF	3393	LLPLTQYNR	3443	WPLIVTALR
3344	LEGSVAYES	3394	FIKQYGDCL	3444	ERSEAGVCV
3345	TILTSLLVL	3395	CVRGTTVLL	3445	RTRSMWSFN

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
3346	HGTILTRPL	3396	NYYKKVDGV	3446	MRVIHFGAG
3347	TCLAYYFMR	3397	TTTLNGLWL	3447	ACFVLAAVY
3348	VAALTNNVA	3398	QKEMATSTL	3448	KDLSPRWYF
3349	LMWLSYFIA	3399	VGELLLLEW	3449	QLQQSMSSA
3350	ALALLLLDR	3400	SMDNSPNLA	3450	LAVHECFVK
3451	SIINNTVYT	3501	ERVRQALLK	3551	GDDYVYLPY
3452	ESIVRFPNI	3502	QVVDADSKI	3552	FMIDVQQWG
3453	ACPLIAAVI	3503	EFLRDGWEI	3553	QPYVFIKRS
3454	RRATRRIRG	3504	CVLAAECTI	3554	SRILGAGCF
3455	QLQAAVGEL	3505	TPFARCCWP	3555	KDCVVLHSY
3456	KAKKGAWNI	3506	AACRKVQHM	3556	EPEEHVQIH
3457	VDRQTAQAA	3507	KSHFAIGLA	3557	KSFTVEKGI
3458	SPLYAFASE	3508	NRDVDTDFV	3558	YPNMFITRE
3459	TKRNVIPTI	3509	FKDASGKPV	3559	ILNNLGVDI
3460	SNSGSDVLY	3510	ELWAKRNIK	3560	KELLVYAAD
3461	SQTSLRCGA	3511	ALRQMSCAA	3561	QWNLVIGFL
3462	YNLPTMCDI	3512	ILSLLSKGR	3562	KPRPPLNRN
3463	EVLTEEVVL	3513	LVDFQVTIA	3563	SYSLFDMSK
3464	HGHVMVELV	3514	NGECPNFVF	3564	LNKEMYLKL
3465	GAEAAVKPL	3515	KPGNFNKDF	3565	DILGPLSAQ
3466	LKTGDLQPL	3516	IRRPFLCCK	3566	GTLIVNSVL
3467	FSTASDTYA	3517	CGACIRRPF	3567	RSLPGVFCG
3468	RFQTLLALH	3518	PQLEQPYVF	3568	NMFITREEA
3469	VRCSFYEDF	3519	NTTKGGRFV	3569	VTLACFVLA
3470	GARRVWTLM	3520	MVMFTPLVP	3570	YRYKPHSLS
3471	FMSEAKCWT	3521	ECIKDLLAR	3571	LVEVEKGVL
3472	LIGLAKRFK	3522	LSEARQHLK	3572	AARYMRSLK
3473	GAKLKALNL	3523	FGEYSHVVA	3573	TFFIYNKIV
3474	NEKTHVQLS	3524	LDKSAGFPF	3574	LRVCVDTVR
3475	GRYMSALNH	3525	TSWQTGDFV	3575	IPLMYKGLP
3476	WDLIISDMY	3526	DELTGHMLD	3576	SYAAFATAQ
3477	SVCRLMKTI	3527	RMLLEKCDL	3577	NGLWLDDVV
3478	TRCNLGGAV	3528	YTERSEKSY	3578	TSDYYQLYS
3479	RNYIAQVDV	3529	VDIAANTVI	3579	VSFSTFEEA
3480	KRTTCFSVA	3530	LNNDYYRSL	3580	TITVEELKK
3481	LSKSLTENK	3531	PAFDKSAFV	3581	MIELSLIDF
3482	KGIMMNVAK	3532	QGSEYDYVI	3582	LLPSLATVA
3483	YDYVIFTQT	3533	CVPQADVEW	3583	KFLVFLGII
3484	MAGNGGDAA	3534	LEQWNLVIG	3584	CNVNRFNVA
3485	SYYKLGASQ	3535	FDYVYNPFM	3585	RRARSVASQ
3486	EKFKEGVEF	3536	NKGAGGHSY	3586	LFLVLIMLI
3487	VTLLPAADL	3537	MKTIGPDMF	3587	AGSKSPIQY
3488	LPETYFTQS	3538	FFTYICGFI	3588	RWFLNRFTT
3489	SAMQTMLFT	3539	TDFATSACV	3589	FNVAITRAK
3490	NTCVGSDNV	3540	CRFDTRVLS	3590	VEKGIYQTS
3491	SLRLIDAMM	3541	SEAFLIGCN	3591	PPLNRNYVF
3492	KCYGVSPTK	3542	GNICYTPSK	3592	YDYCIPYNS
3493	LHFLLFFRA	3543	HKHAFLCLF	3593	ALCADSIII
3494	QASLPFGWL	3544	GDFKLNEEI	3594	QNYGDSATL
3495	LMGHFAWW	3545	TDDNALAYY	3595	LPFTINCQE
3496	YNSVTS SIV	3546	EIPVAYRKV	3596	SHLLLVAAG
3497	NNLVVMAYI	3547	TKYTMADLV	3597	CRHHANEYR
3498	LVPQEHYVR	3548	LYAFASEAA	3598	MDQESFGGA
3499	SVRVLQKAA	3549	PWNVVRIKI	3599	TPGSSRGTS
3500	QVTCGTTTL	3550	VPTGYVDTP	3600	RYPANSIVC
3601	PYEDFQENW	3651	YSDSPCESH	3701	AVNLLTNMF
3602	GARKSAPLI	3652	LTCFSTQFA	3702	YSKWYIRVG
3603	INNTVYTKV	3653	SARIVYTAC	3703	LSVVSKVVK
3604	FKPRSQMEI	3654	NYFLCWHTN	3704	LIMLIIFWF
3605	NSSRVPDLL	3655	MSDRDLYDK	3705	KINAACRKV
3606	ALVYDNKLK	3656	ISRQRLTKY	3706	GILCIMSDR
3607	SIFSRTLET	3657	HMELPTGVH	3707	IKVTLVFLF
3608	NNFCGPDGY	3658	EWLAMAVML	3708	LRCGACIRR
3609	FNAIATCDW	3659	QKSILSPLY	3709	VVQLTSQWL
3610	QLESKMSGK	3660	WNLDYIINL	3710	SQMEIDFLE

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
3611	QPIGALDIS	3661	IELCVDEAG	3711	YALVYFLQS
3612	DPCPIHFYS	3662	LFLAFVVFL	3712	YKLGASQRV
3613	CPNFVFPLN	3663	DQIGYYRRA	3713	VQEIQLQAA
3614	GSEYDYVIF	3664	KDMTYRRLI	3714	RGGSQASSR
3615	AMQRKLEKM	3665	KDGHVETFY	3715	DEPEEHVQI
3616	DGLLLALHF	3666	KDCVMYASA	3716	YTKVDGVDV
3617	ASFSASTSA	3667	YKRDAPAHI	3717	AKVGILCIM
3618	APATVCGPK	3668	VAGDSGFAA	3718	GIAIAMACL
3619	IGNYTVSCL	3669	IAVQMTKLA	3719	YRRATRRIR
3620	GEVPVSIIN	3670	LAVTRMENA	3720	NDVSFLAHI
3621	NNELSPVAL	3671	VGCHNKCAY	3721	LNKHIDAYK
3622	VKCTSVVLL	3672	RSQMEIDFL	3722	WLCWKCRSK
3623	FCAFAVDAA	3673	YAAFATAQE	3723	DNKFALTCF
3624	NHNPQKEMA	3674	TAQEAYEQA	3724	NMRVIHFGA
3625	VLIMLIIFW	3675	PEAGLPYGA	3725	FSTQFAFAC
3626	LKQLPFFYY	3676	IMMNVAKYT	3726	FNICQAVTA
3627	AVINGDRWF	3677	GSEGLNDNL	3727	RCLAVHECF
3628	YQLRARSVS	3678	CNIVNVSLV	3728	TPIAVQMTK
3629	RTTCFSVAA	3679	ILDGISQYS	3729	MESLVPGFN
3630	FHLVDFQVT	3680	WFLNRFTTT	3730	NLAWPLIVT
3631	RSDARTAPH	3681	EQIDGYVMH	3731	MRLWLCWKC
3632	YVFIKRSDA	3682	MHHMELPTG	3732	VTCLAYYFM
3633	RELMRELNG	3683	SDYYQLYST	3733	WNLVIGFLF
3634	FVTNVNASS	3684	ECVRGTTVL	3734	KQLQQSMSS
3635	DCVVLHSYF	3685	MNLKYAISA	3735	ISDMYDPKT
3636	PSLATVAYF	3686	KAIKCVPQA	3736	YQLYSTQLS
3637	VCNSLLTPF	3687	YDFGDFIQT	3737	LPTGTLLVD
3638	PFLYLYALV	3688	RNARNGVLI	3738	KQASLNGVT
3639	VDTVSALVY	3689	IVAAIVFIT	3739	EETIYNLLK
3640	WALIISVTS	3690	SGETLGVLV	3740	WLTYTGAIK
3641	VYSHLLLVA	3691	YDYYRYNLP	3741	PEVKILNNL
3642	YCKSHKPPI	3692	YAYLRKHFS	3742	SVLLSMQGA
3643	SPNECNQMC	3693	RVAGDSGFA	3743	FALTCFSTQ
3644	EETGTLIVN	3694	GIPKDMTYR	3744	KPVPYCYDT
3645	GLPNNTASW	3695	PMDSTVKNY	3745	TENKYSQLD
3646	VQMTKLATT	3696	VCRHHANEY	3746	RDGCVPLNI
3647	KEMATSTLQ	3697	YRRLISMMG	3747	PFLCCKCCY
3648	CATVHTANK	3698	PLADNKFAL	3748	CPIFFITGN
3649	LEQPYVFIK	3699	DFVRATATI	3749	TIKGTHHWL
3650	CSKVITGLH	3700	ASANIGCNH	3750	APLTVFFDG
3751	MVVIPDYNT	3801	KVTIDYTEI	3851	LRIAGHHLG
3752	LFRNARNGV	3802	NSTVLSFCA	3852	HIVYGDFSH
3753	LLEILQKEK	3803	YDCVSFCYM	3853	QLDFIDTKR
3754	EELKKLLEQ	3804	GTTFTYASA	3854	IPDYNTYKN
3755	TTTLNDFNL	3805	YSFVSEETG	3855	SNHNFLVQA
3756	EHDYQIGGY	3806	ESPFELEDF	3856	NKTTLPVNV
3757	GTAVLRQWL	3807	KTVQFCDAM	3857	WHTNCYDYC
3758	NPQKEMATS	3808	KTEGLCVDI	3858	QIAQFAPSA
3759	FARTRSMWS	3809	VDSSQGSEY	3859	FRLFARTRS
3760	EGNFYGPFV	3810	WITGGIAIA	3860	LEDFIPMDS
3761	GYRVTKNSK	3811	REGVFVSNG	3851	LRIAGHHLG
3762	EPIYDEPTT	3812	VYPIILRLG	3852	HIVYGDFSH
3763	VDRYPANSI	3813	IVTTIVYLT	3853	QLDFIDTKR
3764	NGRTILGSA	3814	WAHGFELTS	3854	IPDYNTYKN
3765	YHYQECVRG	3815	LDQAISMWA	3855	SNHNFLVQA
3766	AIRHVRAWI	3816	FNVVNKGHF	3856	NKTTLPVNV
3767	SLLTPFARC	3817	LLLFVTVYS	3857	WHTNCYDYC
3768	LVTLAILTA	3818	AWYTERSEK	3858	QIAQFAPSA
3769	GS SVELKHF	3819	VNGYPNMFI	3859	FRLFARTRS
3770	QLEQPYVFI	3820	TLVFLFVAA	3860	LEDFIPMDS
3771	SRGTSPARM	3821	MQRKLEKMA	3851	LRIAGHHLG
3772	SDEFSSNVA	3822	LCNSQTSLR	3852	HIVYGDFSH
3773	DDNLIDSYF	3823	ATPSDFVRA	3853	QLDFIDTKR
3774	LALLLLDRL	3824	TQFAFACPD	3854	IPDYNTYKN
3775	SQASSRSSS	3825	GAVILRGHL	3855	SNHNFLVQA

Seq. ID	Peptide	Seq. ID	Peptide	Seq. ID	Peptide
3776	LSVLLSMQG	3826	ELLLLEWLA	3856	NKTTLPVNV
3777	NKCAYWVPR	3827	KHLIPLMYK	3857	WHTNCYDYC
3778	IPMDSTVKN	3828	LGGSVAIKI	3858	QIAQFAPSA
3779	FKLVNKFLA	3829	FHQECSLQS	3859	FRLFARTRS
3780	DTYACWHHS	3830	YCRHGTCER	3860	LEDFIPMDS
3781	TPVHVMSKH	3831	ELQTPFEIK
3782	FDMSKFPLK	3832	DDPCPIHFY
3783	TDFNAIATC	3833	CDWTNAGDY
3784	RSMWSFNPE	3834	GSALLEDEF
3785	HCANFNVLF	3835	SKWYIRVGA
3786	RGMVLGSLA	3836	MAVMLLLLC
3787	FRKMAFPSG	3837	KDFYDFAVS
3788	ISASIVAGG	3838	SGVKDCVVL
3789	FAGILIVTT	3839	AQSFLNRVC
3790	YRSLPGVFC	3840	SSSEAFLIG
3791	TLATCELYH	3841	GVEHVTFFI
3792	NQDLNGNWY	3842	MLRKLDNDA
3793	WWTAFVTNV	3843	CVEYCPIFF
3794	LLFVTVYSH	3844	SPIQYIDIG
3795	MDLFMRIFT	3845	LDMYSVMLT
3796	VDTDLTKPY	3846	LNVGDYFVL
3797	DQVILLNKH	3847	TRAGCLIGA
3798	APSASAFFG	3848	MCASLKELL
3799	IKDATPSDF	3849	GQTFSVLAC
3800	RRVVFNGVS	3850	VMSLKEGQI

Table 16. Region-specific peptide pools derived from whole proteome for CD8 T cell assays.

Table 17. Region-specific peptide pools derived from S protein for CD8 T cell assays.

Table 18.. Region-specific peptide pools derived from NSP3 protein for CD8 T cell assays.

Table 19. Region-specific peptide pools derived from NSP4 protein for CD8 T cell assays.

Table 20. Region-specific peptide pools derived from NSP14 protein for CD8 T cell assays.

Table 21. Region-specific peptide pools derived from NSP16 protein for CD8 T cell assays.

Table 22. Region-specific peptide pools derived from N protein for CD8 T cell assays.

Table 23. Region-specific peptide pools derived from M protein for CD8 T cell assays.

Table 24. Region-specific peptide pools derived from NSP5 protein for CD8 T cell assays.

Table 25. Region-specific peptide pools derived from NSP9 protein for CD8 T cell assays.

Table 26. Region-specific peptide pools derived from NSP12 protein for CD8 T cell assays.

Table 27. Region-specific peptide pools derived from NSP6 protein for CD8 T cell assays.

Table 28. Region-specific peptide pools derived from E protein for CD8 T cell assays.

Table 29. Region-specific peptide pools derived from NSP8 protein for CD8 T cell assays.

Table 30. Region-specific peptide pools derived from ORF3a protein for CD8 T cell assays.

Table 31. Region-specific peptide pools derived from NSP1 protein for CD8 T cell assays.

Table 32. Region-specific peptide pools derived from ORF7a protein for CD8 T cell assays.

Table 33. Region-specific peptide pools derived from NSP2 protein for CD8 T cell assays.

Table 34. Region-specific peptide pools derived from NSP13 protein for CD8 T cell assays.

Table 35. Region-specific peptide pools derived from ORF8 protein for CD8 T cell assays.

Table 36. Region-specific peptide pools derived from ORF6 protein for CD8 T cell assays.

Table 37. Region-specific peptide pools derived from NSP15 protein for CD8 T cell assays.

Table 38. Region-specific peptide pools derived from ORF3c protein for CD8 T cell assays.

Table 39. Region-specific peptide pools derived from NSP11 protein for CD8 T cell assays.

Region	Seq. IDs of peptides
Australia	549, 615
Europe	549, 615
North Africa	549, 615, 3145
North America	615
North East Asia	549, 615
Oceania	549, 615
South and Central America	549, 615
South Asia	549, 615, 3145
South East Asia	549, 615
Sub-Saharan Africa	549, 615
Western Asia	549, 615, 3145

Table 40. Region-specific peptide pools derived from ORF3d protein for CD8 T cell assays.

Table 41. Region-specific peptide pools derived from ORF10 protein for CD8 T cell assays.

Table 42. Region-specific peptide pools derived from ORF9b protein for CD8 T cell assays.

Table 43. Region-specific peptide pools derived from NSP10 protein for CD8 T cell assays.

Table 44. Region-specific peptide pools derived from ORF9c protein for CD8 T cell assays.

Table 45. Region-specific peptide pools derived from NSP7 protein for CD8 T cell assays.

Table 46. Region-specific peptide pools derived from ORF3b protein for CD8 T cell assays.

Region	Seq. IDs of peptides
Australia	835, 1199, 1386, 2221, 2335, 3228
Europe	835, 1199, 1386, 2221, 2335, 2948
North Africa	835, 1199, 2221, 2255, 2335, 2505, 2948, 3228, 3813
North America	835, 1386, 2221, 3228, 3789
North East Asia	835, 1199, 1386, 2221, 2255, 2335, 2505, 2948, 3228, 3789
Oceania	835, 1199, 1386, 2221, 2335, 2505, 3228
South and Central America	835, 1199, 1386, 2221, 2255, 2335, 2505
South Asia	835, 1199, 1386, 2221, 2255, 2335, 2948, 3228
South East Asia	835, 1199, 1386, 2221, 2335, 2505, 3789
Sub-Saharan Africa	835, 1199, 1386, 2221, 2335, 2505, 2948, 3228, 3813
Western Asia	835, 1199, 1386, 2221, 2255, 2335, 2505, 2948, 3228

Table 47. Region-specific peptide pools derived from ORF7b protein for CD8 T cell assays.

Discussion

SARS2TPools is a first-of-its-kind software platform that provides optimized SARS-CoV-2 peptide pools for assessing vaccine-induced T cell responses in a geographical region. These pools can be used to explore key questions related to COVID-19 vaccines and the role of T cell immunity. For example, characterizing effects of mutations within T cell epitopes in emerging SARS-CoV-2 variants on vaccine-induced T cell responses, investigating association between targeting specific T cell epitopes and protection from severe disease, characterizing the breadth, diversity, and durability of vaccine-induced T cell responses, and contrasting the T cell responses elicited by different vaccines. These questions are important and relevant for both academic and industry research focused on development and assessment of COVID-19 vaccines. Answering them can help provide pre-emptive indicators of the potential for T-cell escape (e.g., due to variants) for specific vaccines.

At present, HLA alleles commonly found in various regions (e.g., South-east Asia, South Asia, and Oceania) are underrepresented in the available experimental data (FIG. 6B) . T cell responses against SARS-CoV-2 proteins other than S are also understudied (FIG. 6C) . Characterizing these responses is particularly important for inactivated whole-virion based vaccines (already in use in more than 48 countries (Shrotri et al. 2021) ) that have been shown to elicit T cell responses against not only S but also against other SARS-CoV-2 proteins (Bueno et al. 2021) . This characterization will also be important for emerging SARS-CoV-2 vaccines (Hwang et al. 2021; Sohail et al. 2021) that incorporate domains or peptides derived from S as well as other SARS-CoV-2 proteins (e.g., EpiVacCorona involves peptides derived from both S and nucleocapsid (Aleksandr B. Ryzhikov et al. 2021; A. B. Ryzhikov et al. 2021) ) . The peptide pools provided by the developed platform can help with assessing T cell responses in above scenarios.

Analysis of the predictions of our in silico approach revealed that some of the experimentally-determined epitopes may be highly promiscuous and thus may cover a large percentage of the global population. Specifically, we identified 12 epitopes which have been experimentally-determined to be associated with at least 2 HLA alleles, while they are predicted to be associated with at least eight or more additional HLA alleles (Table 14) . For example, in silico predictions suggest that the epitope YLQPRTFLL-the most immunoprevalent SARS-CoV- 2 epitope determined so far (Quadeer et al. 2021) -is associated with a total of 22 HLA alleles, two of which (HLA-A*02: 01 and HLA-B*08: 01) have been determined experimentally thus far. While further experiments are required to validate these predicted associations, such promiscuous epitopes, recognized potentially by a large population, can be of interest in the context of designing robust next generation COVID-19 vaccines.

Table 14. List of epitopes having experimentally-determined association with at least 2 HLA alleles and in silico predicted association with at least 8 additional HLA alleles.

Compared to T cell response assessment using overlapping peptide pools, peptide pools optimized for a specific region comprise of a limited set of peptides in the context of cognate HLA alleles, and would thus be less susceptible to peptide competition (Pala et al. 1988) . This is of practical importance particularly for a virus like SARS-CoV-2 which has a large (～10k residues) proteome. In addition to overlapping peptide pools, generalized peptide pools have also been proposed to assess SARS-CoV-2 T cell responses. Such pools comprise of peptides associated with a few globally prevalent HLA alleles (e.g., the pool proposed in (Grifoni et al. 2020) comprises of peptides associated with 12 most-prevalent HLA-A and -B alleles) . Compared to such generalized peptide pools, a peptide pool optimized for the HLA alleles prevalent in a specific region would be expected to measure T cell responses in a population more comprehensively.

Recent studies have demonstrated that COVID-19 vaccines elicit strong CD4 ⁺ T cell responses, along with CD8 ⁺ responses (Sahin et al. 2020; Tauzin et al. 2021) . The pools for measuring CD4 ⁺ T cell responses, provided on the platform at present, comprise only of experimentally-determined CD4 ⁺ T cell epitopes. In future when sufficient experimentally-determined HLA-resolved CD4 ⁺ T cell epitope data becomes available, CD4 ⁺ pools provided by SARS2TPools may then be supplemented by in silico predictions following an approach similar to the one employed for CD8 ⁺ T cell epitopes. SARS2TPools will be periodically updated to incorporate more experimental CD4 ⁺ and CD8 ⁺ T cell epitope data as it becomes available.

Methods and Materials

Union approach. In this work, we have proposed a union approach based on combining the top-ranked predictions of MHCflurry2.0P and NetMHCpan4.1BA to obtain a set of peptides restricted by a given HLA. This approach was motivated by performance comparison analysis of the 12 in silico epitope prediction methods listed above. Briefly, we ranked peptides in ascending order of their predicted score using each method and compared the histograms of ranks of experimentally-determined SARS-CoV-2 CD8 ⁺ T cell epitopes associated with the 10 HLA class I alleles with the most data. We found that these histograms were bi-modal for all 12 methods. That is, while the top predictions of each method contained a large number of experimentally-determined epitopes, a good number of epitopes were also ranked quite low by each method (FIG. 10) . Exploring the relationships among the set of top 20 ranked peptides per HLA allele predicted by these methods revealed that the predictions of MHCflurry2.0P were most distinct from those of other methods (FIGS. 11A-D) . Consistent results were obtained when this set was constructed by pooling the top 10 to top 25 ranked peptides restricted by each HLA allele (FIGS. 11A-D) . Predictions of MHCflurry2.0P also contained a large number of experimentally-determined SARS-CoV-2 epitopes that were not present in the set of top-ranked peptides predicted by any other method (FIG 7A) . Given the uniqueness of the predictions of MHCflurry2.0P, we asked if a strategy that combines the predictions of MHCflurry2.0P with any of the other 11 methods would work better than any individual method. The union strategy combines the top x predictions of any two methods and provides a set of peptides whose size can vary between x and 2x depending on the number of common peptides predicted by each method. We fixed one of the methods as MHCflurry2.0P and predicted 11 peptide pools by combining predictions of MHCflurry2.0P with those of the other 11 methods. Our analysis showed that the pool predicted by the union approach always had a higher hit-rate (the fraction of experimentally-determined SARS-CoV-2 epitopes present in the set of top-ranked peptides) than those predicted by the individual methods (FIG. 12) . While comparison among the various union approaches did not readily reveal a clear winner, the unions of MHCflurry2.0P with the in silico methods NetMHC4.0, NetMHCpan4.0BA, NetMHCpan4.1BA, and NetMHCpan4.1EL ranked among the top (FIG. 13 Table 13. )

Table 13. Performance comparison of the MHCflurry2.0P-based union methods based on the rank-sum metric, R _i.

*Rank-sum metric R _i is defined as

where r _i (x) is the i-th union method’s rank (assigned based on hit-rate) among the 11 union methods for the set of top x ranked predicted peptides (FIG. 13) . Hit-rate represents the fraction of experimentally known epitopes present in the set of top x ranked predicted peptides.

EXAMPLE 3 –COVIDEP: A WEB-BASED PLATFORM FOR REAL-TIME RESPORTING OF VACCINE TARGET RECOMMENDATIONS FOR SARS-COV-2

Introduction

The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has brought much of the world to a virtual lockdown. As the virus continues to spread rapidly and the pandemic intensifies, the need for an effective vaccine is becoming increasingly apparent. A critical part of vaccine design is to identify targets, or epitopes, that can induce an effective immune response against SARS-CoV-2. This problem is challenged by our limited understanding of this novel coronavirus and of its interplay with the human immune system.

In response to this challenge, we have developed COVIDep (COVIDep. ust. hk) , a first-of-its-kind web-based platform that pools genetic data for SARS-CoV-2 and immunological data for the 2003 SARS virus, SARS-CoV, to identify B-cell and T-cell epitopes to serve as vaccine target recommendations for SARS-CoV-2 (FIG. 14A) . The identified epitopes are experimentally-derived from SARS-CoV and have a close genetic match with the available SARS-CoV-2 sequences (see FIG. 15 for a detailed protocol description) . Briefly, COVIDep periodically pools SARS-CoV-2 sequence data from the GISAID database (gisaid. org) and compares with experimentally-determined T cell and B cell epitopes of SARS-CoV, obtained from the ViPR database (www. viprbrc. org) . The T cell epitopes were determined based on either positive T cell assays or positive MHC binding assays for SARS-CoV. For the B cell epitopes, both linear and discontinuous epitopes were considered. The system outputs those epitopes that are genetically similar in SARS-CoV-2, based on an epitope screening parameter. This user-defined parameter allows the user to select epitopes based on their conservation in the SARS-CoV-2 sequence data, where conservation is defined as the fraction of SARS-CoV-2 sequences with the exact epitope sequence. The value of this parameter is set to 0.95 as default; however, the user may change this value to adjust the stringency of the screening criterion. For example, reducing the value of the parameter will allow for the consideration of epitopes with greater genetic variation, potentially increasing the set of recommended SARS-CoV-2 vaccine targets. For the identified T cell epitopes, the population coverage analysis tool available at IEDB (www. iedb. org) is used to estimate the percentage of a specified population that can elicit a response against them. For T-cell epitopes, it provides estimates of population coverage, globally and for specific regions. COVIDep is flexible and user-friendly, comprising an intuitive graphical interface and interactive visualizations. In addition to producing formatted, exportable lists of the identified B-cell and T-cell epitopes and their basic characteristics, COVIDep includes displays for each of the SARS-CoV-2 proteins, showing the locations of the identified epitopes on the primary structure. Further graphical displays are provided to aid interpretation of the data, including a temporal and geographical breakdown of the analysed sequences, and a display of the observed genetic variation (amino acid mutation frequencies) for each of the SARS-CoV-2 proteins. The platform is updated daily, based on the latest SARS-CoV-2 sequence data in the GISAID database (gisaid. org) . Periodic updates are important since SARS-CoV-2 sequences are being made available at an increasing rate through international data sharing efforts, and the identification of vaccine targets is influenced by newly observed genetic variation.

The vaccine targets recommended by COVIDep exploit the genetic similarities between SARS-CoV-2 and SARS-CoV, along with known immune targets for SARS-CoV that have been determined experimentally (available at the ViPR database; viprbrc. org) . The system implements a protocol that identifies, from among the SARS epitopes that can induce a human immune response, those that are genetically similar in SARS-CoV-2. This approach, proposed and tested in Example 1 [see also 56] based on limited early data, identified known SARS-CoV epitopes that had an identical genetic match in SARS-CoV-2. These epitopes presented initial vaccine target recommendations for potentially eliciting a protective, cross-reactive immune response against SARS-CoV-2. Similar results were reported in a subsequent independent study [57] , where a related approach exploiting genetic similarity between SARS-CoV and SARS-CoV-2 was used to identify potential SARS-CoV-2 vaccine targets.

The use of SARS-CoV immunological data to inform vaccine targets for SARS-CoV-2 is being supported by experimental results. There is evidence of cross-neutralization by SARS-CoV-derived antibodies binding to genetically similar regions of SARS-CoV-2’s spike protein [58-60] . Conversely, studies have demonstrated that specific SARS-CoV-derived antibodies binding to the spike’s receptor binding domain, which has significant genetic differences in SARS-CoV-2, have limited cross-reactivity [61] . T cell responses against spike protein epitopes that are genetically similar in SARS-CoV and SARS-CoV-2 have also been reported in COVID-19 infected patients [62, 63] , and in a preclinical vaccine trial [64] (FIG. 14B) . For instance, FIG. 14B illustrates the T-cell epitopes reported by COVIDep (as of 20 May 2020) for the spike protein of SARS-CoV-2. Here, the Search box (in the top right) was used to select only the HLA-A*02: 01-restricted epitopes. (An explanation of all interactive COVIDep visualizations is incorporated in the “How to use COVIDep page” of the platform. ) . Of the 14 epitopes listed in the display, 9 of them (

IEDB IDs

36724, 54507, 54725, 69657, 71663, 2801, 54680, 16156, and 37289) overlap with epitopes against which cytotoxic CD8+ T cell responses have been observed in peripheral blood mononuclear cells isolated from COVID-19 patients [62, 63] . T cell responses were also recorded against protein regions overlapping with the epitope with IEDB ID 71663 in a pre-clinical trial of a DNA vaccine candidate [64] . Epitopes recommended by COVIDep have notable overlap with the findings in these and other [65, 66] experimental studies [67] (see Figures 2 and 3 in [67] ) .

The recommendations provided by COVIDep may be used to broadly guide vaccine designs and associated experimental studies, and may help to expedite the discovery of an effective vaccine for COVID-19.

Methods and Materials

Data availability. The SARS-CoV-2 full genome sequence data was periodically downloaded from the Global Initiative on Sharing Avian Influenza Database (GISAID; www. gisaid. org) . The SARS-CoV epitope sequence data was downloaded from the Virus Pathogen Database and Analysis Resource (ViPR; viprbrc. org) . The population coverage statistics of HLA alleles were obtained from the Immune Epitope Database and Analysis Resource (IEDB; iedb. org) .

Code availability. The source code for the developed platform is available at the COVIDep GitHub repository github. com/COVIDep) .

All patents, patent applications, and other publications, including GenBank Accession Numbers and equivalents, cited in this application are incorporated by reference in the entirety for all purposes.

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Claims

A peptide of no more than 500 amino acids, comprising (1) at least one T cell epitope set forth in Table 3 or Tables 15-47 or (2) at least one B cell epitope set forth in Table 4, the peptide optionally further comprising at least one heterologous amino acid sequence.
The peptide of claim 1, comprising or consisting of at least one of the T cell epitopes.
The peptide of claim 1, comprising or consisting of at least one of the B cell epitopes.
The peptide of any one of claims 1-3, comprising or consisting of at least one of the T cell epitopes and at least one heterologous amino acid sequence.
The peptide of any one of claims 1-3, comprising or consisting of at least one of the B cell epitopes and at least one heterologous amino acid sequence.
A nucleic acid comprising a polynucleotide sequence encoding the peptide of any one of claims 1-5.
An expression cassette comprising a polynucleotide sequence encoding the peptide of any one of claims 1-5, operably linked to a promoter.
A vector comprising the expression cassette of claim 7.
A host cell comprising the vector of claim 8.
A composition comprising (1) the peptide of any one of claims 1-5, the nucleic acid of claim 6, the expression cassette of claim 7, the vector of claim 8, or the host cell of claim 9; and (2) a pharmaceutically acceptable excipient.
The composition of claim 10, further comprising an adjuvant.
The composition of claim 10, comprising a plurality of peptides each comprising a T cell epitope set forth in Table 3 or Tables 15-47.
A method of eliciting an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising (1) the peptide of any one of claims 1-5, the nucleic acid of claim 6, the expression cassette of claim 7, or the vector of claim 8.
The method of claim 13, wherein the composition is administered to the subject by a route selected from the group consisting of, subcutaneous, intramuscular, and oral.
The method of claim 13 or 14, wherein the subject is at risk of exposure to SARS-CoV or SARS-CoV-2 infection.
A kit for eliciting an immune response in a subject in need thereof, the kit comprising a first container containing the composition comprising (1) the peptide of any one of claims 1-5, the nucleic acid of claim 6, the expression cassette of claim 7, or the vector of claim 8, optionally an additional container containing a therapeutic agent against SARS-CoV-2.
The kit of claim 16, further comprising at least a second container each containing at least one different composition.
A method for detecting T cell immunity against SARS-CoV-2 in a subject, comprising:

(1) contacting T cells obtained from the subject with a T cell epitope set forth in Table 3 or Tables 15-47 and antigen-presenting cells having an HLA allele associated with the epitope; and

(2) detecting activation of the T cells, thereby detecting presence of T cell immunity against SARS-CoV-2 in the subject.
The method of claim 18, wherein step (2) comprises detection of T cell proliferation or T cell secretion of one or more cytokines.
The method of claim 18, wherein step (2) comprises T cell proliferation assay, flow cytometry, ELISPOT, or ELISA.
The method of claim 18, wherein step (1) comprises contacting T cells obtained from the subject with a composition comprising a plurality of peptides each comprising a T cell epitope set forth in Table 3 or Tables 15-47 and antigen-presenting cells having HLA alleles associated with each of the plurality of epitopes.