Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) polypeptide and its use for vaccine purposes

Abstract

We provide SARS-CoV-2 vaccines. [Solution] A SARS-CoV-2 polypeptide (N276-411, "Npep2") is provided, which is derived from the N protein and consists of at least 50 consecutive amino acids of an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 276 to 411 of a specific sequence.

Description

[Technical Field]

[0001] This invention relates to medicine, particularly to the fields of virology and vaccinology. [Background technology]

[0002] Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), which emerged in Wuhan, China in December 2019, has caused a global health threat. On March 11, 2020, the WHO declared COVID-19 a pandemic. Its rapid spread, global rate of dissemination, and rising observed mortality rate have raised public health, socioeconomic, and scientific challenges. It still appears to be spreading extremely rapidly, with over 4.1 million confirmed cases and more than 280,000 deaths in over 185 countries as of May 10, 2020. SARS-CoV-2 presents with clinical manifestations similar to mild upper respiratory tract illness (symptoms similar to the common cold), but can sometimes lead to severe lower respiratory tract illness and extrapulmonary symptoms, causing a respiratory syndrome that can result in multiple organ failure and death. This pandemic follows several highly pathogenic human coronavirus infections, including SARS-CoV in 2002 with a 10% mortality rate and MERS-CoV in 2012 with a 36% mortality rate. There is no cure or vaccine. However, if this virus becomes established in the population, a SARS-CoV-2 vaccine will be essential to reduce morbidity and mortality. [Overview of the Initiative]

[0003] This invention relates to SARS-CoV-2 polypeptides and their use for vaccine purposes.

[0004] Detailed description of the invention The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has undoubtedly emerged as the greatest global health threat to humanity this century. Its rapid spread, global rate of dissemination, and observed rising mortality rates present public health, socioeconomic, and scientific challenges. SARS-CoV-2 causes respiratory syndromes that present with clinical manifestations similar to mild upper respiratory tract illness (symptoms similar to the common cold), sometimes leading to severe lower respiratory tract illness and extrapulmonary symptoms, resulting in multiple organ failure and death. There is no cure or vaccine. However, a SARS-CoV-2 vaccine will be essential to reduce morbidity and mortality if this virus becomes established in the population. To accelerate the development of candidate vaccines, the use of readily available vaccine platforms that are specialized and adapted to emerging and re-emerging pathogens is crucial. We have established a candidate vaccine against SARS-CoV-2. In particular, the inventors identified specific epitopes that should be included in the vaccine candidate by mapping MHC-I and -II epitopes predicted by online software (NetMHC-4.0 and NetMHCII-2.3) and peptide bond prediction software, thanks to in silico analysis of the amino acid sequences of these proteins. They also mapped B cell epitopes using online software (BepiPred-2.0 and Discotope), and mapped regions rich in epitopes with homologous sequences between SARS-CoV-2 and -CoV-1.

[0005] Definition: As used herein, the terms “subject” or “required subject” refer to human or non-human mammals. Generally, patients are infected with or may be infected with SARS-CoV-2.

[0006] As used herein, the term “coronavirus” has its general meaning in the art and refers to any member of the Coronaviridae family. Coronaviruses are viruses whose genome is a positive-strand RNA approximately 27kb to 33kb long, depending on the specific virus. The virion RNA has a cap at the 5' end and a poly-A tail at the 3' end. This RNA length makes coronaviruses the largest of the RNA virus genomes. Specifically, coronavirus RNA encodes (1) RNA-dependent RNA polymerase, (2) N protein, (3) three envelope glycoproteins, and (4) three non-structural proteins. These coronaviruses infect a variety of mammals and birds. Coronaviruses cause respiratory infections (common), enteric infections (primarily in infants 12 months and older), and potentially neurological syndromes. Coronaviruses are transmitted by aerosols of respiratory secretions.

[0007] As used herein, the terms “Severe Acute Respiratory Syndrome Coronavirus 2” or “SARS-CoV-2” have their general meaning in the art and refer to the strain of coronavirus that causes coronavirus disease 2019 (COVID-19), a respiratory syndrome presenting with a clinical condition similar to a mild upper respiratory tract illness (cold-like symptoms) and sometimes with severe lower respiratory tract illness and extrapulmonary symptoms, leading to multiple organ failure and death. In particular, the term refers to the severe acute respiratory syndrome coronavirus 2 isolate 2019-nCoV_HKU-SZ-005b_2020, whose complete genome is accessible under NCBI access number MN975262.

[0008] As used herein, the term "Covid-19" refers to the respiratory illness caused by severe acute respiratory syndrome coronavirus 2.

[0009] As used herein, the term “asymptomatic” refers to an individual who has no detectable symptoms of coronavirus infection. As used herein, the term “symptomatic” refers to an individual who has detectable symptoms of coronavirus infection. Symptoms of coronavirus infection include fatigue, loss of smell, headache, cough, fever, and difficulty breathing.

[0010] As used herein, the terms “polypeptide,” “peptide,” and “protein” are interchangeable to refer to polymers of amino acids of any length. These terms also encompass modified amino acid polymers, such as those resulting from the formation of disulfide bonds, glycosylation, lipidation, phosphorylation, or binding to labeling components. In the context of gene therapy, a polypeptide refers to a fragment or genetically modified derivative of the respective intact polypeptide or protein that retains the desired biochemical function.

[0011] As used herein, the term “polynucleotide” refers to a polymer of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may have non-nucleotide components inserted. Where present, modifications to the nucleotide structure may be conferred before or after the assembly of the polymer. As used herein, the term polynucleotide is interchangeable for double-stranded molecules and single-stranded molecules. Unless otherwise specified or required, any aspect of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0012] As used herein, the expression "derived from" means a process in which a first component (e.g., a first polypeptide) or information from that first component is used to isolate, derive, or produce a different second component (e.g., a second polypeptide different from the first polypeptide).

[0013] As used herein, the "identity percentage" between two sequences is a function of the number of identical positions common to those sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences, and the length of each gap (i.e., identity percentage = number of identical positions / total number of positions × 100). Sequence comparison and determination of the identity percentage between two sequences can be achieved using mathematical algorithms as described below. The identity percentage between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins." Journal of Molecular Biology. 48 (3): 443-53.). The identity percentage between two nucleotides or amino acid sequences can also be determined using algorithms such as EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle can be used with a BLOSUM62 matrix with a "gap open penalty" of 10, a "gap extend penalty" of 0.5, and a "false" "end gap penalty," while the "end gap open penalty" is 10 and the "end gap extend penalty" is 0.5. Generally, the "identity percentage" is a function of dividing the number of matching positions by the number of positions compared and multiplying by 100. For example, if 6 out of 10 sequence positions are identical between two sequences compared after alignment, the identity is 60%. The identity percentage is usually determined over the entire length of the query sequence being analyzed. Two molecules having the same first amino acid sequence or nucleic acid sequence are identical regardless of any chemical and / or biological modifications.According to the present invention, having at least 90% identity with the second amino acid sequence means that the first sequence has 90;91;92;93;94;95;96;97;98;99 or 100% identity with the second amino acid sequence.

[0014] As used herein, the term “mutation” has its general meaning in the art and refers to substitution, deletion, or insertion. In particular, the term “substitution” means that a particular amino acid residue is removed at a particular position and another amino acid residue is inserted at the same position. In this specification, mutations are referred to according to standard mutation nomenclature. In particular, the term “mutation” includes “spontaneous mutations” and “unspontaneous mutations.”

[0015] As used herein, the term “spontaneous mutation” refers to any mutation that may be found in spontaneously occurring variants of the SARS-CoV-2 polypeptide, and generally includes the B.1.1.7 line (also known as 20I / 501Y.V1 Variant of Concern (VOC)202012 / 01), the B.1.351 line (also known as 20H / 501Y.V2), and the P.1 line (also known as 20J / 501Y.V3). Such mutations are well known in the art and include those described in the following references incorporated by reference. (1) Jie Hu et al. The D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity and decreases neutralization sensitivity to individual convalescent sera. bioRxviv (2020). (2) Korber B. et al.Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2. bioRxviv (2020). doi.org / 10.1101 / 2020.04.29.069054. (3) Lizhou Zhang et al. The D614G mutation in the SARS-CoV-2 spike protein reduces S1 shedding and increases infectivity. bioRxviv (2020). doi.org / 10.1101 / 2020.06.12.148726. (4) Junxian Ou et al. Emergence of RBD mutations in circulating SARS-CoV-2 strains enhancing the structural stability and human ACE2 receptor affinity of the spike protein. bioRxiv (2020). doi:10.1101 / 2020.03.15.991844v4 (5) Saha, P. et al.Mutations in Spike Protein of SARS-CoV-2 Modulate Receptor Binding, Membrane Fusion and Immunogenicity: An Insight into Viral Tropism and Pathogenesis of COVID-19. chemRxiv (2020). doi:10.26434 / chemrxiv.12320567.v1 (6) Jian Shang, Yushun Wan, Chuming Luo, Gang Ye, Qibin Geng, Ashley Auerbach, Fang Li. Cell entry mechanisms of SARS-CoV-2. Proceedings of the National Academy of Sciences May 2020, 117 (21) 11727-11734; DOI: 10.1073 / pnas.2003138117 (7) Allison J. Greaney, Andrea N. Loes, Katharine H.D. Crawford, Tyler N. Starr, Keara D. Malone, Helen Y. Chu, Jesse D. Bloom, bioRxiv 2020.12.31.425021; doi: https: / / doi.org / 10.1101 / 2020.12.31.425021 (8) Nicholas G. Davies, Rosanna C. Barnard, Christopher I. Jarvis, Adam J. Kucharski, James Munday, Carl A. B. Pearson, Timothy W. Russell, Damien C. Tully, Sam Abbott, Amy Gimma, William Waites, Kerry LM Wong, Kevin van Zandvoort, CMMID COVID-19 Working Group, Rosalind M. Eggo, Sebastian Funk, Mark Jit, Katherine E. Atkins, W. John Edmunds. Estimated transmissibility and severity of novel SARS-CoV-2 Variant of Concern 202012 / 01 in England. medRxiv 2020.12.24.20248822; doi: https: / / doi.org / 10.1101 / 2020.12.24.20248822 (9) Houriiyah Tegally, Eduan Wilkinson, Marta Giovanetti, et al. Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa. medRxiv 2020.12.21.20248640; doi: https: / / doi.org / 10.1101 / 2020.12.21.20248640 (10) Kim JS, Jang JH, Kim JM, Chung YS, Yoo CK, Han MG. Genome-Wide Identification and Characterization of Point Mutations in the SARS-CoV-2 Genome. Osong Public Health Res Perspect. 2020;11(3):101-111. doi:10.24171 / j.phrp.2020.11.3.05 (11) Nilgiriwala K, Mandal A, Patel G, Mestry T, Vaswani S, Shaikh A, Sriraman K, Parikh S, Udupa S, Chatterjee N, Shastri J, Mistry N. Genome Sequences of Five SARS-CoV-2 Variants from Mumbai, India, Obtained by Nanopore Sequencing. Microbiol Resour Announc. 2021 Apr 15;10(15):e00231-21 (12) Wenjuan Zhang, Brian D Davis, Stephanie S Chen, Jorge M Sincuir Martinez, Jasmine T Plummer, Eric Vail. Emergence of a Novel SARS-CoV-2 Variant in Southern California. JAMA. 2021 Apr 6;325(13):1324-1326

[0016] For example, the N501Y mutation is a non-synonymous mutation in the receptor-binding domain (RBD) of the S protein, common to three SARS-CoV-2 strains first identified in southeastern England, Brazil / Japan, and South Africa: B.1.1.7, P.1 (also known as 20J / 501Y.V3), and 501Y.V2, respectively. This is one of the important contact residues within the RBD and has been shown to enhance binding affinity to human and mouse ACE2. The E484K mutation in the receptor-binding domain (RBD) of the S protein is present in new strains 501Y.S2 and B.1.1.28 from South Africa and Brazil, respectively, and affects residues within the RBD that have been shown to be important for the binding of many neutralizing antibodies. The E484Q mutation in the receptor-binding domain (RBD) of the S protein is present in new strains B.1.617 and B.1.429 from India and Denmark, respectively, and affects the same residues within the RBD. Studies have suggested that the L452R mutation can stabilize the interaction between the spike protein and its human ACE2 receptor, thereby increasing the viral infectivity. Therefore, this mutation affects antibody recognition, enabling immune evasion of SARS-CoV-2. Viruses with this mutation have been shown to evade recognition by antibodies in convalescent serum, thus potentially altering vaccine effectiveness (see, e.g., Allison J. Greaney, Andrea N. Loes, Katharine HD Crawford, Tyler N. Starr, Keara D. Malone, Helen Y. Chu, Jesse D. Bloom, bioRxiv 2020.12.31.425021). Several other mutations have also been discovered. Mutations in the S1 protein K417N, K417T, V367F, N354D, W436R, or V483A have been shown to bind to ACE2 with higher affinity. V483A and G476S mutations have previously been reported to be related to human receptor binding affinity in MERS and SARS-CoV studies. On the other hand, R408I potentially reduces ACE2 binding affinity.According to the present invention, the main natural mutations include the K417N mutation of SEQ ID NO: 3 in which the amino acid residue (K) at position 417 of SEQ ID NO: 3 is substituted with the amino acid residue (N), the K417T mutation of SEQ ID NO: 3 in which the amino acid residue (K) at position 417 of SEQ ID NO: 3 is substituted with the amino acid residue (T), the E484K mutation of SEQ ID NO: 3 in which the amino acid residue (E) at position 484 of SEQ ID NO: 3 is substituted with the amino acid residue (K), the E48Q mutation of SEQ ID NO: 3 in which the amino acid residue (E) at position 484 is substituted with the amino acid residue (Q), the L452N mutation of SEQ ID NO: 3 in which the amino acid residue (L) at position 452 of SEQ ID NO: 3 is substituted with the amino acid residue (N), and the N501Y mutation of SEQ ID NO: 3 in which the amino acid residue (N) at position 501 of SEQ ID NO: 3 is substituted with the amino acid residue (Y).

[0017] As used herein, the term "unnatural mutation" refers to any mutation that is genetically inserted into the polypeptide of the present invention. In particular, the mutation is inserted to facilitate the production of the polypeptide. For example, the mutations include the C136S mutation in which the amino acid residue (C) at position 136 of SEQ ID NO: 3 is substituted by the amino acid residue (S), and the C538S mutation of SEQ ID NO: 3 in which the amino acid residue (C) at position 538 of SEQ ID NO: 3 is substituted with the amino acid residue (S). The mutation is particularly suitable for avoiding the occurrence of disulfide bonds in the polypeptide of the present invention.

[0018] As used herein, the term "membrane protein" or "M protein" refers to a protein of SARS-CoV-2 that is a component of the viral envelope and plays a central role in viral morphogenesis and assembly through interaction with other viral proteins. Generally, the membrane protein has the amino acid sequence shown in SEQ ID NO: 1.

[0019] SEQ ID NO: 1>sp|P0DTC5|VME1_SARS2 membrane protein OS=Severe acute respiratory syndrome coronavirus 2 OX=2697049 PE=3 SV=1. Polypeptides Mpep1 and Mpep2 are underlined.

[0020]

Chem.

[0021] As used herein, the terms "nucleoprotein" or "N protein" refer to the SARS-CoV-2 protein that encapsulates the plus-strand viral genomic RNA into a helical ribonucleocapsid (RNP) and plays a fundamental role during virion assembly through interaction with the viral genome and the membrane protein M.

[0022] SEQ ID NO: 2>sp|P0DTC9|NCAP_SARS2 nucleoprotein OS=severe acute respiratory syndrome coronavirus 2 OX=2697049 GN=N PE=1 SV=1. The polypeptides Npep1 and Npep2 are underlined.

[0023]

Chem.

[0024] As used herein, the terms “spike protein” or “S protein” refer to the SARS-CoV-2 spike glycoprotein, which binds to its cell receptor (i.e., ACE2) and mediates membrane fusion and viral entry. Each monomer of the trimeric S protein is approximately 180 kDa and contains two subunits, S1 and S2, which mediate attachment and membrane fusion, respectively. In particular, spike protein S1 causes virions to attach to the cell membrane by interacting with the host receptor (i.e., the human ACE2 receptor). Spike protein S2 mediates the fusion of virions to the cell membrane by functioning as a class I viral fusion protein. In current models, this protein has at least three conformations: a pre-fusion native state, a pre-hairpin intermediate state, and a post-fusion hairpin state. During membrane fusion between the virus and the target cell, the coiled-coil region (heptad repeat) adopts a trimeric hairpin structure, positioning the fusion peptide close to the C-terminal region of the ectodomain. The formation of this structure is thought to promote the juxtaposition and subsequent fusion of the viral membrane and the target cell membrane. The spike protein S2' functions as a viral fusion peptide that is exposed after S2 cleavage occurs during viral endocytosis. Generally, the spike protein has the amino acid sequence shown in SEQ ID NO: 3.

[0025] Sequence ID 3>sp|P0DTC2|SPIKE_SARS2 spike glycoprotein OS=Severe Acute Respiratory Syndrome Coronavirus 2 OX=2697049 GN=S PE=1 SV=1. Polypeptides Spep1, Spep2, Spep3, and Spep4 are underlined. RBD polypeptides are shown in italics and bold.

[0026] [ka]

[0027] As used herein, the term "RBD polypeptide" refers to a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 3, i.e., the amino acid sequence in the range of amino acid residues 319 to 541 of the spike protein.

[0028] In some embodiments, the RBD polypeptide consists of an amino acid sequence ranging from amino acid residue 319 to amino acid residue 541 of SEQ ID NO: 3.

[0029] In some embodiments, the RBD polypeptide consists of amino acids in the range of amino acid residues 319 to 541 of SEQ ID NO: 3, and containing one or more non-natural mutations. In some embodiments, the RBD polypeptide consists of amino acids in the range of amino acid residues 319 to 541 of SEQ ID NO: 3, and containing a non-natural mutation at position 538. In some embodiments, the RBD polypeptide consists of amino acids in the range of amino acid residues 319 to 541 of SEQ ID NO: 3, and containing a C538S mutation.

[0030] In some embodiments, the RBD polypeptide consists of amino acids in the range of amino acid residues 319 to 541 of SEQ ID NO: 3, and containing one or more spontaneous mutations. In some embodiments, the RBD polypeptide consists of amino acids in the range of amino acid residues 319 to 541 of SEQ ID NO: 3, and containing one or more spontaneous mutations at positions 417, 452, 484, or 501. In some embodiments, the RBD polypeptide consists of amino acids in the range of amino acid residues 319 to 541 of SEQ ID NO: 3, and containing one or more spontaneous mutations at positions selected from the group consisting of K417N, K417T, L452N, E84Q, E484K, and N501Y mutations.

[0031] In some embodiments, the RBD polypeptide consists of amino acids in the range of amino acid residues 319 to 541 of SEQ ID NO: 3, and includes one or more non-natural mutations (particularly the C538S mutation) and one or more natural mutations (preferably one or more of the K417N, K417T, L452N, E84Q, E484K, and N501Y mutations).

[0032] In some embodiments, the RBD polypeptide consists of amino acids in the range of amino acid residues 319 to 541 of SEQ ID NO: 3, and includes the N501Y spontaneous mutation and the C538S non-spontaneous mutation.

[0033] In some embodiments, the RBD polypeptide consists of amino acids in the range of amino acid residues 319 to 541 of SEQ ID NO: 3, and includes the K417T, E484K, N501Y spontaneous mutations and the non-spontaneous mutation C538S.

[0034] In some embodiments, the RBD polypeptide is in the range of amino acid residues 319 to 541 of SEQ ID NO: 3, and includes amino acids including the K417N, E484K, N501Y spontaneous mutations and the non-spontaneous C538S mutation ("RBD South Africa strain" or "RBD"). SA Var」 ) consists of.

[0035] In some embodiments, the RBD polypeptide consists of amino acids in the range of amino acid residues 319 to 541 of SEQ ID NO: 3, and including the E484Q, L452N spontaneous mutation and the non-spontaneous C538S mutation ("RBD Indian strain").

[0036] As used herein, the terms “conjugate” or interchangeable “conjugated polypeptide” are intended to refer to a complex or chimeric molecule formed by the covalent bonding of one or more polypeptides. The terms “covalent bonding” or “conjugation” mean that the polypeptide and non-peptide moieties are directly covalently bonded to each other, or indirectly covalently bonded to each other through one or more intervening parts, such as one or more bridges, spacers, or linking parts. A particular conjugate is a fusion protein.

[0037] As used herein, the term “fusion protein” includes at least one polypeptide of the present invention operably linked to a heterologous polypeptide. Within a fusion protein, the term “operably linked” is intended to indicate that the peptide of the present invention and the heterologous polypeptide are fused to each other in frame.

[0038] As used herein, the term "heterogeneous polypeptide" refers to a polypeptide that does not originate from the same protein with which the heterogeneous polypeptide is fused.

[0039] As used herein, the term "linker" refers to a sequence of at least one amino acid that links the peptide of the present invention to a heterologous polypeptide. Linkers are well known to those skilled in the art and generally consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids.

[0040] As used herein, the term “antibody” refers to an immunoglobulin molecule and a molecule containing the immunologically active portion of an immunoglobulin molecule, i.e., an antigen-binding site that binds immunospecifically to an antigen. In natural antibodies of rodents and primates, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by disulfide bonds. There are two types of light chains: lambda(1) and kappa(k). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinctly different sequence domains. In a typical IgG antibody, the light chain contains two domains: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: one variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant region domains of the light chain (CL) and heavy chain (CH) confer important biological properties, such as antibody chain binding, secretion, placental permeability, complement binding, and binding to the Fc receptor (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of immunoglobulins and consists of the variable regions of one light chain and one heavy chain. Antibody specificity lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is mainly composed of residues from the hypervariable region or complementarity-determining region (CDR). Sometimes, residues from the non-hypervariable region or framework region (FR) may also be involved in the antibody binding site, or may affect the entire domain structure, and therefore the binding site. The complementarity-determining region (CDR) refers to the amino acid sequence that determines both the binding affinity and specificity of the intrinsic Fv region of the immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, called L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, and H-CDR3, respectively. Therefore, the antigen-binding site typically contains six CDRs, including those set from the heavy chain V region and the light chain V region. The framework region (FR) refers to the amino acid sequence sandwiched between the CDRs.Therefore, the variable regions of the light and heavy chains typically include four framework regions and three CDRs in the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Residues in the antibody variable domain are conventionally numbered according to a system devised by Kabat et al. This system is shown in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (Kabat et al., 1992, hereafter “Kabat et al.”). The designation of Kabat residues does not necessarily directly correspond to the linear numbering of amino acid residues in the sequence of the sequence number. The actual linear amino acid sequence, whether the framework of the basic variable domain structure or the complementarity-determining regions (CDRs), may contain fewer or additional amino acids than the strict Kabat code corresponding to the shortening or insertion of structural components. The correct Kabat code for a residue can be determined for a given antibody by aligning homologous residues in the antibody sequence with the "standard" Kabat code sequence. The CDRs for the heavy chain variable domain are located at residues 31-35 (H-CDR1), 50-65 (H-CDR2), and 95-102 (H-CDR3) according to the Kabat code system. The CDRs for the light chain variable domain are located at residues 24-34 (L-CDR1), 50-56 (L-CDR2), and 89-97 (L-CDR3) according to the Kabat code system. For the antibodies listed below, the CDRs were determined using the CDR search algorithm at www.bioinf.org.uk. See the section titled "How to identify the CDRs by looking at a sequence" on the antibody page.

[0041] As used herein, the term “immunoglobulin domain” refers to a globular region of an antibody chain (for example, a chain of a conventional four-chain antibody or a heavy-chain or light-chain antibody), or a polypeptide essentially derived from such a globular region.

[0042] As used herein, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including the native sequence Fc region and the mutant Fc region. The human IgG heavy chain Fc region is generally defined as containing amino acid residues from C226 or P230 to the carboxyl terminus of an IgG antibody. The code numbers of the residues within the Fc region are the code numbers of the Kabat EU index. The C-terminal lysine (residue K447) of the Fc region can be removed, for example, during antibody production or purification. Thus, the antibody compositions of the present invention may include antibody populations from which all K447 residues have been removed, antibody populations from which the K447 residue has not been removed, and antibody populations including mixtures of antibodies containing and not containing the K447 residue.

[0043] As used herein, the term “epitope” has its general meaning in the art and is a fragment of at least eight amino acids recognized by an immune response component. As used herein, the term “immune response component” includes, but is not limited to, macrophages, lymphocytes, T lymphocytes, killer T lymphocytes, immune response modulators, helper T lymphocytes, antigen receptors, antigen-presenting cells, cytotoxic T lymphocytes, T-8 lymphocytes, CD1 molecules, B lymphocytes, antibodies, recombinant antibodies, genetically modified antibodies, chimeric antibodies, monospecific antibodies, bispecific antibodies, multispecific antibodies, diabodies, chimeric antibodies, humanized antibodies, human antibodies, heteroantibodies, monoclonal antibodies, polyclonal antibodies, antibody fragments, and / or synthetic antibodies. The term “epitope” can be used interchangeably with antigen, paratope-binding site, antigenic determinant, and / or determinant.

[0044] As used herein, the term “polyepitope polypeptide” refers to a polypeptide comprising at least two epitopes. In particular, the polyepitope polypeptide of the present invention comprises one or more SARS-CoV-2 polypeptides of the present invention.

[0045] As used herein, the term “antibody epitope” refers to a peptide that can be recognized by a particular antibody or that induces the formation of a particular antibody.

[0046] As used herein, the term “chimeric antibody” refers to an antibody that contains the VH and VL domains of a non-human antibody and the CH and CL domains of a human antibody. In one embodiment, a “chimeric antibody” is an antibody in which the constant region (i.e., the heavy and / or light chain), or a portion thereof, is modified, substituted, or exchanged so that the antigen-binding site (variable region) is a constant region of a different or modified class, effector function, and / or species, or a chimeric antibody in which the constant region (i.e., the heavy and / or light chain), or a portion thereof, is modified, substituted, or exchanged so that it can be bound to an entirely different molecule, such as an enzyme, toxin, agonist molecule, such as a CD40 ligand, hormone, growth factor, drug, etc., conferring new properties to it; or (b) an antibody molecule in which the variable region, or a portion thereof, is modified, substituted, or exchanged so that the variable region has a different or modified antigen specificity. Chimeric antibodies also include primatized, in particular, humanized antibodies. Furthermore, chimeric antibodies may contain residues not found in the recipient antibody or donor antibody. These modifications are made to further refine the performance of the antibody. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992) (US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0047] As used herein, the term “humanized antibody” includes antibodies having six CDRs of a mouse antibody and a humanized framework and constant region. More specifically, as used herein, the term “humanized antibody” may include antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, are grafted onto a human framework sequence.

[0048] As used herein, the term “human monoclonal antibody” is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human immunoglobulin sequences (for example, mutations introduced in vitro by random mutagenesis or site-directed mutagenesis, or in vivo by somatic mutation). However, in one embodiment, the term “human monoclonal antibody” as used herein is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mice, are grafted onto a human framework sequence.

[0049] As used herein, the terms “Human Leukocyte Antigen System” or “HLA” have their general meaning in the art and refer to the human major histocompatibility complex (MHC). The MHC contains many genes that encode cell surface antigen-presenting proteins. Proteins encoded by certain genes are also known as antigens. The major HLA antigens are HLA class I antigens (A, B, and C) and HLA class II antigens (DR, DP, and DQ). HLA class I antigens typically present peptides (8-12 amino acids) derived from inside the cell and attract CD8 cytotoxic T cells that destroy cells. HLA class II antigens typically present peptides derived from outside the cell to CD4 T helper lymphocytes and stimulate B cells and other immune cells.

[0050] As used herein, the term “immune response” refers to the immune system’s reaction to an antigen in a host’s body, including the production of antigen-specific antibodies and / or cytotoxic responses. An immune response to initial antigen exposure (primary immune response) is typically detectable after a time lag of several days to two weeks, while an immune response to subsequent stimulation by the same antigen (secondary immune response) is more rapid than the primary immune response. An immune response to a transgene product may include both humoral (e.g., antibody response) and cellular (e.g., cytolytic T cell response) immune responses that can be induced against the immunogenic product encoded by the transgene. The level of the immune response can be measured by methods known in the art (e.g., measurement of antibody titers).

[0051] As used herein, the terms “APC” or “antigen-presenting cell” refer to, but are not limited to, cells capable of activating T cells, including certain macrophages, B cells, and dendritic cells.

[0052] As used herein, the terms “dendritic cell” or “DC” refer to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their distinctive morphology and high levels of surface MHC class II expression (Steinman, et al, Ann. Rev. Immunol. 9:271 (1991); a description of such cells is incorporated herein by reference).

[0053] As used herein, the term "CD40" has its general meaning in the art and refers to the human CD40 polypeptide receptor. In some embodiments, CD40 is an isoform of the human canonical sequence reported by UniProtKB-P25942 (also known as human TNR5).

[0054] As used herein, the term "CD40L" has its general meaning in the art and refers to the human CD40L polypeptide reported, for example, by UniProtKB-P25942, which includes its CD40-binding domain in SEQ ID NO: 4. CD40L can be represented as a soluble polypeptide and is a native ligand for the CD40 receptor.

[0055] Sequence ID 4>CD40L binding domain [ka]

[0056] As used herein, the term “CD40 agonist antibody” is intended to refer to an antibody that enhances CD40-mediated signaling activity in the absence of CD40L in cell line assays such as B cell proliferation assays. In particular, CD40 agonist antibodies induce B cell proliferation, as measured in vitro by flow cytometry analysis or by analysis of repeated dilutions of CFSE-labeled cells; and / or (ii) induce the secretion of cytokines such as IL-6, IL-12, or IL-15, as measured in vitro by dendritic cell activation assays.

[0057] As used herein, the term "langerin" has its general meaning in the art and refers to a four-member K polypeptide of the human type C lectin domain family. In some embodiments, langerin is an isoform of the human canonical sequence reported by UniProtKB-Q9UJ71 (also referred to as human CD207).

[0058] As used herein, the terms “treatment” or “to treat” include both preventive or protective measures and curative or disease-modifying measures, including the treatment of patients at risk of or suspected of having the disease, as well as patients with the disease or diagnosed with the disease or condition, and include the suppression of clinical relapse. Treatment may be administered to patients with a medical disability or those likely to eventually become disabled, to prevent, cure, delay the onset, reduce the severity of, or improve one or more symptoms of the disability or recurrent disability, or to extend the patient’s survival beyond what would be expected without such treatment. “Treatment regimen” means a pattern of treatment for the disease, e.g., a pattern of dosing used during treatment. A treatment regimen may include an induction regimen and a maintenance regimen. The terms “induction regimen” or “induction period” refer to a treatment regimen (or part of a treatment regimen) used for the initial treatment of the disease. A common goal of an induction regimen is to deliver a high level of medication to the patient during the initial period of a treatment regimen. An induction regimen may employ a “loading regimen” (partially or entirely), which may include administering a larger dose of the drug than the physician uses during the maintenance regimen, administering the drug more frequently than the physician uses during the maintenance regimen, or both. The terms “maintenance therapy” or “maintenance period” refer to a treatment regimen (or part of a treatment regimen) used to maintain a patient during treatment for a disease, for example, used to keep a patient in remission for an extended period (several months or years). Maintenance regimens may be continuous therapy (e.g., administering the drug at regular intervals such as weekly, monthly, or yearly) or intermittent therapy (e.g., interruption therapy, intermittent therapy, therapy on relapse, or therapy when certain predetermined criteria [e.g., pain, disease onset, etc.] are reached).

[0059] As used herein, the term “pharmaceutical composition” refers to the composition described herein, or a pharmaceutically acceptable salt thereof, accompanied by other agents such as carriers and / or excipients. The pharmaceutical compositions provided herein generally include a pharmaceutically acceptable carrier.

[0060] As used herein, the term “pharmaceutically acceptable carrier” includes any solvent, diluent, or other liquid vehicle, dispersion or suspension aid, surfactant, isotonic agent, thickener or emulsifier, preservative, solid binder, lubricant, etc., suitable for a desired specific dosage form. Remington's Pharmaceutical-Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in the preparation of pharmaceutical compositions and known techniques for their preparation.

[0061] As used herein, the terms “vaccination” or “to administer a vaccine” mean, but are not limited to, a method of inducing an immune response to a particular antigen in a subject.

[0062] As used herein, the term “vaccine composition” is intended to mean a composition that can be administered to humans or animals to induce an immune response, which may result in the activation of certain cells, in particular APCs, T lymphocytes, and B lymphocytes.

[0063] As used herein, the term “antigen” refers to a molecule that, when processed by an MHC molecule and presented, can be specifically bound to an antibody or a T cell receptor (TCR). An antigen may also be recognizable by the immune system and / or induce humoral and / or cellular immune responses, resulting in the activation of B lymphocytes and / or T lymphocytes. An antigen may have one or more epitopes or antigenic sites (B-epitopes and T-epitopes).

[0064] As used herein, the term “adjuvant” refers to a compound that, when administered to a subject or animal, can induce and / or enhance an immune response to an antigen. It is also intended to mean a substance that generally acts to accelerate, prolong, or enhance the quality of a specific immune response to a particular antigen. In the context of the present invention, the term “adjuvant” means a compound that enhances the innate immune response by influencing the transient reaction of the innate immune response, as well as enhancing the longer-term effects of the adaptive immune response by activating and maturing antigen-presenting cells (APCs), particularly dendritic cells (DCs).

[0065] As used herein, the expression "therapeutably effective amount" means an amount of the active ingredient of the present invention sufficient to induce an immune response with a reasonable benefit-risk ratio applicable to medical treatment.

[0066] As used herein, the term “immune checkpoint inhibitor” has its general meaning in the art and refers to any compound that inhibits the function of an immunosuppressive checkpoint protein. As used herein, the term “immune checkpoint protein” has its general meaning in the art and refers to a molecule expressed by T cells that either enhances signaling (stimulative checkpoint molecule) or reduces signaling (inhibitory checkpoint molecule). Immune checkpoint molecules are recognized in the art as constituting immune checkpoint pathways similar to the CTLA-4 and PD-1-dependent pathway (see, e.g., Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480-489). Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3, and VISTA.

[0067] The polypeptide of the present invention: One object of the present invention relates to a SARS-CoV-2 polypeptide (M1-110, also referred to as "Mpep1") consisting of at least 50 consecutive amino acids whose amino acid sequence is derived from the M protein and has at least 90% identity with the amino acid sequence in the range of residues 1 to 110 of SEQ ID NO: 1.

[0068] In some embodiments, polypeptide (M1-110) contains 50;51;52;53;54;55;56;57;58;59;60;61;62;63;64;65;66;67;68;69;70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;88;89;90;91;92;93;94;95;96;97;98;99;100;101;102;103;104;105;106;107;108;109; or 110 consecutive amino acids of an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 1 to 110 of SEQ ID NO: 1.

[0069] In some embodiments, polypeptide (M1-110) consists of an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 1 to 110 of SEQ ID NO: 1. In some embodiments, polypeptide (M1-110) contains one or more spontaneous mutations.

[0070] A further object of the present invention relates to a SARS-CoV-2 polypeptide (M132-222, also referred to as "Mpep2") consisting of at least 50 consecutive amino acids of an amino acid sequence derived from the M protein and having at least 90% identity with the amino acid sequence in the range of residues 132 to 222 of SEQ ID NO: 1.

[0071] In some embodiments, the polypeptide (M132-222) contains 50;51;52;53;54;55;56;57;58;59;60;61;62;63;64;65;66;67;68;69;70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;88;89;90; or 91 consecutive amino acids of an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 132 to 222 of SEQ ID NO: 1.

[0072] In some embodiments, the polypeptide (M132-222) consists of an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 132 to 222 of Sequence ID No. 1. In some embodiments, the polypeptide (M132-222) contains one or more spontaneous mutations.

[0073] A further object of the present invention relates to a SARS-CoV-2 polypeptide (also called "Npep1," or N78-206) that is derived from the N protein and consists of at least 50 consecutive amino acids of an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 78 to 206 of SEQ ID NO: 2.

[0074] In some embodiments, polypeptide (N78-206) has at least 90% identity with the amino acid sequence in the range of residues 78 to 206 of SEQ ID NO: 50;51;52;53;54;55;56;57;58;59;60;61;62;63;64;65;66;67;68;69;70;71;72;73;74;75;76;77;78;79;80;81;82;83; Contains 84;85;86;87;88;89;90;91;92;93;94;95;96;97;98;99;100;101;102;103;104;105;106;107;108;109;110;111;112;113;114;115;116;117;118;119;120;121;122;123;124;125;126;127;128; or 129 consecutive amino acids.

[0075] In some embodiments, the polypeptide (N78-206) consists of an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 78 to 206 of SEQ ID NO: 2. In some embodiments, the polypeptide (N78-206) contains one or more spontaneous mutations.

[0076] A further object of the present invention relates to a SARS-CoV-2 polypeptide (also referred to as "Npep2," N276-411) consisting of at least 50 consecutive amino acids of an amino acid sequence derived from the N protein and having at least 90% identity with the amino acid sequence in the range of residues 276 to 411 of SEQ ID NO: 2.

[0077] In some embodiments, polypeptide (N276-411) has an amino acid sequence that is at least 90% identical to the amino acid sequence in the range of residues 276 to 411 of SEQ ID NO: 50;51;52;53;54;55;56;57;58;59;60;61;62;63;64;65;66;67;68;69;70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;8 Contains 8;89;90;91;92;93;94;95;96;97;98;99;100;101;102;103;104;105;106;107;108;109;110;111;112;113;114;115;116;117;118;119;120;121;122;123;124;125;126;127;128;129;130;131;132;133;134;135; or 136 consecutive amino acids.

[0078] In some embodiments, the polypeptide (N276-411) consists of an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 276 to 411 of SEQ ID NO: 2. In some embodiments, the polypeptide (N276-411) contains one or more spontaneous mutations.

[0079] A further object of the present invention relates to a SARS-CoV-2 polypeptide (S125-250, also referred to as "Spep1") consisting of at least 50 consecutive amino acids of an amino acid sequence derived from the S protein and having at least 90% identity with the amino acid sequence in the range of residues 125 to 250 of SEQ ID NO: 3.

[0080] In some embodiments, polypeptide (S125-250) has an amino acid sequence that is at least 90% identical to the amino acid sequence in the range of residues 125 to 250 of SEQ ID NO: 50;51;52;53;54;55;56;57;58;59;60;61;62;63;64;65;66;67;68;69;70;71;72;73;74;75;76;77;78;79;80;81 ;82;83;84;85;86;87;88;89;90;91;92;93;94;95;96;97;98;99;100;101;102;103;104;105;106;107;108;109;110;111;112;113;114;115;116;117;118;119;120;121;122;123;124;125; or containing 126 consecutive amino acids.

[0081] In some embodiments, the polypeptide (S125-250) consists of an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 125 to 250 of SEQ ID NO: 3.

[0082] In some embodiments, the polypeptide (S125-250) contains one or more spontaneous mutations.

[0083] In some embodiments, the polypeptide (S125-250) consists of an amino acid sequence in the range of residues 125 to 250 of SEQ ID NO: 3, and includes a non-natural mutation at position 136. In some embodiments, the polypeptide (S125-250) consists of an amino acid sequence in the range of residues 125 to 250 of SEQ ID NO: 3, and includes a C136S non-natural mutation ("Spep1 C136S It consists of an amino acid sequence containing ''.

[0084] A further object of the present invention relates to a SARS-CoV-2 polypeptide (S280-598, also referred to as "Spep2") derived from the S protein and having at least 90% identity with an amino acid sequence consisting of at least 50 consecutive amino acids in the range of residues 280 to 598 of SEQ ID NO: 3.

[0085] In some embodiments, polypeptide (S280-598) has an amino acid sequence that is at least 90% identical to the amino acid sequence in the range of residues 280 to 598 of SEQ ID NO: 50;51;52;53;54;55;56;57;58;59;60;61;62;63;64;65;66;67;68;69;70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;88;89;90;91;92;93;94;95;96;97;98;99;100;101;102;103;1 04;105;106;107;108;109;110;111;112;113;114;115;116;117;118;119;120;121;122;123;124;125;126;127;128;129;130;131;132;133;134;13 5;136;137;138;139;140;141;142;143;144;145;146;147;148;149;150;151;152;153;154;155;156;157;158;159;160;161;162;163;164;165;166 ;167;168;169;170;171;172;173;174;175;176;177;178;179;180;181;182;183;184;185;186;187;188;189;190;191;192;193;194;195;196;197; 198;199;200;201;202;203;204;205;206;207;208;209;210;211;212;213;214;215;216;217;218;219;220;221;222;223;224;225;226;227;228;2 29;230;231;232;233;234;235;236;237;238;239;240;241;242;243;244;245;246;247;248;249;250;251;252;253;254;255;256;257;258;259;26 0;261;262;263;264;265;266;267;268;269;270;271;272;273;274;275;276;277;278;279;280;281;282;283;284;285;286;287;288;289;290;291;Contains 292;293;294;295;296;297;298;299;300;301;302;303;304;305;306;307;308;309;310;311;312;313;314;315;316;317;318; or 319 consecutive amino acids.

[0086] In some embodiments, the polypeptide (S280-598) consists of an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 280 to 598 of SEQ ID NO: 3.

[0087] In some embodiments, the polypeptide (S280-598) consists of an amino acid sequence that is in the range of residues 280 to 598 of SEQ ID NO: 3 and has at least 90% identity with an amino acid sequence that includes one or more non-natural mutations. In some embodiments, the polypeptide (S280-598) consists of an amino acid sequence that is in the range of residues 280 to 598 of SEQ ID NO: 3 and has at least 90% identity with an amino acid sequence that includes a non-natural mutation at position 538. In some embodiments, the polypeptide (S280-598) consists of an amino acid sequence that is in the range of residues 280 to 598 of SEQ ID NO: 3 and has at least 90% identity with an amino acid sequence that includes a C538S non-natural mutation.

[0088] In some embodiments, the polypeptide (S280-598) consists of an amino acid sequence in the range of residues 280 to 598 of SEQ ID NO: 3, and containing one or more spontaneous mutations. In some embodiments, the polypeptide (S280-598) consists of an amino acid sequence in the range of residues 280 to 598 of SEQ ID NO: 3, and containing one or more spontaneous mutations at positions 417, 438, or 501. In some embodiments, the polypeptide (S280-598) consists of an amino acid sequence in the range of residues 280 to 598 of SEQ ID NO: 3, and containing one or more spontaneous mutations selected from the group consisting of K417N, K417T, E484K, and N501Y mutations.

[0089] In some embodiments, the polypeptide (S280-598) consists of an amino acid sequence in the range of residues 280 to 598 of SEQ ID NO: 3, and includes the N501Y spontaneous mutation and the C538S non-spontaneous mutation.

[0090] In some embodiments, the polypeptide (S280-598) is in the range of residues 280 to 598 of SEQ ID NO: 3 and consists of an amino acid sequence that includes the K417T, E484K, N501Y spontaneous mutations and the C538S non-spontaneous mutation.

[0091] In some embodiments, the polypeptide (S280-598) is in the range of residues 280 to 598 of SEQ ID NO: 3 and consists of an amino acid sequence including the K417N, E484K, N501Y spontaneous mutations and the C538S non-spontaneous mutation.

[0092] A further object of the present invention relates to a SARS-CoV-2 polypeptide (S680-1029, also referred to as "Spep3") consisting of at least 50 consecutive amino acids of an amino acid sequence derived from the S protein and having at least 90% identity with the amino acid sequence in the range of residues 680 to 1029 of SEQ ID NO: 3.

[0093] In some embodiments, polypeptide (S680-1029) has an amino acid sequence that is at least 90% identical to the amino acid sequence in the range of residues 680 to 1029 of SEQ ID NO: 50;51;52;53;54;55;56;57;58;59;60;61;62;63;64;65;66;67;68;69;70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;88;89;90;91;92;93;94;95;96;97;98;99;100;101;102;10 3;104;105;106;107;108;109;110;111;112;113;114;115;116;117;118;119;120;121;122;123;124;125;126;127;128;129;130;131;132;133;134 ;135;136;137;138;139;140;141;142;143;144;145;146;147;148;149;150;151;152;153;154;155;156;157;158;159;160;161;162;163;164;165; 166;167;168;169;170;171;172;173;174;175;176;177;178;179;180;181;182;183;184;185;186;187;188;189;190;191;192;193;194;195;196;1 97;198;199;200;201;202;203;204;205;206;207;208;209;210;211;212;213;214;215;216;217;218;219;220;221;222;223;224;225;226;227;22 8;229;230;231;232;233;234;235;236;237;238;239;240;241;242;243;244;245;246;247;248;249;250;251;252;253;254;255;256;257;258;259 ;260;261;262;263;264;265;266;267;268;269;270;271;272;273;274;275;276;277;278;279;280;281;282;283;284;285;286;287;288;289;290;291;292;293;294;295;296;297;298;299;300;301;302;303;304;305;306;307;308;309;310;311;312;313;314;315;316;317;318;319;320;321;322; Contains 323;324;325;326;327;328;329;330;331;332;333;334;335;336;337;338;339;340;341;342;343;344;345;346;347;348;349; or 350 consecutive amino acids.

[0094] In some embodiments, the polypeptide (S680-1029) comprises an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 680 to 1029 of SEQ ID NO: 3. In some embodiments, the polypeptide (S680-1029) contains one or more spontaneous mutations. A further object of the present invention relates to a SARS-CoV-2 polypeptide (S1056-1209, also referred to as "Spep4") derived from the S protein and comprising at least 50 consecutive amino acids of an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 1056 to 1209 of SEQ ID NO: 3.

[0095] In some embodiments, polypeptide (S1056-1209) has an amino acid sequence that is at least 90% identical to the amino acid sequence in the range of residues 1056 to 1209 of SEQ ID NO: 50;51;52;53;54;55;56;57;58;59;60;61;62;63;64;65;66;67;68;69;70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;88;89;90;91;92;93;94;95;96;97;98;99 ;100;101;102;103;104;105;106;107;108;109;110;111;112;113;114;115;116;117;118;119;120;121;122;123;124;125;126;127;128;129;130;131;132;133;134;135;136;137;138;139;140;141;142;143;144;145;146;147;148;149;150;151;152;153; or 154 consecutive amino acids.

[0096] In some embodiments, the polypeptide (S1056-1209) consists of an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 1056 to 1209 of SEQ ID NO: 3. In some embodiments, the polypeptide (S10565-1209) contains one or more spontaneous mutations.

[0097] Conjugates and Fusion Proteins In some embodiments, the present invention relates to a conjugate in which a heterologous polypeptide is conjugated or fused to one or more SARS-CoV-2 polypeptides of the present invention.

[0098] In some embodiments, the conjugate of the present invention comprises 2, 3, 4, 5, 6, 7, or 8 of the SARS-CoV-2 polypeptides of the present invention. In some embodiments, the conjugate of the present invention comprises polypeptide (M1-110, "Mpep1"), polypeptide (M132-222, "Mpep2"), polypeptide (N78-206, "Npep1"), polypeptide (N276-411, "Npep2"), polypeptide (S125-250, "Spep1"), polypeptide (S280-598, "Spep2"), polypeptide (S680-1029, "pep3"), and polypeptide (S1056-1209, "Spep4").

[0099] In some embodiments, the conjugate of the present invention comprises one or more sequences derived from restriction cloning sites present in the polynucleotide encoding the conjugate. Generally, the sequences may consist of two amino acid residues and generally include AP, AS, AR, PR, SA, TR, and TS sequences.

[0100] In some embodiments, the conjugate of the present invention comprises a sequence of signal peptides. As used herein, the term “signal peptide” has its general meaning in the art and refers to a prepeptide that exists as an N-terminal peptide in the form of a protein precursor. The function of the signal peptide is to facilitate the transport of the expressed polypeptide to which it is attached into the endoplasmic reticulum. The signal peptide is typically cleaved in this process. The signal peptide may be heterogeneous or homogeneous to the organism used to produce the polypeptide.

[0101] In some embodiments, the conjugate of the present invention comprises polypeptide (N276-411 "Npep2"), polypeptide (S125-250 "Spep1"), and polypeptide (S1056-1209 "Spep4"). In some embodiments, the conjugate comprises a polyepitope polypeptide comprising, from the N-terminus to the C-terminus, a fusion of polypeptide (N276-411 "Npep2"), polypeptide (S125-250 "Spep1"), and polypeptide (S1056-1209 "Spep4"), and optionally one or more linkers described herein for separating Npep2 from Spep1 and / or Spep1 from Spep4. In some embodiments, the conjugate comprises a polyepitope polypeptide having the formula Npep2-Spep1-f2-Spep4, where f2 represents a linker described herein. In some embodiments, the conjugate comprises a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO: 5.

[0102] [ka]

[0103] In some embodiments, the conjugate of the present invention comprises a polypeptide (S125-250, "Spep1") and a polypeptide (S1056-1209, "Spep4"). In some embodiments, the conjugate comprises a polyepitope polypeptide comprising a fusion of polypeptide (N276-411, "Npep2") and polypeptide (S125-250, "Spep1") from the N-terminus to the C-terminus, and optionally a linker described herein for separating Npep2 from Spep1. In some embodiments, the conjugate comprises a polyepitope polypeptide having the formula Spep1-f2-Spep4, where f2 represents a linker described herein. In some embodiments, the conjugate comprises a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO: 6.

[0104] [ka]

[0105] In some embodiments, the conjugate of the present invention comprises a polypeptide (N276-411, "Npep2") and an RBD polypeptide. In some embodiments, the conjugate comprises a polyepitope polypeptide comprising a fusion of the polypeptide (N276-411, "Npep2") and the RBD polypeptide, and optionally a linker, as described later herein, for separating Npep2 from RBD, from the N-terminus to the C-terminus. In some embodiments, the conjugate comprises a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO: 7.

[0106] [ka]

[0107] In some embodiments, the conjugate of the present invention is RBD SA VAR The present invention comprises polypeptides and polypeptides (S1056-1209, "Spep4"). In some embodiments, the conjugate comprises a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO: 58.

[0108] [ka]

[0109] In some embodiments, the conjugate of the present invention comprises a polypeptide (N276-411, "Npep2") and a polypeptide (S125-250, "Spep1"). In some embodiments, the conjugate comprises a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO: 63.

[0110] [ka]

[0111] In some embodiments, the heterologous polypeptide is the immunoglobulin domain of the antibody, particularly the Fc region. More specifically, the heterologous polypeptide is the heavy and / or light chain of the antibody.

[0112] In some embodiments, the antibody is an IgG antibody, preferably an IgG1 or IgG4 antibody, or more preferably an IgG4 antibody.

[0113] In some embodiments, the antibody is a chimeric antibody, particularly a chimeric mouse / human antibody.

[0114] In some embodiments, the antibody is a humanized antibody.

[0115] Chimeric or humanized antibodies can be prepared based on the sequence of a mouse monoclonal antibody prepared as described above. The DNA encoding heavy and light chain immunoglobulins can be obtained from the target mouse hybridoma and manipulated using standard molecular biological techniques to include non-mouse (e.g., human) immunoglobulin sequences. For example, to produce a chimeric antibody, the mouse variable region can be ligated to the human constant region using methods known in the art (see, for example, Cabilly et al., U.S. Patent No. 4,816,567). To produce a humanized antibody, the mouse CDR region can be inserted into the human framework using methods known in the art. See, for example, Winter, U.S. Patent No. 5,225,539; Queen et al., U.S. Patents No. 5,530,101; No. 5,585,089; No. 5,693,762 and No. 6,180,370.

[0116] In some embodiments, the antibody is a human antibody. In some embodiments, the human antibody can be identified using a transgenic or transchromosomal mouse that has part of the human immune system rather than the mouse immune system. These transgenic and transchromosomal mice are referred to herein as HuMAb mice and KM mice, respectively, and include mice collectively referred to herein as "human Ig mice." HuMAb mice® (Medarex, Inc.) contain a human immunoglobulin gene minilosophyll that encodes unreconstituted human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, along with targeted mutations that inactivate endogenous μ and κ chain loci (see, e.g., Lonberg, et al., 1994 Nature 368(6474): 856-859). In other embodiments, the human antibody can be produced using a mouse having human immunoglobulin sequences on a transgene and transchromosome, such as a mouse having a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to as "KM mice" in this specification, are described in detail in Ishida et al.'s PCT publication WO02 / 43478.

[0117] In some embodiments, the antibody is against the surface antigen of the APC, and therefore the SARS-CoV-2 polypeptide of the present invention is targeted to the cell in order to elicit an immune response.

[0118] In some embodiments, the antibody is selected from antibodies that specifically bind to DC immune receptor (DCIR), MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11, CD15, CD16, CD19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1, Fey receptor, LOX-1, and ASPGR. In some embodiments, the antibody is specific to the cell surface marker of professional APC. Preferably, the antibody is specific to a cell surface marker of DC, such as CD83, CMRF-44, or CMRF-56. The antibody may also be specific to a cell surface marker of another professional APC, such as B cells or macrophages.

[0119] In some embodiments, the antibody is specific to CD40, which is expressed in both DCs, B cells, and other APCs, and therefore a larger number of APCs are recruited.

[0120] In some embodiments, the CD40 antibody is derived from the 12E12 antibody. • A heavy chain comprising complementarity-determining regions CDR1H, CDR2H, and CDR3H, wherein CDR1H has the amino acid sequence GFTFSDYYMY (SEQ ID NO: 8), CDR2H has the amino acid sequence YINSGGGSTYYPDTVKG (SEQ ID NO: 9), and CDR3H has the amino acid sequence RGLPFHAMDY (SEQ ID NO: 10), and • A light chain containing complementarity-determining regions CDR1L, CDR2L, and CDR3L, wherein CDR1L has the amino acid sequence SASQGISNYLN (SEQ ID NO: 11), CDR2L has the amino acid sequence YTSILHS (SEQ ID NO: 12), and CDR3L has the amino acid sequence QQFNKLPPT (SEQ ID NO: 13). Includes.

[0121] In some embodiments, the CD40 antibody is derived from the 11B6 antibody. • A heavy chain comprising complementarity-determining regions CDR1H, CDR2H, and CDR3H, wherein CDR1H has the amino acid sequence GYSFTGYYMH (SEQ ID NO: 14), CDR2H has the amino acid sequence RINPYNGATSYNQNFKD (SEQ ID NO: 15), and CDR3H has the amino acid sequence EDYVY (SEQ ID NO: 16), and • A light chain containing complementarity-determining regions CDR1L, CDR2L, and CDR3L, wherein CDR1L has the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO: 17), CDR2L has the amino acid sequence KVSNRFS (SEQ ID NO: 18), and CDR3L has the amino acid sequence SQSTHVPWT (SEQ ID NO: 19). Includes.

[0122] In some embodiments, the CD40 antibody is derived from the 12B4 antibody. • A heavy chain comprising complementarity-determining regions CDR1H, CDR2H, and CDR3H, wherein CDR1H has the amino acid sequence GYTFTDYVLH (SEQ ID NO: 20), CDR2H has the amino acid sequence YINPYNDGTKYNEKFKG (SEQ ID NO: 21), and CDR3H has the amino acid sequence GYPAYSGYAMDY (SEQ ID NO: 22), and • A light chain containing complementarity-determining regions CDR1L, CDR2L, and CDR3L, wherein CDR1L has the amino acid sequence RASQDISNYLN (SEQ ID NO: 23), CDR2L has the amino acid sequence YTSRLHS (SEQ ID NO: 24), and CDR3L has the amino acid sequence HHGNTLPWT (SEQ ID NO: 25). Includes.

[0123] In some embodiments, the CD40 antibody is selected from the group consisting of mAb1, mAb2, mAb3, mAb4, mAb5, and mAb6 listed in Table A.

[0124] [Table 1]

[0125] Sequence ID 26 (amino acid sequence of the heavy chain variable region (VH)(v2) of humanized 11B6)

[0126] [ka]

[0127] Sequence ID 27 (amino acid sequence of the light chain variable chain (VL) Vk(v2) of humanized 11B6 VL) [ka]

[0128] Sequence ID 28 (amino acid sequence of the heavy chain variable region VH(v3) of humanized 11B6) [ka]

[0129] Sequence ID 29 (VH amino acid sequence of mAb3(12B4)) [ka]

[0130] Sequence ID 30 (VL amino acid sequence of mAb3(12B4)) [ka]

[0131] Sequence ID 31 (VH amino acid sequence of mAb4(24A3 HC)) [ka]

[0132] Sequence ID 32 (VL amino acid sequence of mAb4(24A3 KC)) [ka]

[0133] Sequence ID 33 (VH amino acid sequence of mAb5) [ka]

[0134] Sequence ID 34 (VL amino acid sequence of mAb5) [ka]

[0135] Sequence ID 35 (VH amino acid sequence of mAb6(12E12 H3 humanized HC)) [ka]

[0136] Sequence ID 36 (VL amino acid sequence of mAb6 (humanized K2 12E12)) [ka]

[0137] In some embodiments, CD40 antibodies are CD40 agonist antibodies. CD40 agonist antibodies are described in WO2010 / 009346, WO2010 / 104747, and WO2010 / 104749. Other anti-CD40 agonist antibodies under development include CP-870,893, a fully human IgG2 CD40 agonist antibody developed by Pfizer. It binds to CD40 at a KD of 3.48 × 10⁻¹⁰ M, but does not block the binding of CD40L (see, e.g., U.S. Patent No. 7,338,660) and SGN-40, a humanized IgG1 antibody developed by Seattle Genetics from the mouse antibody clone S2C6, which was produced using human bladder cancer cell lines as immunogens. This binds to CD40 at KD 1.0 × 10⁻⁹ M and acts by enhancing the interaction between CD40 and CD40L, thus exhibiting a partial agonist effect (Francisco JA, et al., Cancer Res, 60: 3225-31, 2000). More specifically, the CD40 agonist antibodies are selected from the group consisting of mAb1, mAb2, mAb3, mAb4, mAb5, and mAb6, as listed in Table A.

[0138] In some embodiments, the heavy or light chain of the CD40 agonist antibody (i.e., a chain that is not conjugated or fused to one or more SARS-CoV-2 polypeptides of the present invention) is conjugated or fused to the CD40 binding domain of CD40L.

[0139] In some embodiments, the CD40-binding domain of CD40L is fused to the C-terminus of the light or heavy chain of the CD40 agonist antibody, optionally via a linker, preferably a FlexV1 linker as described later herein.

[0140] In some embodiments, the antibody of the present invention comprises a CD40 agonist antibody, wherein the heavy chain of the antibody is fused or conjugated to one or more SARS-CoV-2 polypeptides of the present invention, and the light chain is conjugated or fused to the CD40-binding domain of CD40L (SEQ ID NO: 4).

[0141] In some embodiments, the antibody is specific to Langerin.

[0142] In some embodiments, the antibody is derived from antibody 15B10 having ATCC accession number PTA-9852. In some embodiments, the antibody is derived from antibody 2G3 having ATCC accession number PTA-9853. In some embodiments, the antibody is derived from antibody 91E7, 37C1, or 4C7 described in publication WO2011032161.

[0143] In some embodiments, the anti-Langerin antibody comprises a heavy chain containing complementarity-determining regions CDR1H, CDR2H, and CDR3H of the 15B10 antibody and a light chain containing complementarity-determining regions CDR1L, CDR2L, and CDR3L of the 15B10 antibody.

[0144] In some embodiments, the anti-Langerin antibody comprises a heavy chain containing complementarity-determining regions CDR1H, CDR2H, and CDR3H of the 2G3 antibody, and a light chain containing complementarity-determining regions CDR1L, CDR2L, and CDR3L of the 2G3 antibody.

[0145] In some embodiments, the anti-Langerin antibody comprises a heavy chain containing the complementarity-determining regions CDR1H, CDR2H, and CDR3H of the 4C7 antibody and a light chain containing the complementarity-determining regions CDR1L, CDR2L, and CDR3L of the 4C7 antibody.

[0146] In some embodiments, resistance is Table B It is selected from the group consisting of mAb7, mAb8, mAb9, mAb10, mAb11, and mAb12 as described in [reference].

[0147] [Table 2]

[0148] Sequence ID 37 (amino acid sequence of the heavy chain variable region (VH) of 15B10) [ka]

[0149] Sequence ID 38 (amino acid sequence of light chain variable chain (VL) 15B10) [ka]

[0150] Sequence ID 39 (amino acid sequence of the heavy chain variable region (VH) of 2G3) [ka]

[0151] Sequence ID No. 40 (amino acid sequence of light chain variable chain (VL) 2G3) [ka]

[0152] Sequence ID 41 (4C7 heavy chain amino acid sequence) [ka]

[0153] Sequence ID 42 (amino acid sequence of the 4C7 light chain) [ka]

[0154] In some embodiments, heterologous polypeptides are conjugated with one or more SARS-CoV-2 polypeptides of the present invention by using chemical coupling. Several methods are known in the art for attachment or conjugation of an antibody to its conjugated portion. Examples of linker types used to conjugate a portion to an antibody include, but are not limited to, hydrazone, thioether, ester, disulfide, and peptide-containing linkers, e.g., valine-citrulline linkers. For example, linkers that are sensitive to cleavage by low pH in a lysosomal compartment or to cleavage by proteases such as cathepsins (e.g., cathepsins B, C, D) that are preferentially expressed in tumor tissues may be selected.Techniques for conjugating polypeptides are well known, particularly in this field (e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” Monoclonal Antibodies And Cancer Therapy (edited by Reisfeld et al., Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,” Controlled Drug Delivery (edited by Robinson et al., Marcel Deiker, Inc., 2nd edition 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” Monoclonal Antibodies '84: Biological And Clinical Applications (edited by Pinchera et al., 1985); “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy,” Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al.) See also al., ed., Academic Press, 1985; and Thorpe et al., 1982, Immunol. Rev. 62:119-58; see, for example, PCT Publication WO89 / 12624). Generally, peptides are covalently bonded to lysine or cysteine ​​residues on antibodies via N-hydroxysuccinimide ester or maleimide functional groups, respectively.Methods of conjugation using engineered cysteine ​​or incorporation of unnatural amino acids have been reported to improve the homogeneity of conjugates (Axup, JY, Bajjuri, KM, Ritland, M., Hutchins, BM, Kim, CH, Kazane, SA, Halder, R., Forsyth, JS, Santidrian, AF, Stafin, K., et al. (2012). Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. USA 109, 16101-16106.; Junutula, JR, Flagella, KM, Graham, RA, Parsons, KL, Ha, E., Raab, H., Bhakta, S., Nguyen, T., Dugger, DL, Li, G., et al. (2010). Engineered (thio-trastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin. Cancer Res. 16, 4769-4778). Junutula et al. (Nat Biotechnol. 2008; 26:925-32) developed a cysteine-based site-directed conjugation called "THIOMAB" (TDC), which is claimed to show an improved therapeutic index compared to conventional conjugation methods. Conjugation to non-natural amino acids incorporated into antibodies is also being explored for ADCs, but the generality of this approach has not yet been established (Axup et al., 2012).In particular, those skilled in the art also envision Fc-containing polypeptides manipulated with acyl-donor glutamine-containing tags (e.g., Gin-containing peptide tags or Q-tags) or endogenous glutamine made reactive by polypeptide manipulation (e.g., amino acid deletion, insertion, substitution, or mutation on the polypeptide). Transglutaminase can then covalently crosslink with amine donors (e.g., small molecules containing or bound to reactive amines) to form a stable and homogeneous population of manipulated Fc-containing polypeptide conjugates in which the amine donors site-specifically conjugate to the Fc-containing polypeptide via acyl-donor glutamine-containing tags or accessible / exposed / reactive endogenous glutamine (WO2012059882).

[0155] In some embodiments, heterologous polypeptides (in particular any antibody disclosed in the present invention, in particular any CD40 antibody derived from the 12E12 antibody as defined above) are conjugated with one or more SARS-CoV-2 polypeptides of the present invention by docklin domains or multiple domains to enable non-covalent coupling to cohesin fusion proteins described in US20160031988A1 and US20120039916A1.

[0156] In some embodiments, heterologous polypeptides (in particular any antibodies disclosed in the present invention, especially any CD40 antibodies derived from the 12E12 antibody as defined above) fuse with one or more SARS-CoV-2 polypeptides of the present invention to form a fusion protein.

[0157] In some embodiments, heterologous polypeptides (in particular any antibody disclosed in the present invention, especially any CD40 antibody derived from the 12E12 antibody as defined above) can be fused to the N-terminus or C-terminus of the SARS-CoV-2 polypeptide of the present invention.

[0158] In some embodiments, the SARS-CoV-2 polypeptide of the present invention is fused directly or via a linker to a heterologous polypeptide (in particular, any antibody disclosed in the present invention, in particular, any CD40 antibody derived from the 12E12 antibody as defined above). As used herein, the term “directly” means that the terminal (N-terminus or C-terminus) (first or last) amino acid of the SARS-CoV-2 polypeptide of the present invention is fused with the terminal (N-terminus or C-terminus) (first or last) amino acid of the heterologous polypeptide. This direct fusion can be carried out as essentially described in (Vigneron et al., Science 2004, PMID 15001714), (Warren et al., Science 2006, PMID 16960008), (Berkers et al., J. Immunol. 2015a, PMID 26401000), (Berkers et al., J. Immunol. 2015b, PMID 26401003), (Delong et al., Science 2016, PMID 26912858), (Liepe et al., Science 2016, PMID 27846572), and (Babon et al., Nat. Med. 2016, PMID 27798614).

[0159] In some embodiments, the linker is selected from the group consisting of FlexV1, f1, f2, f3, or f4 as described below.

[0160] [ka]

[0161] In some embodiments, the fusion protein of the present invention comprises two, three, four, five, six, seven, or eight SARS-CoV-2 polypeptides of the present invention that can be fused to each other directly or indirectly by a linker. In some embodiments, the fusion protein of the present invention comprises polypeptides (M1-110), (M132-222), (N78-206), (N276-411), (S125-250), (S280-598), (S680-1029), and (S1056-1209) that can be fused to each other directly or indirectly by a linker.

[0162] In some embodiments, heterologous polypeptides (in particular any antibodies disclosed in the present invention, in particular any CD40 antibodies derived from the 12E12 antibody as defined above) are not conjugated with polyepitope polypeptides, including RBD polypeptides fused to polypeptides (S125-250, "Spep1").

[0163] In some embodiments, heterologous polypeptides (in particular any antibodies disclosed in the present invention, in particular any CD40 antibodies derived from the 12E12 antibody as defined above) are not conjugated with polyepitope polypeptides, including a polypeptide (N276-411 "Npep2") fused to a polypeptide (S1056-1209 "Spep4").

[0164] In some embodiments, the conjugate of the present invention (referred to as "Gen2a" or "CD40.CoV2") comprises a CD40 antibody (in particular any antibody disclosed in the present invention, in particular a CD40 antibody derived from the 12E12 antibody as defined above), • The antibody heavy chain is fused to the RDB polypeptide, and The antibody light chain is fused to polyepitope polypeptides, including polypeptide (N276-411 "Npep2"), polypeptide (S125-250 "Spep1"), and polypeptide (S1056-1209 "Spep4").

[0165] In some embodiments, the light chain of the Gen2a antibody is fused to a polyepitope polypeptide having the formula Npep2-Spep1-f2-Spep4, where f2 represents a linker as described herein. In some embodiments, the light chain of the Gen2a antibody is fused to a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO: 5.

[0166] In some embodiments, the Gen2a antibody comprises a heavy chain shown in SEQ ID NO: 48 and a light chain having the amino acid sequence shown in SEQ ID NO: 49.

[0167] SEQ ID NO:48>[hAnti-CD40VH3-LV-hIgG4H-C-ViralSARS-CoV-2-Spike-RBD] [ka]

[0168] SEQ ID NO:49>[hAnti-CD40VK2-LV-hIgGK-C-f4-ViralSARS-CoV-2-Npep2-Spep1-f2-Spep4] [ka]

[0169] In some embodiments, the conjugate of the present invention (referred to as "Gen2b") comprises a CD40 antibody (in particular any antibody disclosed in the present invention, in particular any CD40 antibody derived from the 12E12 antibody as defined above), The antibody heavy chain is fused to a polyepitope polypeptide containing polypeptide (S125-250, "Spep1") and polypeptide (S1056-1209, "Spep4"), The antibody light chain is fused to a polyepitope polypeptide, including polypeptides (N276-411, "Npep2") and RBD polypeptides.

[0170] In some embodiments, the heavy chain of the Gen2b antibody is fused to a polyepitope polypeptide having the formula Spep1-f2-Spep4, where f2 represents a linker as described herein. In some embodiments, the heavy chain of the Gen2b antibody is fused to a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO: 6.

[0171] In some embodiments, the Gen2b light chain is fused to a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the Gen2b antibody comprises a heavy chain shown in SEQ ID NO: 50 and a light chain having the amino acid sequence shown in SEQ ID NO: 51.

[0172] SEQ ID NO:50>[hAnti-D40VH3-LV-hIgG4H-C-Flex-v1-ViralSARS-CoV-2-Spep1-f2-pep4] [ka]

[0173] SEQ ID NO:51>[hANTICD40VK2-LV-hIgGK-C-f4-ViralSARS-CoV-2-Npep2-Spike-RBD] [ka]

[0174] In some embodiments, the conjugate of the present invention (referred to as "Gen2c") comprises a CD40 antibody (in particular any antibody disclosed in the present invention, in particular any CD40 antibody derived from the 12E12 antibody as defined above), The antibody heavy chain is fused to a polyepitope polypeptide including polypeptide (N276-411 "Npep2"), polypeptide (S125-250 "Spep1"), and polypeptide (S1056-1209 "Spep4"), and • The antibody light chain is fused to the RBD polypeptide.

[0175] In some embodiments, the heavy chain of the Gen2c antibody is fused to a polyepitope polypeptide having the formula Npep2-Spep1-f2-Spep4, where f2 represents a linker as described herein. In some embodiments, the heavy chain of the Gen2c antibody is fused to a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO: 5.

[0176] In some embodiments, the Gen2c antibody comprises a heavy chain shown in SEQ ID NO: 52 and a light chain having the amino acid sequence shown in SEQ ID NO: 53.

[0177] SEQ ID NO:52>[hAnti-CD40VH3-LV-hIgG4H-C-f4-ViralSARS-CoV-2-Npep2-Spep1-f2-Spep4] [ka]

[0178] SEQ ID NO:53>[hAnti-CD40VK2-LV-hIgGK-C-ViralSARS-CoV-2-Spike-RBD] [ka]

[0179] In some embodiments, the conjugate of the present invention (referred to as "CD40.CoV2v") comprises a CD40 antibody (in particular any antibody disclosed in the present invention, in particular any CD40 antibody derived from the 12E12 antibody as defined above), • The antibody heavy chain is fused to the RBD polypeptide, and The antibody light chain is fused to polyepitope polypeptides, including polypeptide (N276-411 "Npep2"), polypeptide (S125-250 "Spep1"), and polypeptide (S1056-1209 "Spep4").

[0180] In some embodiments, the light chain of the CD40.CoV2v antibody is fused to a polyepitope polypeptide having the formula Npep2-Spep1-f2-Spep4, where f2 represents a linker as described herein. In some embodiments, the light chain of the CD40.CoV2v antibody is fused to a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO: 5.

[0181] In some embodiments, the CD40.CoV2v antibody comprises a heavy chain shown in SEQ ID NO: 54 and a light chain having the amino acid sequence shown in SEQ ID NO: 55.

[0182] Sequence ID 54>[hAnti-CD40VH3-LV-hIgGK-C-ViralSARS-CoV-2-Spike-RBDC221S mutant (South Africa mutation 20H / 501Y.V2)]

[0183] [ka]

[0184] SEQ ID NO:55>[hAnti-CD40VK2-LV-hIgGK-C-f4-ViralSARS-Cov-2-Npep2-Spep1C12S-f2-Spep4] [ka]

[0185] In some embodiments, the conjugate of the present invention (referred to as "CD40.N2.RBDv") consists of a CD40 antibody (in particular, any CD40 antibody derived from the 12E12 antibody as defined above), • The heavy chain of the antibody is fused to the polypeptide (N276-411 "Npep2"), and • The antibody light chain is fused to the RBD polypeptide.

[0186] In some embodiments, CD40.N2.RBDv comprises a heavy chain shown in SEQ ID NO: 56 and a light chain having the amino acid sequence shown in SEQ ID NO: 57.

[0187] SEQ ID NO:56>[hAnti-CD40VH3-LV-hIgG4H-C-f4-ViralSARS-Cov-2-Npep2] [ka]

[0188] SEQ ID NO:57>[hAnti-CD40VK2-LV-hIgGK-C-ViralSARS-CoV-2-Spike-RBD C221S SA var] [ka]

[0189] In some embodiments, the conjugate of the present invention consists of a CD40 antibody (referred to as "CD40.N2.RBDv-2") (in particular, any CD40 antibody derived from the 12E12 antibody as defined above), • The antibody heavy chain is fused to the RBD polypeptide, and The antibody's light chain is fused to the polypeptide (N276-411 "Npep2").

[0190] In some embodiments, the conjugate of the present invention (referred to as "CD40.RBDvS4.N2") consists of a CD40 antibody (in particular, any CD40 antibody derived from the 12E12 antibody as defined above), • The antibody heavy chain is fused to a polyepitope polypeptide containing the RBD polypeptide and the polypeptide (S1056-1209, "Spep4"), and The antibody's light chain is fused to the polypeptide (N276-411 "Npep2").

[0191] In some embodiments, the heavy chain of the CD40.RBDvS4.N2 antibody is fused to a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO: 58.

[0192] In some embodiments, CD40.RBDvS4.N2 comprises a heavy chain shown in SEQ ID NO: 59 and a light chain having the amino acid sequence shown in SEQ ID NO: 60.

[0193] SEQ ID NO:59>[hAnti-CD40VH3-LV-hIgG4H-C-ViralSARS-CoV-2-RBD C221S SA var-Spep4] [ka]

[0194] SEQ ID NO:60>[hAnti-CD40VK2-LV-hIgGK-C-f4-ViralSARS-CoV-2-Npep2] [ka]

[0195] In some embodiments, the conjugate of the present invention (referred to as "CD40.N2S1.RBDvS4") consists of a CD40 antibody (in particular, any CD40 antibody derived from the 12E12 antibody as defined above), The antibody heavy chain is fused to a polyepitope polypeptide containing polypeptide (N276-411 "Npep2") and polypeptide (S125-250 "Spep1"), The antibody light chain is fused to a polyepitope polypeptide, including the RBD polypeptide and the polypeptide (S1056-1209, "Spep4").

[0196] In some embodiments, the light chain of the CD40.N2S1.RBDvS4 antibody is fused to a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO: 58. In some embodiments, the heavy chain of the CD40.N2S1.RBDvS4 antibody is fused to a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO: 61.

[0197] In some embodiments, the CD40.N2S1.RBDvS4 antibody comprises a heavy chain shown in SEQ ID NO: 62 and a light chain having the amino acid sequence shown in SEQ ID NO: 63.

[0198] SEQ ID NO:62>[hAnti-CD40VH3-LV-hIgG4H-C-f4-ViralSARS-CoV-2-Npep2-Spep1 C12S ] [ka]

[0199] SEQ ID NO:63>[hAnti-CD40VK2-LV-hIgGK-C-ViralSARS-CoV-2-RBD C221S SA var-Spep4] [ka]

[0200] In some embodiments, the RBD polypeptide contained in the conjugates Gen2a, Gen2b, Gen2c, CD40.CoV2v, CD40.N2.RBDv, CD40.N2.RBDv-2, CD40.RBDvS4.N2, or CD40.N2S1.RBDvS4 as defined above is in the range of amino acid residues 319 to 541 of SEQ ID NO: 3, and consists of amino acids that include a non-natural C538S mutation and at least one natural mutation, particularly among 417, 452, 484, or 501 of SEQ ID NO: 3.

[0201] In particular, the RBD polypeptides included in the conjugates Gen2a, Gen2b, Gen2c, CD40.CoV2v, CD40.N2.RBDv, CD40.N2.RBDv-2, CD40.RBDvS4.N2, or CD40.N2S1.RBDvS4 as defined above are • Amino acids in the range of amino acid residues 319 to 541 of Sequence ID No. 3, including the N501Y spontaneous mutation and the C538S non-spontaneous mutation; • Amino acids in the range of amino acid residues 319 to 541 of Sequence ID No. 3, including the K417T, E484K, N501Y spontaneous mutations and the non-spontaneous C538S mutation; • Amino acids in the range of amino acid residues 319 to 541 of SEQ ID NO: 3, and including the K417N, E484K, N501Y spontaneous mutations and the non-spontaneous C538S mutation ("RBD South African strain"); or The amino acids in the range of amino acid residues 319 to 541 of Sequence ID No. 3, and which may include E484Q, L452N spontaneous mutations and non-spontaneous C538S mutations ("RBD India strain").

[0202] The RBD polypeptide contained in the conjugates Gen2a, Gen2b, Gen2c, CD40.CoV2v, CD40.N2.RBDv, CD40.N2.RBDv-2, CD40.RBDvS4.N2, or CD40.N2S1.RBDvS4 as defined above is preferably in the range of amino acid residues 319 to 541 of SEQ ID NO: 3, and may consist of amino acids including the K417N, E484K, N501Y spontaneous mutations and the non-spontaneous C538S mutation ("RBD South Africa strain").

[0203] Method for producing polypeptides of the present invention: The SARS-CoV-2 polypeptides, fusion proteins, and antibodies of the present invention can be produced by any technique known in the art, either alone or in combination, including but not limited to chemical, biological, genetic, or enzymatic techniques. If the amino acid sequence of the desired sequence is known, those skilled in the art can readily produce the polypeptide by standard techniques for polypeptide production. For example, they can be synthesized using well-known solid-phase methods, preferably using commercially available peptide synthesizers (e.g., manufactured by Applied Biosystems, Foster City, California) in accordance with the manufacturer's instructions. Alternatively, the polypeptides and fusion proteins of the present invention can be synthesized by recombinant DNA techniques currently known in the art. For example, these fragments can be obtained as DNA expression products after the incorporation of the DNA sequence encoding the desired (poly)peptide into an expression vector and the introduction of this vector into a suitable eukaryotic or prokaryotic host expressing the desired polypeptide, after which these fragments can be isolated from these hosts using well-known techniques.

[0204] For recombinant expression, the DNA construct preferably includes sequences encoding signal peptides at the N-terminuses of the heavy and light chains of the conjugate according to the present invention.

[0205] Pharmaceutical compositions and vaccine compositions: The fusion proteins and antibodies and SARS-CoV-2 polypeptides and conjugates containing antibodies described herein may be administered as part of one or more pharmaceutical compositions. Unless any conventional carrier medium is incompatible with the polypeptides of the present invention due to producing undesirable biological effects or otherwise interacting in a detrimental manner with other components of the pharmaceutical composition, its use is intended to be within the scope of the present invention. Some examples of materials that can act as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; and propylene glycol. Examples of suitable lubricants include glycols such as chol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogenic substance-removed water; isotonic saline solution; Ringer's solution; ethyl alcohol and phosphate buffer solution; and other non-toxic suitable lubricants such as sodium lauryl sulfate and magnesium stearate. At the discretion of the compounder, colorants, release agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives and antioxidants may also be present in this composition.

[0206] The SARS-CoV-2 polypeptides and conjugates, including the fusion proteins and antibodies described herein, are particularly suitable for preparing vaccine compositions.

[0207] In some embodiments, the vaccine composition of the present invention comprises one or more SARS-CoV-2 polypeptides as antigens. One advantage of the SARS-CoV-2 polypeptide of the present invention is that it comprises multiple epitopes, including an HLA class I restriction epitope and / or an HLA class II restriction epitope and / or an antibody epitope.

[0208] In some embodiments, the vaccine composition of the present invention comprises 2, 3, 4, 5, 6, 7 or 8 SARS-CoV-2 polypeptides of the present invention. In some embodiments, the vaccine composition of the present invention preferably comprises polypeptide (M1-110), polypeptide (M132-222), polypeptide (N78-206), polypeptide (N276-411), polypeptide (S125-250), polypeptide (S280-598), polypeptide (S680-1029) and polypeptide (S1056-1209).

[0209] In some embodiments, the vaccine composition of the present invention comprises, as an antigen, one or more conjugates of the present invention (including the fusion proteins and antibodies described herein).

[0210] Thus, in some embodiments, the vaccine composition of the present invention comprises an adjuvant. In some embodiments, the adjuvant is alum. In some embodiments, the adjuvant is incomplete Freund's adjuvant (IFA) or other oil-based adjuvant present at 30-70% weight (w / w), preferably 40-60% weight, more preferably 45-55% weight. In some embodiments, the vaccine composition of the present invention comprises at least one toll-like receptor (TLR) agonist selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR8 agonists.[[ID=X12]]

[0211] Polynucleotides and their uses: A further object of the present invention relates to a polynucleotide encoding a SARS-CoV-2 polypeptide or conjugate of the present invention (including the fusion proteins and antibodies of the present invention). The polynucleotide preferably comprises a sequence encoding a signal peptide at the N-terminus of the heavy and light chains of the conjugate according to the present invention, whereby the conjugate can be expressed and secreted in vivo.

[0212] Generally, the polynucleotide is a DNA or RNA molecule that can be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector.

[0213] The polynucleotides of the present invention are particularly suitable for administering vaccines to targets that require them.

[0214] As defined herein, a wide variety of methods exist for delivering polynucleotides. For example, the polynucleotides of the present invention can be formulated using cationic polymers, including cationic liposomes. Other liposomes also serve as effective means for formulating and delivering autoacidic nucleic acid molecules. Alternatively, DNA can be incorporated into viral vectors, viral particles, or bacteria for pharmacological delivery. Viral vectors may have infectivity, attenuation (having mutations that reduce their ability to induce disease), or replication defects. Methods utilizing DNA to prevent the deposition, accumulation, or activity of pathogenic autoproteins can be enhanced by the use of viral vectors or other delivery systems that increase the humoral response to encoded autoantigens. In some embodiments, DNA can be conjugated with solid supports containing gold particles, polysaccharide-based supports, or other particles or beads that can be delivered by injection, inhalation, or particle impact (ballistic delivery). Methods for delivering nucleic acid formulations are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466. Several virus-based systems have been developed for introduction into mammalian cells. For example, retroviral systems have been described (U.S. Patent No. 5,219,740; Miller et al, Biotechniques 7:980-990 (1989); Miller, Human Gene Therapy 1:5-14, (1990); Scarpa et al, Virology 180:849-852 (1991); Burns et al, Proc. Natl Acad. Sci. USA 90:8033-8037 (1993); and Boris-Lawrie and Temin, Cur. Opin. Genet. Develop. 3: 102-109 (1993). For nucleic acid delivery, adeno-associated virus (AAV) vector systems have also been developed. AAV vectors can be readily constructed using techniques well known in the art.For example, U.S. Patent Nos. 5,173,414 and 5,139,941; International Publication Nos. WO92 / 01070 and WO93 / 03769; Lebkowski et al, Molec. Cell Biol. 8:3988-3996 (1988); Vincent et al, Vaccines 90 (Cold Spring Harbor Laboratory Press) (1990); Carter, Current Opinion in Biotechnology 3:533-539 (1992); Muzyczka, Current Topics in Microbiol. And Immunol. 158:97-129 (1992); Kotin, Human Gene Therapy 5:793-801 (1994); Shelling et al., Gene Therapy 1: 165-169 (1994); and Zhou et al., J. Exp. Med. See 179: 1867–1875 (1994).

[0215] In some embodiments, the polynucleotides of the present invention are delivered without the use of viral vectors. For example, the polynucleotides can be encapsulated in liposomes before delivery to the target. Lipid encapsulation is generally achieved using liposomes that can stably bind to or retain nucleic acids. For reviews on the use of liposomes as carriers for nucleic acid delivery, see, for example, Hug et al, Biochim. Biophys. Acta. 1097: 1-17 (1991); Straubinger et al., Methods of Enzymology, 101: 512-527 (1983). Alternatively, the polynucleotides are delivered by electroporation (i.e., intramuscular delivery by electroporation).

[0216] In some embodiments, polynucleotides are delivered by intramuscular ("IM") injection. In some embodiments, the nucleic acid molecules of the present invention are delivered intranasally, orally, subcutaneously, intradermally, intravenously, mucousally, transdermally, or by binding to particles delivered to the dermis, or by delivery via the dermis. Alternatively, polynucleotides can be delivered to skin cells by topical application, with or without the use of liposomes and charged lipids. A further alternative is to deliver the nucleic acid as an inhaled agent.

[0217] Treatment method: The SARS-CoV-2 polypeptides and conjugates (including fusion proteins and antibodies) and pharmaceutical or vaccine compositions described herein are particularly suitable for inducing an immune response to SARS-CoV-2 and can therefore be used for vaccine purposes.

[0218] Therefore, a further object of the present invention is a method for administering SARS-CoV-2 as a vaccine to a target in need, comprising administering one or more SARS-CoV-2 polypeptides of the present invention in a therapeutically effective amount.

[0219] A further object of the present invention relates to a method for administering SARS-CoV-2 as a vaccine to a target in need, comprising administering a therapeutically effective amount of one or more conjugates described herein.

[0220] A further object of the present invention relates to a method for administering SARS-CoV-2 as a vaccine to a target in need, comprising administering a therapeutically effective amount of the pharmaceutical or vaccine composition described herein.

[0221] A further object of the present invention relates to a method for inoculating a target in need of SARS-CoV-2 as a vaccine, comprising administering a therapeutically effective amount of the polynucleotide described herein.

[0222] In some embodiments, the SARS-CoV-2 polypeptides and conjugates (including fusion proteins and antibodies) and pharmaceutical or vaccine compositions described herein are particularly suitable for the treatment of Covid-19.

[0223] In some embodiments, the subject may be a human or any other animal susceptible to coronavirus infection (e.g., birds and mammals) (e.g., domestic animals such as cats and dogs; livestock and farm animals such as horses, cattle, pigs, and chickens). Generally, the subject is a mammal, including non-primates (e.g., camels, donkeys, zebras, cattle, pigs, horses, goats, sheep, cats, dogs, rats, and mice) and primates (e.g., monkeys, chimpanzees, and humans). In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a farm animal or pet. In some embodiments, the subject is a human. In some embodiments, the subject is a human infant. In some embodiments, the subject is a human child. In some embodiments, the subject is a human adult. In some embodiments, the subject is a human elderly. In some embodiments, the subject is a human premature infant.

[0224] In some embodiments, the subjects may be symptomatic or asymptomatic.

[0225] Generally, the active ingredients of the present invention (i.e., the SARS-CoV-2 polypeptides and conjugates (including fusion proteins and antibodies) and pharmaceutical or vaccine compositions described herein) are administered to a subject in a therapeutically effective amount. The total daily dose of the compounds and compositions of the present invention is understood to be determined by the attending physician within the bounds of sound medical judgment. A specific therapeutically effective dose level or any particular subject will vary depending on various factors well known in the medical field, including the disorder being treated and its severity; the activity of the specific compound used; the specific composition used, the subject's age, weight, health status, sex, and diet; the timing of administration, route of administration, and excretion rate of the specific compound used; the duration of treatment; and drugs used in combination or concurrently with the specific polypeptide used. For example, it is well within the skill of those skilled in the art to start administration of a compound at a level lower than necessary to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. However, the daily dose of the product can be varied over a wide range of 0.01 to 1,000 mg / adult / day. In particular, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the active ingredient to allow for dose adjustment tailored to the symptoms of the target patient. The drugs generally contain approximately 0.01 mg to approximately 500 mg of the active ingredient, particularly 1 mg to approximately 100 mg of the active ingredient. Effective doses of the drug are typically supplied at dose levels of 0.0002 mg / kg to approximately 20 mg / kg body weight / day, particularly approximately 0.001 mg / kg to 7 mg / kg body weight / day.

[0226] The SARS-CoV-2 polypeptides and conjugates (including fusion proteins and antibodies) and pharmaceutical or vaccine compositions described herein may be administered to a target by any route of administration, particularly by oral, nasal, rectal, topical, oral (e.g., sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous) and transdermal administration, but the most preferred route in any given case will depend on the nature and severity of the condition being treated and the specific activator used.

[0227] In some embodiments, the SARS-CoV-2 polypeptides and conjugates (including fusion proteins and antibodies) and pharmaceutical or vaccine compositions described herein can be administered to a target in combination with, for example, known therapeutic agents or vaccination methods against the SARS-CoV-2 coronavirus. Examples of such known therapeutic agents, though not limited to, include antiviral agents such as remdesivir, lopinavir, ritonavir, hydroxychloroquine, and chloroquine. In some embodiments, the SARS-CoV-2 polypeptides and conjugates (including fusion proteins and antibodies) and pharmaceutical or vaccine compositions described herein are administered in combination with immune checkpoint inhibitors. Examples of immune checkpoint inhibitors include PD-1 antagonists, PD-L1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, and BTLA antagonists. In some embodiments, PD-1 (programmed cell death-1) axis antagonists include PD-1 antagonists (e.g., anti-PD-1 antibodies), PD-L1 (programmed cell death ligand-1) antagonists (e.g., anti-PD-L1 antibodies), and PD-L2 (programmed cell death ligand-2) antagonists (e.g., anti-PD-L2 antibodies). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (also known as nivolumab, MDX-1106-04, ONO-4538, BMS-936558, and Opdivo®), Merck 3475 (also known as pembrolizumab, MK-3475, lambrolizumab, Keytruda®, and SCH-900475), and CT-011 (also known as pidilizumab, hBAT, and hBAT-1). In some embodiments, the PD-1 conjugated antagonist is AMP-224 (also known as B7-DCIg).In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. MDX-1105, also known as BMS-936559, is the anti-PD-L1 antibody described in publication WO2007 / 005874. Antibody YW243.55.S70 is the anti-PD-L1 antibody described in WO2010 / 077634 A1. MEDI4736 is the anti-PD-L1 antibody described in publications WO2011 / 066389 and US2013 / 034559. MDX-1106, also known as MDX-1106-04, ONO-4538, or BMS-936558, is an anti-PD-1 antibody described in U.S. Patent No. 8,008,449 and WO2006 / 121168. Merck 3745, also known as MK-3475 or SCH-900475, is an anti-PD-1 antibody described in U.S. Patent No. 8,345,509 and WO2009 / 114335. CT-011 (pizidilumab), also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in U.S. Patent No. 2010 / 027827 and U.S. Patent No. 2011 / 066342. Atezolimmab is an anti-PD-L1 antibody described in U.S. Patent No. 8,217,149. Avelumab is an anti-PD-L1 antibody described in U.S. Patent No. 20140341917. CA-170 is a PD-1 antagonist described in U.S. Patent No. 2015033301 and U.S. Patent No. 2015033299. Other anti-PD-1 antibodies are disclosed in U.S. Patent No. 8,609,089, U.S.2010028330, and / or U.S.20120114649. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody selected from nivolumab, pembrolizumab, or pidilizumab.In some embodiments, the PD-L1 antagonist is selected from the group consisting of avelumab, BMS-936559, CA-170, durvalumab, MCLA-145, SP142, STI-A1011, STIA1012, STI-A1010, STI-A1014, A110, KY1003, and atezolizumab, and preferably is avelumab, durvalumab or atezolizumab.

[0228] The present invention will be further described by the following figures and examples. However, these examples and figures should not be construed as limiting the scope of the present invention in any way.

Brief Description of Drawings

[0229] [Figure 1A] Design and physical properties of anti-CD40.SARS-CoV-2 Gen2a, 2b, 2c fusion proteins. A) Schematic diagrams of anti-CD40.SARS-CoV-2 Gen2a, 2b, 2c, CD40.CoV2v, CD40.N2.RBDv, CD40.N2.RBDv-2, CD40.RBDvS4.N2, CD40.N2S1.RBDvS4 fusion proteins. B) Analysis of three different batches of anti-CD40.SARS-CoV-2 Gen2a, 2b, 2c fusion proteins purified by protein-A and stained with Coomassie Brilliant Blue R250 by reducing and non-reducing SDS PAGE. Molecular weight markers are shown in the rightmost lanes of both gels (BenchMark Pre-Stained Prptein Ladder; 180, 115, 82, 64, 49, 37, 26, 19, 15, 6 kDa; ThermoFisher). [Figure 1B]Design and physical properties of anti-CD40.SARS-CoV-2 Gen2a, 2b, and 2c fusion proteins. A) Schematic diagrams of anti-CD40.SARS-CoV-2 Gen2a, 2b, 2c, CD40.CoV2v, CD40.N2.RBDv, CD40.N2.RBDv-2, CD40.RBDvS4.N2, and CD40.N2S1.RBDvS4 fusion proteins. B) Analysis of three different batches of anti-CD40.SARS-CoV-2 Gen2a, 2b, and 2c fusion proteins purified by protein-A via reduced and unreduced SDS-PAGE, stained with Coomassie Brilliant Blue R250. Molecular weight markers are shown in the rightmost lane of both gels (BenchMark Pre-Stained Prptein Ladder; 180, 115, 82, 64, 49, 37, 26, 19, 15, 6 kDa; ThermoFisher). [Figure 2] A) Binding of anti-CD40 Gen2a, Gen2b, and Gen2c fusion proteins to human CD40 and B) human IgG. The fusion proteins corresponding to the shown constructs were normalized by molar concentration, and the dilution system was added to a surface coated with either human CD40 ectodomain protein (upper panel) or goat anti-human IgG polyclonal serum, incubated to bind, washed, then incubated with anti-human IgG reagent conjugated with horseradish peroxidase (HRP), washed, and developed with an HRP chromogenic substrate. [Figure 3]CD40-targeted SARS-CoV-2pep antigen in Gen2a, Gen2b, and Gen2c fusion proteins, as determined by in vitro proliferation of SARS-CoV-2-specific T cells in SARS-CoV-2-recovered donor PBMC cultures. PBMCs from SARS-CoV-2+ donor patients 1 were cultured for 9 days with IL-2 and anti-CD40 Gen2a, Gen2b, and Gen2c fusion proteins (1nM or 10nM), then stimulated with brefelzin-A and a peptide pool specific to the SARS-CoV-2 N and S regions for 6 hours, followed by analysis by intracellular cytokine staining (ICS). 1=DMSO stimulation; 2, 3, 4=Stimulation with a peptide pool containing the N region; 5, 6, 7=Stimulation with a peptide pool containing the RBD region; 8=SEB polyclonal stimulation; 9, 10=Stimulation with a peptide pool containing the Spep1 region; 11, 12=Stimulation with a peptide pool containing the Spep4 region. The bar graph shows the cumulative percentage of CD4+ T cells possessing intracellular interferon-γ (I+), intracellular tumor necrosis factor α (T+), or both (I+T+). [Figure 4] An example of evaluating the relative expression of various H and L chain constructs in simultaneous transfection experiments using CD40-conjugated ELISA. ELISA plates were coated with 1 μg / ml human CD40 ectodomain protein, and the conjugated antibody was detected using an anti-human IgG-HRP reagent. The CHO-S transfection supernatant was started undiluted (1 μg / ml point for the control anti-human CD40 12E12 hIgG4 antibody batch PAB2220). The construct naming is detailed in Table 2. [Figure 5]Multifunctional CD4+ T cell response in COVID-19 recovered patients induced by anti-CD40.Gen2 vaccine. PBMCs from 13 COVID patients were stimulated on day 0 with either 1 nM anti-CD40.Gen2a vaccine or an equimolar peptide pool overlapping each antigen (RBD, S1, S4, N2) contained in the vaccine. After culturing with IL-2 for 8 days, cells were restimulated or unstimulated with each peptide pool (1 μg / ml). In vitro T cell proliferation was analyzed by flow cytometry. Mean values ​​for individual CD4+ T cell responses (sum of IFNγ, TNFα, and IL-2) and vaccine-stimulated PBMCs are given. Multiple comparison one-way ANOVA trials were used for statistical analysis (ns, not significant; *p<0.05; ***p<0.001; ****p<0.0001). [Figure 6] Frequency of SARS-CoV-2 S protein-specific IgG-switched human B- cells in hu- mouse spleens at 44 days post-prime injection. A. Schematic outline of the vaccination strategy for NSG humanized (hu) mice, including four test groups of 7-9 mice / group. B. Hu-mouse splenocytes from 44 days post-immunization were first incubated with PE-SARS-CoV-2 S protein at 4°C for 1 hour. After washing, cells were stained with anti-mouse CD45, anti-human (h)CD45, anti-hCD19, anti-hCD20, and anti-hIgG at 4°C for 30 minutes. Staining on splenocytes also included a survival marker (LiveDead aqua). Cells were washed twice with FACS buffer (PBS 1% FCS) and acquired using an LSRII flow cytometer (BD Biosciences). Analysis was performed using FlowJo v.10.7.1. [Examples]

[0230] Example 1: The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has undoubtedly emerged as the greatest global health threat to humanity this century. The rapid spread, global rate of dissemination, and observed high mortality rate of SARS-CoV-2 have raised public health, socioeconomic, and scientific challenges. SARS-CoV-2 causes respiratory syndromes that present with a clinical picture similar to a mild upper respiratory tract illness (symptoms similar to the common cold), but can sometimes lead to severe lower respiratory tract illness and extrapulmonary symptoms, resulting in multiple organ failure and death.

[0231] There is no cure or vaccine. However, a SARS-CoV-2 vaccine will be essential to reduce morbidity and mortality if the virus becomes established in a population. To accelerate the development of candidate vaccines, it is crucial to use a ready-to-use vaccine platform that is specialized and adapted for emerging and re-emerging pathogens. The inventors have positioned a candidate vaccine against SARS-CoV-2 within their ready-to-use DC-targeted vaccine platform, which is specialized and adapted for emerging and re-emerging pathogens.

[0232] The inventors have developed a vaccine-specific platform for delivering targeted antigens (public, dominant, and cross-reactive) to endogenous DCs using specific humanized monoclonal antibodies (mAbs) against specific DC endocytosis receptors. The inventors intend to promote antigen presentation and activation of antigen-specific immune responses, inducing potent humoral and cellular immune responses even with low doses of antigen. In Phase I trials, the inventors obtained a vast amount of in vitro and in vivo data that could lead to the clinical development of vaccines targeting HIV or HPV [1-6]. The inventors have previously demonstrated that anti-CD40 mAbs fused to HIV T cell epitope stretches induce polyepitope T cell responses in human[3], NHP[2], or humanized mice[1]. Furthermore, combining an anti-CD40 vaccine fused with HIV.Env gp140 with a Pox vector, or using it as a booster for DNA or DREP HIV vaccines, induced a potent and robust T-cell and B-cell response [6] (P Liljestrom / Y Levy, personal data). This DC-targeting platform is currently being validated in preclinical stages for several infections (Ebola, influenza, chlamydia, and tuberculosis) and is scheduled to advance to a Phase I clinical trial this year for HIV prevention and HPV-related head and neck cancer.

[0233] The inventors analyzed the amino acid sequences of these proteins in silico and identified specific epitopes included in the vaccine candidate by mapping MHC-I and MHC-II epitopes predicted by online software (NetMHC-4.0 and NetMHCII-2.3) and peptide bond prediction software. In addition, B cell epitopes were also mapped using online software (BepiPred-2.0 and Discotope), and regions rich in epitopes with homologous sequences between SARS-CoV-2 and -CoV-1 were also mapped.

[0234] The four structural proteins of SARS-CoV-2 (S, N, M, and E) were each identified using NetMHC 4.0. https: / / services.healthtech.dtu.dk / service.php?NetMHC-4.0) and NetMHCII 2.3( https: / / services.healthtech.dtu.dk / service.php?NetMHCII-2.3 The data was analyzed using MHC class-I and MHC class-II / peptide bond prediction software.

[0235] Linear B cell epitopes are found in BepiPred 2.0( https: / / services.healthtech.dtu.dk / service.php?BepiPred-2.0 The prediction was made using ).

[0236] 80 HLA class I molecules (in the case of 9-mer peptides) and 54 HLA class II molecules (in the case of 15-mer peptides) were used for T cell epitope prediction.

[0237] We listed the T cell and B cell epitopes of SARS-CoV-1 and examined their homology percentage with SARS-CoV-2, as well as the sequence conservation between different β-coronaviruses.

[0238] We selected regions containing predicted B, CD8, and CD4 SARS-CoV-2 epitopes, as well as clusters of described B, CD8, and CD4 SARS-CoV-1 epitopes (particularly regions conserved between different β-coronaviruses).

[0239] The SARS-CoV-1 sequence targeted by NAb was specifically selected to be included in the vaccine region.

[0240] We defined the region considering the recent cartography of the SARS-CoV-2 B-cell response published by Dahlke et al.

[0241] Furthermore, we adjusted the regions according to T cell and B cell predictive epitopes published by other groups (Prachar M et al bioRxiv pre-print, Bojin F et al pre-print, Fast et al, [7-10]).

[0242] Based on the method described above, the inventors identified the following target areas (see Table 1).

[0243] [Table 3]

[0244] Example 2: method: The methods for purifying and producing the expression vector and protein, as well as quality assurance including CD40 binding specificity, are as described [1;2;3]. Protein expression was performed by transient transfection of CHO-S (Chinese hamster ovary cells) using the TransIT®-CHO transfection kit (Mirus). Cloning was performed using synthetic DNA cassettes encoding various SARS-Co-V2 antigen regions with CHO-optimized codons, which typically contain adjacent restriction sites convenient for ligation to vectors in various combinations.

[0245] [1] Flamar AL., S. Zurawski, F. Scholz, I. Gayet, L. Ni, XH. Li, E. Klechevsky, J. Quinn, S. Oh, DH Kaplan, J. Banchereau and G. Zurawski. 2012. Noncovalent assembly of anti-Dendritic Cell antibodies and antigens for evoking immune responses in vitro and in vivo. J. Immunl. 189: 2645-55. [2] Flamar AL., Y. Xue, S. M. Zurawski, M. Montes, B. King, L. Sloan, S. Oh, J. Banchereau, Y. Levy and G. Zurawski. 2013. Targeting concatenated HIV antigens to human CD40 expands a broad repertoire of multifunctional CD4+ and CD8+ T Cells. AIDS. 27: 2041-51. [3] Zurawski G., X. Shen, S. Zurawaski, G. D. Tomaras, D. C. Montafiori, M. Roederer, G. Ferrari, C. Lacabaratz, P. Klucar, Z. Wang, K. E. Foulds, SF. Kao, X. Yu, A. Sato, N. L. Yates, C. LaBranche, S. Stanfield-Oakley, K. Kibler, B. Jacobs, A. Salazar, S. Self, E. Fulp, R. Gottardo, L. Galmin, D. Weiss, A. Cristillo, G. Pantaleo and Y. Levy. 2017. Superiority in rhesus macaques of targeting HIV-1 Env gp140 to CD40 versus LOX-1 in combination with replication-competent NYVAC-KC for induction of Env-specific antibody and T Cell responses. J. Virol. 91: 1-20.

[0246] result: The inventors have previously patented compositions and methods relating to the expression, secretion, and use of novel recombinant antigen antibodies against a panel of human DC receptors: CD40, DCIR, Langerin, and LOX-1. The DC targeting technology is based on the engineering direct fusion of pathogen antigens to the C-terminus of the heavy and / or light chains of anti-DC receptor monoclonal antibodies. Thus, the coding sequence of a selected CoV antigen region is fused by molecular cloning as a linked string of antigen regions on the heavy or light chain, flanked by a proprietary linker peptide.

[0247] Expression constructs of anti-COVID-19 DC-targeted vaccine candidates were constructed using various SARS-CoV-2 antigen-coding regions or combinations of regions, with or without flexible linker regions at the heavy and light chain C-terminuses of humanized anti-human CD40 12E12 IgG4 antibodies.

[0248] Heavy and light chain expression constructs were transiently co-transfected into Expi CHO-S cells, and the expression of the secreted recombinant fusion antibody protein was then tested by ELISA to measure the relative production of hIgG4 levels and / or human CD40 binding. ELISA measurements were evaluated by comparison with a 1 μg / ml standard curve for humanized anti-human CD40 12E12 IgG4 antibody (see Figure 4). This initial assay was intended to determine which construct combinations produced a product within a range potentially useful for large-scale vaccine production. This reflects the production capacity shown in Table 2.

[0249] The quality of the produced vaccine candidates shown in Table 2 was evaluated by (i) SDS-PAGE analysis and / or (ii) size exclusion chromatography analysis of purified rAb antigen fusion proteins separated by SDS-PAGE under reducing and non-reducing conditions and stained with Coomassie blue.

[0250] [Table 4]

[0251] In particular, the inventors produced eight constructs with the best production capacity and quality as follows: Figure 1 : · Anti-CD40.COVID-19 construct "Gen2a" having one peptide (ViralSARS-CoV-2 Spike-RBD) in the heavy chain (h anti-CD40 VH3-LV-hIgG4H) and three peptides (ViralSARS-CoV-2 Npep2, Spep1, Spep4) in the light chain (h anti-CD40 VK2-LV-hIgGK) · Anti-CD40.COVID-19 construct "Gen2b" having two peptides (ViralSARS-CoV-2 Spep1, Spep4) in the heavy chain (h anti-CD40 VH3-LV-hIgG4H) and two peptides (ViralSARS-CoV-2 Npep2, Spike-RBD) in the light chain (h anti-CD40 VK2-LV-hIgGK) · Anti-CD40.COVID-19 construct "Gen2c" having three peptides (ViralSARS-CoV-2 Npep2, Spep1, Spep4) in the heavy chain (h anti-CD40 VH3-LV-hIgG4H) and one peptide (ViralSARS-CoV-2 Spike-RBD) in the light chain (h anti-CD40 VK2-LV-hIgGK) · Anti-CD40.COVID-19 construct "Gen2a" having one peptide (ViralSARS-CoV-2 Spike-RBD SA VAR ) in the heavy chain (h anti-CD40 VH3-LV-hIgG4H) and three peptides (ViralSARS-CoV-2 Npep2, Spep1 C136S , Spep4) in the light chain (h anti-CD40 VK2-LV-hIgGK) · Anti-CD40.COVID-19 construct "CD40.CoV2v" having one peptide (ViralSARS-CoV-2 Npep2) in the heavy chain (h anti-CD40 VH3-LV-hIgG4H) and one peptide (ViralSARS-CoV-2 Spike-RBD SA VAR ) in the light chain (h anti-CD40 VK2-LV-hIgGK), • Heavy chain (h anti-CD40VH3-LV-hIgG4H) contains one peptide (ViralSARS-CoV-2 Spike-RBD) SA VAR The anti-CD40.COVID-19 construct "CD40.N2.RBDv-2" has a light chain (hanti-CD40VK2-LV-hIgGK) containing one peptide (ViralSARS-CoV-2 Npep2). • The heavy chain (h anti-CD40VH3-LV-hIgG4H) contains two peptides (ViralSARS-CoV-2 Spike-RBD) SA VAR The anti-CD40.COVID-19 construct "CD40.RBDvS4.N2" has Spep4 and one peptide (ViralSARS-CoV-2 Npep2) in its light chain (hantiCD40VK2-LV-hIgGK). • The heavy chain (h anti-CD40VH3-LV-hIgG4H) contains two peptides (ViralSARS-CoV-2 Npep2, Spep1) C136S It has a light chain (h anti-CD40VK2-LV-hIgGK) and two peptides (ViralSARS-CoV-2 Spike-RBD SA VAR The anti-CD40.COVID-19 construct "CD40.N2S1.RBDvS4" possesses Spep4.

[0252] The inventors confirmed that anti-CD40 Gen2a, Gen2b, and Gen2c fusion proteins bind to human CD40 (Figure 2). Next, the CD40-targeting SARS-CoV2pep antigen in the Gen2a, Gen2b, and Gen2c fusion proteins was tested by in vitro expansion culture of SARS-CoV-2-specific T cells in SARS-CoV-2-infected recovered donor PBMC cultures (Figure 3).

[0253] The inventors show in Figure 5 that the anti-CD40.Gen2a vaccine induced a multifunctional CD4+ T cell response in PBMCs of COVID-19 recovered patients.

[0254] The immunogenicity of anti-CD40 Gen2a vaccines administered using homogeneous or heterogeneous prime / boost vaccination strategies was investigated according to the protocol described in Figure 6A. The results are shown in Figure 6B. We demonstrate that the vaccine induces S-specific IgG+ hu-B cells (Figure 6B). Interestingly, it is shown that the addition of an adjuvant is not necessary to induce S-specific IgG+ hu-B cells (Figure 6B).

[0255] Throughout this application, various references illustrate the state of the art to which this invention belongs. The disclosures of these references are incorporated by reference into this disclosure. References TIFF2026062799000046.tif155151TIFF2026062799000047.tif76151

Claims

1. SARS-CoV-2 polypeptide (N276-411, "Npep2"), derived from N protein and consisting of at least 50 consecutive amino acids having at least 90% identity with the amino acid sequence in the range of residues 276 to 411 of SEQ ID NO:

2.

2. The amino acid sequence 50;51;52;53;54;55;56;57;58;59;60;61;62;63;64;65;66;67;68;69;70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;88;89;90;91;92;93;94;95;96;9 Polypeptide according to claim 1 (N276-411), comprising 7; 98; 99; 100; 101; 102; 103; 104; 105; 106; 107; 108; 109; 110; 111; 112; 113; 114; 115; 116; 117; 118; 119; 120; 121; 122; 123; 124; 125; 126; 127; 128; 129; 130; 131; 132; 133; 134; 135; or 136 consecutive amino acids.

3. Polypeptide (N276-411) according to claim 1, comprising an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 276 to 411 of SEQ ID NO:

2.

4. SARS-CoV-2 polypeptide (S125-250, "Spep1"), derived from the S protein and consisting of at least 50 consecutive amino acids having at least 90% identity with the amino acid sequence in the range of residues 125 to 250 of SEQ ID NO:

3.

5. The amino acid sequence 50;51;52;53;54;55;56;57;58;59;60;61;62;63;64;65;66;67;68;69;70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;88;89;90 Polypeptide according to claim 4 (S125-250), comprising 91;92;93;94;95;96;97;98;99;100;101;102;103;104;105;106;107;108;109;110;111;112;113;114;115;116;117;118;119;120;121;122;123;124;125; or 126 consecutive amino acids.

6. The polypeptide (S125-250) according to claim 4, comprising an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 125 to 250 of SEQ ID NO:

3.

7. The polypeptide (S125-250) according to claim 4, comprising an amino acid sequence in the range of residues 125 to 250 of SEQ ID NO: 3, and including the C136S non-natural mutation.

8. SARS-CoV-2 polypeptide (S1056-1209, "Spep4"), derived from the S protein and consisting of at least 50 consecutive amino acids whose amino acid sequence is at least 90% identical to the amino acid sequence in the range of residues 1056 to 1209 of SEQ ID NO:

3.

9. The amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 1056 to 1209 of Sequence ID No. 3 is 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100; 101; 102; 103; 104; 105; 106; Polypeptide according to claim 8 (S1056-1209), comprising 107; 108; 109; 110; 111; 112; 113; 114; 115; 116; 117; 118; 119; 120; 121; 122; 123; 124; 125; 126; 127; 128; 129; 130; 131; 132; 133; 134; 135; 136; 137; 138; 139; 140; 141; 142; 143; 144; 145; 146; 147; 148; 149; 150; 151; 152; 153; or 154 consecutive amino acids.

10. The polypeptide (S1056-1209) according to claim 9, comprising an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 1056 to 1209 of SEQ ID NO:

3.

11. SARS-CoV-2 polypeptide (M1-110) is derived from M protein and consists of at least 50 consecutive amino acids whose amino acid sequence is at least 90% identical to the amino acid sequence in the range of residues 1 to 110 of SEQ ID NO:

1.

12. Polypeptide (M1-110) according to claim 11, comprising 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100; 101; 102; 103; 104; 105; 106; 107; 108; 109; or 110 consecutive amino acids having at least 90% identity with the amino acid sequence in the range of residues 1 to 110 of Sequence ID No.

1.

13. The polypeptide (M1-110) according to claim 11, comprising an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 1 to 110 of SEQ ID NO:

1.

14. SARS-CoV-2 polypeptide (M132-222) is derived from M protein and consists of at least 50 consecutive amino acids whose amino acid sequence has at least 90% identity with the amino acid sequence in the range of residues 132 to 222 of SEQ ID NO:

1.

15. Polypeptide (M132-222) according to claim 14, comprising 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; or 91 consecutive amino acids, having at least 90% identity with the amino acid sequence in the range of residues 132 to 222 of Sequence ID No.

1.

16. The polypeptide (M132-222) according to claim 14, comprising an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 132 to 222 of SEQ ID NO:

1.

17. SARS-CoV-2 polypeptide (N78-206) is derived from the N protein and consists of at least 50 consecutive amino acids whose amino acid sequence is at least 90% identical to the amino acid sequence in the range of residues 78 to 206 of SEQ ID NO:

2.

18. The amino acid sequence 50;51;52;53;54;55;56;57;58;59;60;61;62;63;64;65;66;67;68;69;70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;88;89;90;91;92 Polypeptide (N78-206) according to claim 17, comprising 93;94;95;96;97;98;99;100;101;102;103;104;105;106;107;108;109;110;111;112;113;114;115;116;117;118;119;120;121;122;123;124;125;126;127;128; or 129 consecutive amino acids.

19. The polypeptide (N78-206) according to claim 17, comprising an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 78 to 206 of SEQ ID NO:

2.

20. SARS-CoV-2 polypeptide (S280-598) is derived from the S protein and consists of at least 50 consecutive amino acids whose amino acid sequence is at least 90% identical to the amino acid sequence in the range of residues 280 to 598 of SEQ ID NO:

3.

21. The amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 280 to 598 of Sequence ID No. 3 is 50;51;52;53;54;55;56;57;58;59;60;61;62;63;64;65;66;67;68;69;70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;88;89;90;91;92;93;94;95;96;97;98;99;100;101;102;103;104;105;106;107;108;109;110;1 11; 112; 113; 114; 115; 116; 117; 118; 119; 120; 121; 122; 123; 124; 125; 126; 127; 128; 129; 130; 131; 132; 133; 134; 135; 136; 137; 138; 139; 140; 141; 14 2;143;144;145;146;147;148;149;150;151;152;153;154;155;156;157;158;159;160;161;162;163;164;165;166;167;168;169;170;171;172;173 ;174;175;176;177;178;179;180;181;182;183;184;185;186;187;188;189;190;191;192;193;194;195;196;197;198;199;200;201;202;203;204; 205; 206; 207; 208; 209; 210; 211; 212; 213; 214; 215; 216; 217; 218; 219; 220; 221; 222; 223; 224; 225; 226; 227; 228; 229; 230; 231; 232; 233; 234; 235; 2 36;237;238;239;240;241;242;243;244;245;246;247;248;249;250;251;252;253;254;255;256;257;258;259;260;261;262;263;264;265;266;26 7;268;269;270;271;272;273;274;275;276;277;278;279;280;281;282;283;284;285;286;287;288;289;290;291;292;293;294;295;296;297;298;A polypeptide according to claim 20 (S280-598), comprising 299; 300; 301; 302; 303; 304; 305; 306; 307; 308; 309; 310; 311; 312; 313; 314; 315; 316; 317; 318; or 319 consecutive amino acids.

22. The polypeptide (S280-598) according to claim 20, comprising an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 280 to 598 of SEQ ID NO:

3.

23. The polypeptide (S280-598) according to claim 20, comprising an amino acid sequence in the range of residues 280 to 598 of Sequence ID No. 3, and including one or more spontaneous mutations selected from the group consisting of K417N, K417T, E484K, and N501Y mutations.

24. The polypeptide (S280-598) according to claim 20, comprising an amino acid sequence in the range of residues 280 to 598 of SEQ ID NO: 3, and including the K417N, E484K, and N501Y spontaneous mutations and the C538S non-spontaneous mutation.

25. SARS-CoV-2 polypeptide (S680-1029) is derived from the S protein and consists of at least 50 consecutive amino acids whose amino acid sequence is at least 90% identical to the amino acid sequence in the range of residues 680 to 1029 of SEQ ID NO:

3.

26. The amino acid sequence 50;51;52;53;54;55;56;57;58;59;60;61;62;63;64;65;66;67;68;69;70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;88;89;90;91;92;93;94;95;96;97;98;99;100;101;102;103;104;105;106;107;108;109;110 ;111;112;113;114;115;116;117;118;119;120;121;122;123;124;125;126;127;128;129;130;131;132;133;134;135;136;137;138;139;140;141; 142; 143; 144; 145; 146; 147; 148; 149; 150; 151; 152; 153; 154; 155; 156; 157; 158; 159; 160; 161; 162; 163; 164; 165; 166; 167; 168; 169; 170; 171; 172; 1 73; 174; 175; 176; 177; 178; 179; 180; 181; 182; 183; 184; 185; 186; 187; 188; 189; 190; 191; 192; 193; 194; 195; 196; 197; 198; 199; 200; 201; 202; 203; 2 04;205;206;207;208;209;210;211;212;213;214;215;216;217;218;219;220;221;222;223;224;225;226;227;228;229;230;231;232;233;234;23 5;236;237;238;239;240;241;242;243;244;245;246;247;248;249;250;251;252;253;254;255;256;257;258;259;260;261;262;263;264;265;266 ;267;268;269;270;271;272;273;274;275;276;277;278;279;280;281;282;283;284;285;286;287;288;289;290;291;292;293;294;295;296;297;Polypeptide according to claim 25 (S680-1029), comprising 298; 299; 300; 301; 302; 303; 304; 305; 306; 307; 308; 309; 310; 311; 312; 313; 314; 315; 316; 317; 318; 319; 320; 321; 322; 323; 324; 325; 326; 327; 328; 329; 330; 331; 332; 333; 334; 335; 336; 337; 338; 339; 340; 341; 342; 343; 344; 345; 346; 347; 348; 349; or 350 consecutive amino acids.

27. The polypeptide (S680-1029) according to claim 25, comprising an amino acid sequence having at least 90% identity with the amino acid sequence in the range of residues 680 to 1029 of SEQ ID NO:

3.

28. A conjugate in which a heterogeneous polypeptide is conjugated or fused to one or more SARS-CoV-2 polypeptides according to any one of claims 1 to 27.

29. The conjugate according to claim 28, comprising the polypeptide (N276-411, "Npep2") according to claim 1.

30. The conjugate according to claim 29, comprising polypeptide (N276-411 "Np2"), polypeptide (S125-250 "Sp1"), and polypeptide (S1056-1209 "Sp4").

31. The conjugate according to claim 30, comprising a polyepitope polypeptide having the amino acid sequence shown in Sequence ID No.

5.

32. The conjugate according to claim 29, comprising polypeptide (S125-250, "Sp1") and polypeptide (S1056-1209, "Sp4").

33. The conjugate according to claim 32, comprising a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO:

6.

34. The conjugate according to claim 29, comprising a polypeptide (N276-411, "Npep2") and an RBD polypeptide.

35. The conjugate according to claim 34, comprising a polyepitope polypeptide having the amino acid sequence shown in Sequence ID No.

7.

36. The conjugate according to claim 29, wherein the heterologous polypeptide is an immunoglobulin domain, in particular, the heavy chain or light chain of an antibody.

37. The conjugate according to claim 36, wherein the antibody is an IgG antibody, preferably an IgG1 or IgG4 antibody, or more preferably an IgG4 antibody.

38. The conjugate according to claim 36, wherein the antibody is against a surface antigen of APC selected from the group consisting of DC immune receptor (DCIR), MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD3lb, CD14, CD15, CD16, CD19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1, Fey receptor, LOX-1, and ASPGR.

39. The conjugate according to claim 38, wherein the antibody is against CD40.

40. CD40 antibody ・12E12 antibody containing the following: - A heavy chain comprising complementarity-determining regions CDR1H, CDR2H, and CDR3H, wherein CDR1H has the amino acid sequence GTFFSDYYMY (SEQ ID NO: 8), CDR2H has the amino acid sequence YINSGGGSTYYPDTVKG (SEQ ID NO: 9), and CDR3H has the amino acid sequence RGLPFHAMDY (SEQ ID NO: 10), and - A light chain comprising complementarity-determining regions CDR1L, CDR2L, and CDR3L, wherein CDR1L has the amino acid sequence SASQGISNYLN (SEQ ID NO: 11), CDR2L has the amino acid sequence YTSILHS (SEQ ID NO: 12), and CDR3L has the amino acid sequence QQFNKLPPT (SEQ ID NO: 13), or 11B6 antibody containing the following: - A heavy chain comprising complementarity-determining regions CDR1H, CDR2H, and CDR3H, wherein CDR1H has the amino acid sequence GYSFTGYYMH (SEQ ID NO: 14), CDR2H has the amino acid sequence RINPYNGATSYNQNFKD (SEQ ID NO: 15), and CDR3H has the amino acid sequence EDYVY (SEQ ID NO: 16); and - A light chain comprising complementarity-determining regions CDR1L, CDR2L, and CDR3L, wherein CDR1L has the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO: 17), CDR2L has the amino acid sequence KVSNRFS (SEQ ID NO: 18), and CDR3L has the amino acid sequence SQSTHVPWT (SEQ ID NO: 19), or ・12B4 antibody containing the following: - A heavy chain comprising complementarity-determining regions CDR1H, CDR2H, and CDR3H, wherein CDR1H has the amino acid sequence GYTFTDYVLH (SEQ ID NO: 20), CDR2H has the amino acid sequence YINPYNDGTKYNEKFKG (SEQ ID NO: 21), and CDR3H has the amino acid sequence GYPAYSGYAMDY (SEQ ID NO: 22), and - A light chain comprising complementarity-determining regions CDR1L, CDR2L, and CDR3L, wherein CDR1L has the amino acid sequence RASQDISNYLN (SEQ ID NO: 23), CDR2L has the amino acid sequence YTSRLHS (SEQ ID NO: 24), and CDR3L has the amino acid sequence HHGNTLPWT (SEQ ID NO: 25). A conjugate according to claim 39, derived from the present invention.

41. The conjugate according to claim 39, wherein the CD40 antibody is selected from the group consisting of mAb1, mAb2, mAb3, mAb4, mAb5 and mAb6 as listed in Table A.

42. The conjugate according to claim 38, wherein the antibody is specific to Langerin.

43. The conjugate according to claim 42, wherein the antibody is derived from antibody 15B10 having ATCC accession number PTA-9852, or from antibody 2G3 having ATCC accession number PTA-9853, or from antibody 91E7, 37C1, or 4C7 described in WO2011032161.

44. The conjugate according to claim 43, wherein the antibody is selected from the group consisting of mAb7, mAb8, mAb9, mAb10, mAb11, and mAb12 as listed in Table B.

45. The conjugate according to claim 28, wherein a heterologous polypeptide is fused with one or more SARS-CoV-2 polypeptides according to any one of claims 1 to 27 to form a fusion protein.

46. The conjugate according to claim 45, wherein a SARS-CoV-2 polypeptide is fused to a heterogeneous polypeptide directly or via a linker.

47. The conjugate according to claim 46, wherein the linker is selected from the group consisting of FlexV1, f1, f2, f3, or f4.

48. It consists of CD40 antibody (referred to as "Gen2a"), - The heavy chain of the antibody fuses with the RDB polypeptide, and The antibody light chain is fused to a polyepitope polypeptide comprising polypeptide (N276-411 "Npep2"), polypeptide (S125-250 "Spep1"), and polypeptide (S1056-1209 "Spep4"). The conjugate according to any one of claims 39 to 41.

49. The conjugate according to claim 48, wherein the light chain of the Gen2a antibody is fused to a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO:

5.

50. The conjugate according to claim 48, wherein the Gen2a antibody comprises a heavy chain shown in SEQ ID NO: 48 and a light chain having the amino acid sequence shown in SEQ ID NO:

49.

51. A conjugate according to any one of claims 39 to 41, comprising a CD40 antibody (referred to as "Gen2b"), - The heavy chain of the antibody is fused to a polyepitope polypeptide comprising polypeptide (S125-250, "Sp1") and polypeptide (S1056-1209, "Sp4"), - The antibody light chain is fused to a polyepitope polypeptide comprising a polypeptide (N276-411, "Npep2") and an RBD polypeptide. Conjugate.

52. The conjugate according to claim 51, wherein the heavy chain of the Gen2b antibody is fused to a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO:

6.

53. The conjugate according to claim 51, wherein the light chain of Gen2b is fused to a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO:

7.

54. The conjugate according to claim 51, wherein the Gen2b antibody comprises a heavy chain shown in SEQ ID NO: 50 and a light chain having the amino acid sequence shown in SEQ ID NO:

51.

55. A conjugate according to any one of claims 39 to 41, comprising a CD40 antibody (referred to as "Gen2c"), - The heavy chain of the antibody is fused to a polyepitope polypeptide comprising polypeptide (N276-411 "Npep2"), polypeptide (S125-250 "Spep1"), and polypeptide (S1056-1209 "Spep4"), and - The antibody light chain is fused to the RDB polypeptide. Conjugate.

56. The conjugate according to claim 55, wherein the heavy chain of the Gen2c antibody is fused to a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO:

5.

57. The conjugate according to claim 55, wherein the Gen2c antibody comprises a heavy chain shown in SEQ ID NO: 52 and a light chain having the amino acid sequence shown in SEQ ID NO:

53.

58. A conjugate according to any one of claims 39 to 41, comprising a CD40 antibody (referred to as "CD40.CoV2v"), - The heavy chain of the antibody fuses with the RDB polypeptide, and The antibody light chain is fused to a polyepitope polypeptide comprising polypeptide (N276-411 "Npep2"), polypeptide (S125-250 "Spep1"), and polypeptide (S1056-1209 "Spep4"). Conjugate.

59. The conjugate according to claim 58, wherein the light chain of CD40 CoV2 antibody is fused to a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO:

5.

60. The conjugate according to claim 58, comprising a heavy chain shown in SEQ ID NO: 54 and a light chain having the amino acid sequence shown in SEQ ID NO:

55.

61. A conjugate according to any one of claims 39 to 41, comprising a CD40 antibody (referred to as "CD40.N2.RBDv"), - The heavy chain of the antibody fuses with the polypeptide (N276-411 "Npep2"), The antibody light chain is fused to the RBD polypeptide. Conjugate.

62. The conjugate according to claim 61, comprising a heavy chain shown in SEQ ID NO: 56 and a light chain having the amino acid sequence shown in SEQ ID NO:

57.

63. A conjugate according to any one of claims 39 to 41, comprising a CD40 antibody (referred to as "CD40.N2.RBDv-2"), - The heavy chain of the antibody fuses with the RBD polypeptide, - The antibody's light chain is fused to the polypeptide (N276-411 "Npep2"). Conjugate.

64. A conjugate according to any one of claims 39 to 41, comprising a CD40 antibody (referred to as "CD40.RBDv.S4.N2"), - The heavy chain of the antibody is fused to a polyepitope polypeptide comprising an RBD polypeptide and a polypeptide (S1056-1209, "Spep4"), - The antibody's light chain is fused to the polypeptide (N276-411 "Npep2"). Conjugate.

65. The conjugate according to claim 64, wherein the heavy chain of the CD40. RBDv. S4. N2 antibody is fused to a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO:

58.

66. The conjugate according to claim 64, comprising a heavy chain shown in SEQ ID NO: 59 and a light chain having the amino acid sequence shown in SEQ ID NO:

60.

67. A conjugate according to any one of claims 39 to 41, comprising a CD40 antibody (referred to as "CD40.N2S1.RBDvS4"), - The heavy chain of the antibody is fused to a polyepitope polypeptide comprising polypeptide (N276-411 "Npep2") and polypeptide (S125-250 "Spep1"), - The light chain of the antibody is fused to a polyepitope polypeptide comprising an RBD polypeptide and a polypeptide (S1056-1209, "Spep4"). Conjugate.

68. The conjugate according to claim 67, wherein the light chain of the CD40.N2S1.RBDvS4 antibody is fused to a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO:

58.

69. The conjugate according to claim 67, wherein the heavy chain of the CD40.N2S1.RBDvS4 antibody is fused to a polyepitope polypeptide having the amino acid sequence shown in SEQ ID NO:

61.

70. The conjugate according to claim 67, comprising a heavy chain shown in SEQ ID NO: 62 and a light chain having the amino acid sequence shown in SEQ ID NO:

63.

71. The conjugate according to claim 58, 61, 63, 64, or 67, wherein the RBD polypeptide comprises amino acids in the range of amino acid residues 319 to 541 of SEQ ID NO: 3, and including the K417N, E484K, N501Y spontaneous mutation and the non-spontaneous C538S mutation ("RBD South African strain").

72. A vaccine composition comprising one or more SARS-CoV-2 polypeptides as described in any one of claims 1 to 27 and / or a conjugate as described in any one of claims 28 to 71 as an antigen.

73. The vaccine composition according to claim 72, comprising 2, 3, 4, 5, 6, 7, or 8 SARS-CoV-2 polypeptides as described in any one of claims 1 to 27.

74. A polynucleotide encoding one or more SARS-CoV-2 polypeptides according to any one of claims 1 to 27 and / or a conjugate according to any one of claims 28 to 71.

75. A method for administering SARS-CoV-2 vaccination to a target in need, comprising administering a therapeutically effective amount of one or more SARS-CoV-2 polypeptides according to any one of claims 1 to 27 and / or one or more conjugates according to any one of claims 28 to 71, and / or the vaccine composition according to claim 72, and / or the polynucleotide according to claim 74.

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