Epitope combinations and uses thereof, vaccine constructs, methods for inducing an immune response, methods for identifying epitopes

Abstract

The present invention relates to a combination of epitopes and the use of said combinations ("sets of epitopes") that contain at least eight T cell epitopes from SARS-CoV-2 that are broadly recognized by the vast majority of CD4+ T lymphocytes in convalescent COVID-19 individuals.

Description

[Technical field]

[0001] The present invention relates to new epitopes recognized by CD4+ T lymphocytes in combination. In addition, the present invention relates to the use of such epitopes and combinations thereof, in particular for the treatment or prevention of disorders caused by the SARS-CoV-2 virus.

[0002] Furthermore, the present invention relates to methods for identifying epitopes for use in combination and methods for preventing infection caused by SARS-CoV-2.

[0003] In particular, the present invention relates to epitope combinations containing at least eight T cell epitopes derived from SARS-CoV-2 in a construct comprising a SARS-CoV-2 spike protein receptor binding domain (RBD) monomer or dimer.

[0004] In addition, the present invention can be used to induce enhanced T cell responses against COVID-19 vaccine antigens and breakthrough infections. Thus, the present invention also describes the use of said combination for the manufacture of a vaccine. [Background technology]

[0005] Vaccines against Covid-19 are already in use as an important response to the Covid-19 pandemic, however, communities around the world are aware that the currently available vaccines are not sufficient to eradicate the virus, let alone prevent reinfection or transmission.

[0006] Moreover, since the start of vaccination at the end of 2021, the world faces further challenges regarding the Covid-19 pandemic. It remains true that manufacturing, sales and distribution to achieve global coverage remain daunting logistical and financial challenges, especially in low- and middle-income countries and remote areas. Thus, unequal distribution of vaccines among countries will lead to insufficient vaccination coverage far from the proportion of immunized people required to control the pandemic, which will also encourage the emergence of new mutant strains.

[0007] Therefore, there remains a pressing need for a vaccine that can induce a better immune response against the SARS-CoV-2 virus that is reliable, low-cost, easy to handle and store, but most importantly, highly antigenic / immunogenic, including a better cellular (T cell-mediated) immune response that lasts longer than antibodies in coronavirus infections.

[0008] The prior art has attempted to provide technical solutions regarding immune responses to control the Covid-19 pandemic.

[0009] WO2021195286 discloses compositions and methods for treating and preventing coronaviruses using RBD polypeptides.

[0010] IN202011018845 discloses a codon-optimized nucleotide sequence designed to express the RBD of the spike protein of SARS-CoV-2. The technical solution described in this document aims to identify small molecules or peptides with antiviral potential for the development of antigen-antibody based diagnostics and for the discovery and validation of antibodies targeting the SARS-CoV-2 RBD.

[0011] None of these documents could provide the technical solution.

[0012] It is therefore a goal of the present invention to provide vaccine constructs that induce better cellular immune responses.

[0013] To achieve this goal, it is necessary to identify a set of SARS-CoV-2 T cell epitopes with high population coverage that, in combination with the monomeric or dimeric RBD of the spike protein derived from the SARS-CoV-2 virus, can induce a relatively high cellular immune response in all of the virus-exposed population.

[0014] Identifying such immunodominant peptides is not trivial, and furthermore, given the nature of T cell epitope recognition, it is unlikely that a single T cell epitope will be recognized by all individuals, so a combination of immunodominant T cell epitopes must be used to maximize vaccine coverage.

[0015] In silico analysis shows that increasing the number of epitopes allows the recognition of more peptides per HLA-DRB1 and per individual, reaching a minimum of 10 peptides / HLA-DRB1 when using the complete set of peptides from SEQ ID NOs: 1-19. Increasing the number of recognized epitopes per individual increases the spread and coverage of vaccination. The more peptides recognized by the vaccinee, the less likely it is that the vaccine protection against new mutant viruses will be compromised by mutations. A sufficient spread and complete coverage (the number of T cell epitopes recognized by each individual) is a novel property expected from a wide variety of combinations of HLA-binding epitopes. In a genetically heterogeneous population, each epitope will not be as antigenic / immunogenic as a wide variety of combinations of epitopes that bind to HLA-DR.

[0016] Therefore, the present invention discloses a technical solution, which is the identification and use of a set of SARS-CoV-2 peptides capable of binding to multiple HLA class II molecules in a vaccine construct against the SARS-CoV-2 virus.

[0017] As a result, each individual carries an HLA that can present multiple such SARS-CoV2 peptides to T cells, allowing the vast majority of the vaccinated population to mount T cell responses to multiple epitopes.

[0018] Furthermore, in vaccines, CD8+ T cell epitopes are essential for the destruction of virus-infected cells and are beneficial for the control of subsequent infection or reinfection. A method to identify CD8+ T cell epitopes with high population coverage is to search for peptides that stably bind to HLA class I molecules / alleles that cover the vast majority of the population.

[0019] The identification method of the present invention recognizes hundreds of peptides equivalent to those taught in the state of the art, but because the present invention employs a small number of peptides rather than hundreds, it is possible to identify a number of peptides with much fewer resources.

[0020] Brief Description of the Sequence Listing The present invention will be more clearly understood upon reading the following non-limiting detailed description and the sequence listing provided herein. Summary of the Invention

[0021] The present invention relates to a combination of epitopes and the use of said combinations ("sets of epitopes") that contain at least eight T cell epitopes derived from SARS-CoV-2, which epitopes are widely recognized by the vast majority of CD4+ T lymphocytes in convalescent COVID-19 individuals.

[0022] Another object of the invention is a vaccine construct that contains the amino acid sequence of a single polypeptide.

[0023] Furthermore, the present invention also embodies a method of inducing an immune response comprising administering said vaccine construct.

[0024] Furthermore, the present invention provides a method for identifying the best combination of T cell epitopes of SARS-CoV-2 for a vaccine, thereby allowing high coverage of the world population, comprising: a) selecting peptides from the entire SARS-CoV-2 proteome sequence, wherein said peptides bind to at least 50% of the HLA-DR molecules in the bioinformatics server https: / / webs.iiitd.edu.in / raghava / propred / ; b) extending the sequence of the selected peptide at the N-terminus and C-terminus; c) searching the website iedb.org for world population coverage based on the frequency of HLA molecules predicted to bind to the peptide; d) synthesizing the corresponding peptide; e) identifying and testing SARS-CoV-2 specific responses in sensitized individuals who suffered from mild (non-hospitalized) COVID-19 disease and recovered well; The present invention relates to a method comprising the steps of:

[0025] A further object of the present invention is the use of a set / combination of epitopes to prepare a vaccine construct using the dimeric or monomeric RBD of the spike protein of the SARS-CoV-2 virus. [Brief description of the drawings]

[0026] [Figure 1] Figure 1 shows the global population coverage across 19 peptides based on the frequency of HLA molecules predicted to bind to the peptides as assessed by IEDB population coverage at iedb.org. The in silico analysis results show that 99.6% of the world's population can recognize at least one epitope. Average hit means that each individual recognizes, on average, 29 HLA / epitope combinations. Pc90 means that 90% of the world's population recognizes at least 15 HLA / epitope combinations. [Diagram 2]Figure 2 shows a cytokine release-based whole blood cytokine secretion high-throughput T cell assay using the 19 SARS-CoV-2 CD4+ T cell epitopes of the invention. The assay shows that 42 of 45 SARS-CoV-2 seropositive subjects (94%) showed peptide-induced cytokine secretion indicative of T cell recognition of the T cell epitopes, and none of the 16 seronegative subjects showed a T cell response, demonstrating the specificity of the peptides for COVID-19 convalescent individuals. [Diagram 3] FIG. 3 shows the ROC curve - IL2 and IFNγ production in whole blood in response to SARS-CoV-2 epitopes of the invention. [Figure 4] Figure 4 shows the dimeric RBD protein derived from the Wuhan strain. Figure 4A is a schematic diagram of the dimeric RBD protein. Figure 4B is a map of the pcDNA3.1 plasmid containing two tandem sequences of RBD. [Diagram 5] Figure 5 shows the purification of dimeric RBD protein by nickel resin affinity chromatography. Figure 5A is a purification chromatogram using IMAC-Ni resin. Figure 5B is a 7.5% SDS-PAGE gel in non-reducing conditions containing different fractions: 1. molecular weight markers; 2. pre-column dialyzed supernatant; 3. eluate after loading onto the column; 4. eluate after the washing step; 5. eluate from elution fraction 1; 6. eluate from elution fraction 2; 7. eluate after column regeneration. [Figure 6] Figure 6 shows purification of dimeric RBD protein by ion exchange. Figure 6A is an ion exchange purification chromatogram on S-Sepharose using a saline gradient up to 500 mM. Figure 6B is a 7.5% SDS-PAGE gel in non-reducing conditions containing various fractions: 1. Pre-column dialyzed supernatant; 2. Eluate after loading on the column; 3. Eluate after the washing step; 4. Molecular weight markers; 5. Eluate from elution fraction 1; 6. Eluate from elution fraction 2; 7. Eluate from elution fraction 3; 8. Eluate from elution fraction 4; 9. Eluate from elution fraction 5; 10. Eluate from elution fraction 6. [Figure 7]Figure 7 shows the stability of dimeric RBD protein in culture supernatant. Figure 7A shows protein purification chromatography using IMAC-Ni resin after waiting for 4 days and a 7.5% SDS-PAGE gel under non-reducing conditions containing purified protein fractions. 1. Molecular weight markers (kDa); 2. Purified dimeric RBD protein. Figure 7B shows protein purification chromatogram using IMAC-Ni resin after waiting for 5 days and a 7.5% SDS-PAGE gel under non-reducing conditions containing purified protein fractions. 1. Molecular weight markers (kDa); 2. Purified dimeric RBD protein. [Figure 8] Figure 8 shows ELIspot responses of PBMCs to a pool of 19 CD4+ T cell epitopes from a convalescent SARS-CoV-2 patient, 40 days after symptom onset. [Figure 9] Figure 9 shows ELIspot responses of PBMCs to a pool of 26 CD8+ T cell epitopes from a convalescent SARS-CoV-2 patient, 40 days after symptom onset. [Figure 10] FIG. 10 shows anti-RBD titers in C57Bl / 6 mice immunized with recombinant Wuhan strain dimeric RBD or RBD plus eight synthetic peptides encoding CD4+ T cell epitopes. [Figure 11] FIG. 11 shows the cellular immune responses of mice immunized with dimeric Wuhan strain RBD or dimeric Wuhan strain RBD and pooled SARS-CoV-2 dipeptides in response to individual SARS-CoV-2 dipeptides. [Figure 12] Figure 12 shows the cellular immune response of mice. The graph shows the measurement of the cellular immune response to RBD stimulation for the following immunization groups: negative control (adjuvant), RBD, and RBD plus dipeptide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description of the Invention A series of literature data has shown that the main target of neutralizing antibodies against the SARS-CoV2 virus is directed against the spike protein (S). More specifically, antibodies with high neutralizing capacity target the binding domain (receptor binding domain, RBD) for the ACE2 receptor (angiotensin 2 converting enzyme). Therefore, the present invention uses RBD in a vaccine construct against Covid-19 infection.

[0028] Based on the design-by-structure data, which showed that the dimeric form of the RBD is more immunogenic than the monomeric form, a dimeric vaccine antigen is constructed using the strategy described by Dai et al. (2020). In this strategy, two types of sequences of the RBD of the Wuhan strain (amino acids R319 to K537) were depicted in tandem (Figure 4A / B). To ensure the expression of the recombinant protein, the signal peptide sequence of the gene encoding human IgE (YAN et al., 2007) was added, as well as a sequence of six histidines at the C-terminal part (His tag) required for purification of the vaccine antigen for preclinical trials (Figure 4A). This sequence was synthesized with codon optimization for expression in mammalian cells and cloned into the expression vector pcDNA3.1 (Figure 4B) of GenScript Inc. (Piscataway, USA).

[0029] The pcDNA3.1 plasmid containing the dimeric RBD sequence was reconstituted in ultrapure water and transformed into chemocompetent E. coli TOP-10 by heat shock. As can be seen from Figure 4B, the pcDNA3.1 plasmid carries a resistance gene to ampicillin, which is used at a concentration of 100 μg / mL for the selection of transformed bacteria. The isolated colonies were inoculated into liquid Luria-Bertani (LB) medium containing ampicillin and stirred at a constant speed (approximately 300 pm) at 37 °C. Plasmid DNA was extracted using the PureLink HiPure Plasmid Maxiprep Kit (Thermo Fisher Scientific). The quality of the obtained DNA was assessed by optical spectrometry at 260 nm and 280 nm.

[0030] Plasmid DNA was transfected into Chinese hamster ovary cells (ExpiCHO-S) using the ExpiCHOTM Expression System Kit (Thermo Fisher Scientific), which contains the media and reagents for performing the transfection.

[0031] Transient transfections were performed using ExpiFectamine CHO reagent and plasmid DNA (both pre-diluted in OptiPRO SFM medium). Bottles were kept under agitation in a humidified oven at 37°C and 8% CO2 partial pressure. After 16-22 hours of transfection, ExpiCHO feed medium with ExpiFectamine CHO enhancer was added to the cultures, the oven temperature was changed to 32°C, the CO2 partial pressure was reduced to 5%, and shaking was continued as on the previous day. After 10 days of expression, culture supernatants were clarified by centrifugation and subsequently dialyzed for purification by affinity chromatography on nickel resin (IMAC-Ni, Cytiva) or by ion exchange using the cationic resin S-Sepharose (Cytiva).

[0032] Figure 5A / B shows the purification chromatogram of dimeric RBD protein by IMAC-Ni column (Figure 5A) and 7.5% SDS-PAGE gel containing various fractions (Figure 5B). For this purification, the culture supernatant was dialyzed against 20 mM NaH2PO4, 500 mM NaCl and 50 mM imidazole buffer (pH 7.4). The same buffer was used to equilibrate the IMAC-Ni column and the supernatant containing the dimeric RBD protein was loaded onto the column. Washing was also performed with the same buffer and the protein was eluted with 20 mM NaH2PO4, 500 mM NaCl and 300 mM imidazole (pH 7.4). As can be seen from Figure 5B, the dimeric RBD protein was eluted in elution fraction 1 (column 5). After elution, the eluate was diafiltered and concentrated into PBS using an Amicon Ultra 10 MWCO (Merck Sigma).

[0033] The results of purification of the dimeric RBD protein by ion exchange are shown in Figure 6A / B. Here, the culture supernatant was dialyzed with a buffer containing 50 mM Tris-HCl (pH 7.4). This same buffer was used to equilibrate and wash the S-Sepharose column. Elution was performed with a concentration gradient of 0 to 500 mM NaCl (pH 7.4) (Figure 6A). Figure 6B shows a 7.5% SDS-PAGE gel containing the various fractions, where it can be seen that the dimeric RBD protein was successfully eluted in fractions 3 to 6 (columns 7 to 10). In the same way as was done for the elution of the affinity column, the various fractions containing the protein were mixed, diafiltered and concentrated in PBS using an Amicon Ultra 10 MWCO (Merck Sigma).

[0034] The dimeric RBD protein was also expressed at other time points with consistent results. To test the stability of the protein in the culture supernatant, transient transfection was performed and the supernatant was subsequently stored at 4°C for 4 or 5 days before dialysis and purification. After this, the dimeric RBD protein was purified by nickel resin affinity chromatography exactly as described previously. Figure 7A / B shows the chromatograms and 7.5% SDS-PAGE gels containing the purified protein after 4 days (Figure 7A) and 5 days (Figure 7B).

[0035] These results led to the conclusion that the dimeric RBD protein was stable in the culture supernatant for at least 5 days before purification at 4°C.

[0036] Thus, the present invention relates to a combination of at least eight synthetic peptides that bind to multiple HLA-DR molecules in a wide variety of ways, that are recognized by CD4+ T lymphocytes of patients infected with the SARS-CoV-2 virus, that were selected from the entire SARS-CoV-2 proteome sequence (GenBank MN908947.3), that are referred to in this specification as epitopes, and that are simple or have covalent modifications such as myristoylation and other forms of lipopeptides, added terminal cysteines, or other forms that can allow polymerization, including the following sequences:

[0037] The epitope is selected from the group consisting of the sequences in Table 1:

[0038] [Table 1]

[0039] Furthermore, said epitope is selected from the group consisting of the sequences of Table 1, in particular from the group consisting of SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:14 and SEQ ID NO:16.

[0040] The epitopes selected were those predicted to bind above a selected threshold (3%) to the maximum number of possible HLA-DR molecules in a "diverse" manner (at a threshold of 3%, sequences predicted to bind to at least 26 of the 51 HLA-DR molecules available to the algorithm select epitopes likely to bind to HLA-DR molecules with high avidity).

[0041] To achieve the final peptide sequences in Table 1, N- and C-terminal flanking residues were added, thereby increasing the proportion of individuals recognizing each peptide. The synthetic peptide combinations were designed to induce or detect SARS-CoV-2 specific responses in more than 99% of the world's population according to HLA frequency data in the iedb.org server. Each HLA-DR molecule was predicted to bind at least 10 different peptides according to in silico analysis at https: / / webs.iiitd.edu.in / raghava / propred / .

[0042] In silico analysis shows that increasing the number of epitopes allows the recognition of more peptides per HLA-DRB1 and per individual, reaching a minimum of 10 peptides / HLA-DRB1 when using the complete set of peptides from SEQ ID NOs: 1-19. Increasing the number of epitopes per individual increases the spread of vaccination. The more peptides recognized by the vaccinee, the less likely it is that the vaccine protection against new mutant viruses will be compromised by mutations. Full coverage of sufficient spread (the number of T cell epitopes recognized by each individual) is a novel property expected from a wide variety of combinations of HLA-binding epitopes. Each epitope individually will not be as antigenic / immunogenic in a genetically heterogeneous population as the combination of these individual epitopes.

[0043] Furthermore, the epitopes of the present invention to be used in combination are derived, in particular, from spike proteins, envelope proteins, membrane proteins, nucleocapsid proteins and other SARS-CoV-2 ORFs.

[0044] One advantage of the present invention is the novel property of a wide variety of combinations of epitopes that can bind to multiple HLA-DR molecules, resulting in combinatorial recognition of said epitopes by CD4+ T cells. Another advantage is the targeting of a number of non-spike viral proteins that are less susceptible to mutations and antibody-induced immune pressure.

[0045] According to the present invention, "epitope" means the above-mentioned epitopes, their functional equivalents and their mimetic sequences.

[0046] "Functional equivalents" refers to structurally distinct sequences, fragments, analogs, derivatives, or conjugates that perform the same function and achieve equivalent results. It is understood that any modification made by one skilled in the art that clearly produces an equivalent effect is considered to be part of the present invention. More specifically, functional equivalents are sequences that exhibit at least 12 amino acid homology to the above epitopes and perform the same function of said epitopes with equal or similar results.

[0047] According to the present invention, a "mimetic sequence" is understood to be a non-natural amino acid sequence that has been modified so as to exhibit a function and result equal to or similar to the sequence of the epitope of the invention.

[0048] The epitopes are putative immunodominant SARS-CoV-2 CD4+ T cell epitopes. The epitopes were selected from the entire SARS-CoV-2 proteome sequence (GenBank MN908947.3). Such synthetic peptides were designed to bind to at least 50% of the 51 HLA-DR molecules in the bioinformatics server https: / / webs.iiitd.edu.in / raghava / propred / .

[0049] In particular, combinations of eight or more epitopes of the invention, including the SEQ ID NOs in Table 2, can allow for sufficient coverage. In silico studies suggested that with at least the following ten peptides, T cell responses against at least five peptides could be detected in all individuals:

[0050] To evaluate the ability of a combination of at least eight selected epitopes to recognize individual T lymphocytes, the epitopes (listed in Table 1) were solid-phase synthesized by amidating the C-terminus using Fmoc chemistry. Synthetic peptide stocks were diluted to 5 mg / mL in dimethyl sulfoxide and ELISPOT assays were performed to detect IFNγ-producing cells responding to the epitopes using peripheral blood mononuclear cells (PBMCs) from 98 COVID-19 convalescent patients (patients with mild symptoms who did not require hospitalization) at least 2 months after signs of infection (Figure 8). Synthetic peptides were diluted to prepare pools of 5 mg / mL and added to the wells (total peptide concentration 5 μg / mL, individual concentration 0.25 μg / ml). Cryopreserved cells were cultured for 16 hours, washed, and placed in ELISPOT plates with the epitopes and incubated for an additional 18 hours. ELISPOT plates were developed for identification of spot-forming / interferon-gamma producing cells (IFNγ SFCs) and then counted in an automated counter (Zeiss KS ELISpot / Axioplan 2).Background was calculated using PBMCs from 17 seronegative control individuals obtained prior to the COVID-19 pandemic.

[0051] Responses in background wells without peptide were subtracted from the peptide-induced responses of the 98 tested individuals and the results are plotted in the graph in Figure 8. The response cutoff (103 spot forming cells (SFC) / 10 6 The T cell response (n=10 PBMCs) was calculated as the mean + 3 standard deviations of the ELISPOT responses to the 20 synthetic peptides in Table 1. Individuals with responses above the cutoff were considered to possess a T cell response to the SARS-CoV-2 peptide, which demonstrated 97% recognition.

[0052] All epitope combinations of the invention are used in diagnostic methods and in clinical trials to evaluate the immune response of CD4+ T lymphocytes against the SARS-CoV-2 virus. For example, diagnostic methods that allow the in vivo, ex vivo or in vitro identification of breakthrough infection in individuals immunized with a SARS-CoV-2 subunit vaccine by detecting or monitoring the cellular response to infection or vaccination. Other diagnostic methods, such as in vitro cellular immune response assays for high volume (scalable to hundreds of reactions per day) diagnostics, are also aspects of the invention. Another example is the use in in vivo diagnostics, where a response is observed after administration of the peptides.

[0053] Furthermore, the combination of at least eight epitopes of the present invention is useful for the production of a vaccine, which aids T cells and increases their immunogenicity and protective properties, and the vaccine may be more effective than those of the prior art, since the combination of at least eight epitopes of the present invention is recognized by T cells of the majority of individuals and covers a significant proportion of the population exposed to the virus.

[0054] An object of the present invention is also a composition containing a combination of at least eight epitopes of SEQ ID NO: 1 to SEQ ID NO: 19. Said composition further comprises a pharma- ceutically acceptable carrier or base.

[0055] The compositions of the present invention may be in solid or liquid form. The compositions may be formulated for immediate or sustained release of their ingredients and may further contain compounds that stimulate and / or inhibit the immune system. The compositions may be prepared according to conventional methods.

[0056] A further object of the present invention is the use of said composition, including in diagnostic methods and tests for evaluating the immune response of CD4+ T lymphocytes against the SARS-CoV-2 virus, in the manufacture of a vaccine, as previously described for the combination of at least eight epitopes listed in Table 1 as SEQ ID NO: 1 to SEQ ID NO: 19.

[0057] The present invention also relates to vaccine constructs that contain CD4+ T cell epitopes in the form of synthetic peptides combined with RBD monomers or dimers.

[0058] The vaccine construct of the present invention contains a recombinant protein defined by a single polypeptide chain formed by linking one to three novel beta coronavirus (SARS-CoV-2) spike (S) protein receptor binding domains (RBDs) linked in tandem and / or via linkers to a peptide string defined by a CD4+ epitope or a CD4+ and CD8+ T cell epitope.

[0059] The CD8+ T cell epitope is selected from the group consisting of the sequences in Table 2:

[0060] [Table 2]

[0061] The vaccine construct of the present invention is illustrated by the following technical scheme: The amino acid sequence of a single polypeptide chain is: (A) RBD1-RBD2 or RBD1-RBD2-RBD3 as set forth in SEQ ID NOs: 54, 55, 56 and 57; (B) RBD1-linker-peptide strings as shown in SEQ ID NOs: 58, 59 and 60; (C) RBD1-RBD2-linker-peptide strings as shown in SEQ ID NOs: 61, 62, 63, 64, 65, 66 and 67; (D) RBD1-linker-RBD2 as shown in SEQ ID NOs: 68 and 69; (E) RBD1-linker-peptide string-linker-RBD2 shown in SEQ ID NOs: 70, 71, 72, 73, 74, 75, 76 and 77 is expressed as (1) the amino acid sequence of the spike (S) protein receptor binding domain (RBD) of a novel β-coronavirus (SARS-CoV-2) contains residues 319 to 537 (SEQ ID NOs: 54 to 67, SEQ ID NOs: 70 to 75) and 330 to 528 (SEQ ID NOs: 68, 69, 76, and 77) of the S protein; (2) RBD1, RBD2, and RBD3 are the amino acid sequences of the spike (S) protein receptor-binding domain (RBD) of the original Wuhan strain of novel β-coronavirus (SARS-CoV-2) and the β-mutant (B.1.1.529, K417N, E484K, and N501Y), γ-mutant (P1, K417T, E484K, and N501Y), and δ-mutant (P2, K417T, E484K, and N501Y) strains. representing the substitutions corresponding to the variants (B.1.617.2, L452R and T478K) and ο variants (B.1.1.159, BA1, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y and Y505H); (3) the spike (S) protein receptor binding domain (RBD) of a novel beta coronavirus (SARS-CoV-2) and a peptide string are linked in tandem (SEQ ID NOs: 54, 55, 56, and 57) and / or linked via a peptide linker (SEQ ID NOs: 58-77), specifically, linked by the sequence EAAAKEAAAKEAAAK (SEQ ID NOs: 58-67), KPKPKP (SEQ ID NOs: 70, 72, 74, and 75), GGGGS (SEQ ID NOs: 71 and 73), or PKPK (SEQ ID NOs: 68, 69, 76, and 77); (4) Peptide strings were constructed by CD4+ and / or CD8+ T cell epitopes linked via GPGPG linkers (see SEQ ID NOs: 1-45 for a list of CD4+ and CD8+ T cell epitopes), and the list of CD4+ and CD8+ T cell epitopes was used to construct dipeptides linked by GPGPG sequences (SEQ ID NOs: 46-53), CD4+ T cell epitope strings 1-4 (SEQ ID NO: 54), CD4+ T cell epitope strings 5-8 (SEQ ID NO: 55), CD4+ T cell epitope string 08 (SEQ ID NO: 56), CD8+ T cell epitope string 11 (SEQ ID NO: 57), and CD4+ and CD8+ T cell epitope string 22 (SEQ ID NO: 58).

[0062] In addition, the present invention also provides a vaccine construct in which RBD1, RBD2 and RBD3 in (2) above represent the amino acid sequence of the spike (S) protein receptor binding domain (RBD) of a novel mutant strain of beta coronavirus (SARS-CoV-2) of interest.

[0063] The vaccine construct contains a combination of at least eight epitopes listed in Table 1 and one or more pharma- ceutically acceptable adjuvants, bases, excipients, binders, carriers or preservatives.

[0064] The vaccine construct according to the present invention can be formulated according to the following forms, including, but not limited to, combinations of eight or more epitopes set forth in Table 1 as SEQ ID NO:1 to SEQ ID NO:19: a) combining eight or more epitopes set forth in SEQ ID NOs: 1-19 with an adjuvant; b) recombinant or synthetic DNA or RNA constructs containing sequences of eight or more new epitopes, particularly including other protein products; c) recombinant protein or synthetic peptide constructs carrying new epitope sequences, especially in combination with adjuvants; d) a viral vector containing the sequence of the new epitope; e) a virus-like particle expressing the epitope in an encoded protein; or f) Combination of said new epitopes with SARS-CoV-2 epitopes and immunogens known in the state of the art or as boosters to such known SARS-CoV-2 immunogens.

[0065] Another object of the invention is a method for identifying new epitopes for the combinations of the invention allowing a high coverage of the world's population (Figure 1), a) selecting peptides from the entire SARS-CoV-2 proteome sequence, wherein said peptides bind to at least 50% of the HLA-DR molecules in the bioinformatics server https: / / webs.iiitd.edu.in / raghava / propred / ; b) extending the sequence of the selected peptide at the N-terminus and C-terminus; c) searching the website iedb.org for world population coverage based on the frequency of HLA molecules predicted to bind to the peptide; d) synthesizing the corresponding peptide; e) identifying and testing SARS-CoV-2 specific responses in sensitized individuals who suffered from mild (non-hospitalized) COVID-19 disease and recovered well; The method comprises the steps of:

[0066] In silico population coverage analysis was also performed for the selected 8 and 11 CD4+ peptide combinations that are part of the constructs described herein. The world population coverage of the selected 11 CD4+ peptide combinations that are part of the constructs, also evaluated by IEDB population coverage at iedb.org, shows that 90.6% of the world's population can recognize at least one of the epitopes, with each individual recognizing an average of 9.8 HLAs per epitope combination. The world population coverage of one combination of 8 CD4+ T cell epitopes that are part of the constructs also covers 90.6% of the population, with each individual recognizing an average of 9.5 HLAs per epitope combination. The world population coverage of one combination of 8 CD4+ T cell epitopes that are part of the constructs also covers 90.6% of the population, with each individual recognizing an average of 6.3 HLAs per epitope combination. These in silico results indicate that subsets of 8 or 11 CD4+ epitopes provide high population coverage with a high number of available peptides per HLA molecule.

[0067] The following examples are illustrative and provide a clear and consistent understanding of the present invention, but are not intended to limit its scope. EXAMPLES

[0068] Example 1 T cell responses of SARS-CoV-2 convalescent patients to selected CD4+ and CD8+ T cell epitopes To assess cellular responses to predicted T cell epitopes, we used samples from 110 convalescent participants taken approximately 40 days after infection. Blood was processed to obtain peripheral blood mononuclear cells (PBMCs) that were stored frozen in liquid nitrogen until use.

[0069] After in silico analysis of the SARS-CoV-2 sequence and selection of potentially more diverse CD4+ T cell epitopes, selected peptides were synthesized. In total, 19 peptides derived from CD4+ T cells and 26 peptides derived from CD8+ T cells were synthesized. Synthetic peptides were used to stimulate PBMCs for 24 h and analyzed by ELISPOT to evaluate IFNγ production.

[0070] Analysis showed that synthetic peptides corresponding to CD4+ T cell epitopes demonstrated sufficient immune coverage in convalescent subjects, including inducing T lymphocyte responses in 92% of samples tested (Figure 8).

[0071] Subsequently, to analyze the overall immunodominance of CD4+ T cell epitopes, PBMCs were stimulated with each peptide individually. Again, the magnitude of responses varied between subjects, but each peptide alone induced IFNγ-producing T cells in 60%-80% of patients tested. T cells from each patient recognized an average of 14 of the 19 CD4+ T cell peptides, or 74% of the epitopes tested, with 93% of patients recognizing at least one CD4+ T cell epitope (data not shown), demonstrating a wide diversity of HLA class II binding and T cell responses.

[0072] A similar analysis of T cell recognition of 26 CD8+ T cell epitopes was performed in the same convalescent cohort. 26 synthetic peptides corresponding to CD8+ T cell epitopes demonstrated substantial immune coverage in convalescent subjects, including induction of T lymphocyte responses in 91% of samples tested (Figure 9).

[0073] To analyze the overall immunodominance of CD8+ T cell epitopes, PBMCs were stimulated with each CD8+ peptide epitope individually. Although the magnitude of responses varied between subjects, T cells from each patient recognized an average of 20 of the 26 CD4+ T cell peptides, or 77% of the epitopes tested, and 95% of patients recognized at least one CD4+ T cell epitope (data not shown), demonstrating a remarkable diversity of HLA class I binding and T cell responses.

[0074] Results showed that in silico predicted peptides were highly recognized by cells from convalescent patients, with no pattern of immunodominance for peptides derived from different regions of the virus.

[0075] Example 2 Humoral responses to vaccines and synthetic peptides encoding the RBD: induction of specific antibodies To identify the best vaccine candidates and immunization strategies, C57BL / 6 mice were immunized as shown in Table 3. The animals used were obtained from the Center for Development of Experimental Models for Medicine and Biology (CEDEME) and were kept pathogen-free in a biomimetic breeding facility in the Immunology Department with a controlled light / dark cycle (12:12) and free access to water and food (DMIP / UNIFESP). The project was approved in advance by the Ethics Committee for Animal Use (CEUA-UNIFESP) under number 4813280820.

[0076] [Table 3]

[0077] Animals were immunized subcutaneously (SC) with 100 μL in the presence of AS03 adjuvant (1:1 v / v) at dosing intervals of 15 days with 5 μg of Wuhan strain dimeric RBD protein or 5 μg of Wuhan strain dimeric RBD protein and 50 μg of eight synthetic dipeptides set forth in SEQ ID NOs: 46-53, each encompassing one CD4+ and one CD8+ T cell epitope as shown in Table 4. A control group received only the ASO3 adjuvant via the SC route.

[0078] [Table 4]

[0079] For the analysis of humoral immune responses, the blood of the animals was taken 14 days after dosing and the titer of dimer-specific RBD antibodies was determined in an ELISA assay. For this, ELISA plates (high binding, Costar) were used and 250 ng of dimeric RBD recombinant protein, prepared and characterized as described in the previous section, was added to each well. After 16-18 hours of incubation at room temperature, the plates were washed with 0.02% PBS-Tween® 20 (PBS-T0.02) and blocked for 1 hour at room temperature (PBS-T0.02 / 1% BSA). After 2 hours of incubation with serial dilutions of sera of immunized mice, the contents of the wells were discarded and the plates were washed with PBS-T0.02. A secondary antibody (peroxidase-linked anti-mouse IgG-KPL) diluted 1:10,000 in PBS-T0.02 / BSA1% was added and incubated for another 2 hours at room temperature. After three further washes with PBS-T0.02, detection was performed with a solution of OPD (Sigma) dissolved in 0.2 M NaH2PO4 and 0.1 M citric acid (pH 4.7) plus 10 μL of 30% H2O2. After 15 min, the reaction was stopped with 50 μL of 4N H2SO4. The plates were then analyzed at 492 nm in a 96-well plate reader (PerkinElmer).

[0080] Analysis of serum antibody production showed that IgG production was comparable between animals that received RBD and those that received RBD and eight synthetic peptides encoding SARS-CoV-2 CD4+ and CD8+ T cell epitopes (Figure 10).

[0081] Example 3 Cellular immune responses to vaccines encoding SARS-CoV-2 RBD and selected SARS-CoV-2 synthetic peptides: Induction of potent peptide-specific T cell responses In addition to humoral immune responses, cellular immune responses play an important role in protection against SARS-CoV-2. Evaluation of T lymphocyte differentiation specific for recombinant SARS-CoV-2 RBD antigen in mice immunized with 5 μg of Wuhan strain dimeric RBD protein or 5 μg of Wuhan strain dimeric RBD protein and 50 μg of a pool of 8 synthetic dipeptides set forth in SEQ ID NOs: 46-53, each encompassing one CD4+ and one CD8+ T cell epitope (see Table 4 in Example 2).

[0082] The profile of IFNγ-producing cells in the spleens of immunized mice was analyzed by ELIspot. After euthanasia, the spleen of each animal was collected using surgical instruments under laminar flow. The cells were obtained and washed by centrifugation with 10 ml of supplemented RPMI medium (Gibco). Subsequently, the cells were treated with ACK hemolysis buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA) and washed twice with 10 ml of RPMI medium. Finally, the cells were resuspended in 1 ml of R10 medium (RPMI medium containing 10% fetal bovine serum - Gibco).

[0083] ELISpot assays were performed using a mouse IFNγ ELISpot kit (BD Biosciences) according to the manufacturer's instructions. Briefly, capture antibody (purified IFNγ anti-mouse) was added to the plate (Millipore Multi-Screen IP-MAIPS 4510) at a concentration of 5 μg / mL in a final volume of 100 μL, and the plate was stored at 4° C. overnight. The wells were then washed with PBS and blocked for 2 h with RPMI medium (R10) containing 10% fetal bovine serum at room temperature. Stimuli (5 μg / mL of each dipeptide) and cell suspension were then added. The plate was incubated overnight at 37° C. in an oven containing 5% CO2. The plate was then washed with deionized water and PBS-0.05% Tween® 20 (PBS-T). Detection antibody (biotinylated anti-IFNγ) was diluted in PBS-10% FBS and added to the wells at a final concentration of 2 μg / mL, and the plate was incubated at room temperature for 2 h. After washing the wells three times with PBS-T, the enzyme conjugate (streptavidin-peroxidase) was diluted in PBS-10% FBS and added to the wells (100 μL / well) followed by further incubation for 1 h at room temperature. For color development, the wells were washed four times with PBS-T followed by two washes with 1×PBS. 100 μL of AEC (3-amino-9-ethylcarbazole-BD) solution was added to each well to monitor spot formation. The reaction was stopped by washing the plate five times with deionized water. The number of UFS (spot forming units) was performed with a Reader AID ELISpot Reader System (Autoimmun Diagnostika GmbH, Germany).

[0084] Figure 11 shows the cellular immune response to each dipeptide in mice immunized with the Wuhan strain dimeric RBD protein or the dimeric RBD protein and eight dipeptides. All peptides induced strong immune responses, and the group immunized simultaneously with synthetic peptides induced 1,000 SFU / 10 6 It can be seen that the expression of SARS-CoV-2 peptides translocated across cells, indicating that the selected SARS-CoV-2 peptides were highly immunogenic.

[0085] Further results showing additional non-cognate effects on cellular immune responses using dimeric RBD recombinant protein and the eight dipeptides set forth in SEQ ID NOs: 46-53 are shown in FIG.

[0086] An increase in the cellular immune response of immunized groups using adjuvant, RBD and dipeptides can be identified, said increase being directly caused by the booster immunization with the dipeptides set forth in SEQ ID NOs: 46-53, which do not share an epitope with the RBD protein.

[0087] References DAI, Lianpan et al. A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS. Cell, v. 182, n. 3, p. 722- 733.e11, ago. 2020. SCHMIDT, Fabian et al. Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses. Journal of Experimental Medicine, v. 217, n. 11, 2 nov. 2020. YAN, Jian et al. Enhanced Cellular Immune Responses Elicited by an Engineered HIV-1 Subtype B Consensus-based Envelope DNA Vaccine. Molecular Therapy, v. 15, n. 2, p. 411-421, fev. 2007.

Claims

1. A combination of epitopes, said combination containing at least eight T cell epitopes derived from SARS-CoV-2.

2. The combination of epitopes according to claim 1 , wherein the epitopes are broadly recognized by CD4+ T lymphocytes.

3. The combination of epitopes according to claim 1 or 2, wherein the T cell epitopes are selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:

19.

4. 4. The combination of epitopes according to claim 3, wherein the T cell epitopes are selected from the group consisting of SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14 and SEQ ID NO:

16.

5. (A) RBD1-RBD2 or RBD1-RBD2-RBD3 as set forth in SEQ ID NOs: 54, 55, 56 and 57; (B) RBD1-linker-peptide strings as shown in SEQ ID NOs: 58, 59 and 60; (C) RBD1-RBD2-linker-peptide strings as shown in SEQ ID NOs: 61, 62, 63, 64, 65, 66 and 67; (D) RBD1-linker-RBD2 as set forth in SEQ ID NOs:68 and 69; (E) RBD1-linker-peptide string-linker-RBD2 as set forth in SEQ ID NOs:70, 71, 72, 73, 74, 75, 76 and 77; A vaccine construct comprising the amino acid sequence of a single polypeptide chain represented as: (1) the amino acid sequence of the spike (S) protein receptor binding domain (RBD) of a novel beta coronavirus (SARS-CoV-2) containing residues 319-537 (SEQ ID NOs:54-67, SEQ ID NOs:70-75) and 330-528 (SEQ ID NOs:68, 69, 76, and 77) of the S protein; (2) RBD1, RBD2 and RBD3 are identical to the amino acid sequence of the spike (S) protein receptor-binding domain (RBD) of the original Wuhan strain of novel β-coronavirus (SARS-CoV-2) and the amino acid sequence of the β-mutant (B.1.1.529, K417N, E484K and N501Y), γ-mutant (P1, K417T, E484K and N501Y), δ-mutant (P2, K417T, E484K and N501Y), and β-mutant (B.1.1.529, K417N, E484K and N501Y), γ-mutant (P1, K417T, E484K and N501Y), and δ-mutant (P1, K417T, E484K and N501Y), respectively. representing the substitutions corresponding to the variants (B.1.617.2, L452R and T478K) and o variants (B.1.1.159, BA1, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y and Y505H); (3) the novel beta coronavirus (SARS-CoV-2) spike (S) protein receptor binding domain (RBD) and the peptide string are linked in tandem (SEQ ID NOs: 54, 55, 56, and 57) and / or linked via the peptide linker (SEQ ID NOs: 58-77); (4) the peptide string is constructed by CD4+ and / or CD8+ T cell epitopes linked via a GPGPG linker (see SEQ ID NOs: 1-45 for a list of CD4+ and CD8+ T cell epitopes); where from the list of CD4+ and CD8+ T cell epitopes, dipeptides linked by GPGPG sequences (SEQ ID NOs: 46-53), CD4+ T cell epitope strings 1-4 (SEQ ID NO: 54), CD4+ T cell epitope strings 5-8 (SEQ ID NO: 55), CD4+ T cell epitope string 08 (SEQ ID NO: 56), CD8+ T cell epitope string 11 (SEQ ID NO: 57), and CD4+ and CD8+ T cell epitope string 22 (SEQ ID NO: 58) are constructed; Vaccine constructs.

6. 6. The vaccine construct of claim 5, wherein the novel beta coronavirus (SARS-CoV-2) spike (S) protein receptor binding domain (RBD) and the peptide string are linked by the sequence EAAAKEAAAKEAAAK (SEQ ID NOs: 58-67), KPKPKP (SEQ ID NOs: 70, 72, 74 and 75), GGGGS (SEQ ID NOs: 71 and 73) or PKPK (SEQ ID NOs: 68, 69, 76 and 77).

7. The vaccine construct of claim 5, wherein (2) RBD1, RBD2 and RBD3 represent the amino acid sequence of the spike (S) protein receptor binding domain (RBD) of a new variant strain of beta coronavirus (SARS-CoV-2) of interest.

8. A vaccine construct containing a CD4+ T cell epitope in the form of a synthetic peptide combined with a monomer or dimer of the RBD.

9. The vaccine construct of claim 6, wherein the combined peptides are set forth in SEQ ID NOs: 46-53.

10. a) combining eight or more epitopes set forth in SEQ ID NOs: 1-19 with an adjuvant; b) recombinant or synthetic DNA or RNA constructs containing sequences of eight or more new epitopes, particularly including other protein products; c) a recombinant protein or synthetic peptide construct carrying the sequence of said new epitope, in particular in combination with an adjuvant; d) a viral vector comprising the sequence of said new epitope; e) a virus-like particle expressing the epitope in an encoded protein; or f) combination of said new epitopes with SARS-CoV-2 epitopes and immunogens known in the state of the art or as boosters to such known SARS-CoV-2 immunogens; 23. A vaccine construct comprising:

11. The vaccine construct of any one of claims 5 to 10, further comprising one or more pharma- ceutically acceptable adjuvants, bases, excipients, binders, carriers or preservatives.

12. A method for inducing an immune response comprising administering a vaccine construct according to any one of claims 5 to 11.

13. 1. A method for identifying T cell epitopes of SARS-CoV-2 for use in a vaccine, comprising: a. selecting peptides from the entire SARS-CoV-2 proteome sequence, wherein said peptides bind to at least 50% of the HLA-DR molecules in the bioinformatics server https: / / webs.iiitd.edu.in / raghava / propred / ; b. extending the sequence of the selected peptide at the N-terminus and C-terminus; c. searching the website iedb.org for world population coverage based on the frequency of HLA molecules predicted to bind to the peptide; d. synthesizing the corresponding peptide; e. testing and confirming SARS-CoV-2 specific responses in sensitized individuals who have had mild (non-hospitalized) COVID-19 disease and recovered well; A method comprising:

14. Use of a combination according to any one of claims 1 to 4 for the manufacture of a vaccine construct according to any one of claims 5 to 11.

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