WO2022244831A1

WO2022244831A1 - Coronavirus vaccine suitable for elderly individuals

Info

Publication number: WO2022244831A1
Application number: PCT/JP2022/020772
Authority: WO
Inventors: Hiroki Ishikawa; Mary Katharine Levinge Collins
Original assignee: Okinawa Institute Of Science And Technology School Corporation
Priority date: 2021-05-21
Filing date: 2022-05-19
Publication date: 2022-11-24

Abstract

The present invention provides a vaccine or an immunogenic composition that can be useful in an elderly human subject.

Description

CORONAVIRUS VACCINE SUITABLE FOR ELDERLY INDIVIDUALS

TECHINICAL FIELD

The invention relates to a method of inducing antigen-specific immune response (preferably, T cell immune response) in a subject, and a composition for use in the method.

Background

There is extensive individual variation in severity of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), ranging from asymptomatic infection to fatal pneumonia ¹. Various factors, including age, sex, and comorbidities such as obesity and diabetes, influence the risk of severe COVID-19 ^2-4. For example, morbidity and mortality among the elderly are significantly higher than among the young². Consideration of protective measures for individuals vulnerable to COVID-19 should be particularly important to control the pandemic⁵. However, the cellular and molecular bases of the variable risk of COVID-19 remain poorly understood.

T cells are assumed to mediate both protective and pathogenic immune responses to SARS-CoV-2 infection ^6-7. The magnitude and quality of T cell responses induced by SARS-CoV-2 infection are highly heterogeneous and are likely associated with COVID-19 clinical outcomes. For example, SARS-CoV-2-specific T cell numbers and their interferon-γ (IFN-γ) expression in severe COVID-19 patients are lower than in mild COVID-19 patients^8-10. Furthermore, asymptomatic COVID-19 patients tend to have increased SARS-CoV-2-specific T cells expressing higher levels of IFN-γ compared to symptomatic patients¹¹. This individual variation in T cell responses may be partly explained by heterogeneity in levels of pre-existing SARS-CoV-2-reactive T cells.

Some individuals who have not been exposed to SARS-CoV-2 have nonetheless acquired SARS-CoV-2-reactive T cells, probably through exposure to other common cold coronaviruses^12-14. Pre-existing CD4 and CD8 memory T cells, specific to various SARS-CoV-2 proteins, including the structural proteins, Spike (S), Membrane (M), and Nucleoprotein (N), have been detected with significant individual variation¹⁵. These pre-existing SARS-CoV-2-reacive T cells are associated with immune protection against COVID-19¹⁶; however, in other cases, they may exacerbate COVID-19 severity^17-18. As many of the current vaccines express the SARS-CoV-2 S protein, only pre-existing S-reactive T cells are activated by these vaccines^19-20. Several studies have reported age-related differences in SARS-CoV-2-specific T cell responses in COVID-19 patients^9,21; however, the effect of age on pre-existing SARS-CoV-2-reactive T cells remains unknown.

BRIEF SUMMARY

In this study, the inventors compared frequencies of T cells reactive to SARS-CoV-2 S, N, and M antigens between young and elderly donors. The inventors found that pre-existing T cell responses to S and N antigens are significantly impaired in elderly donors compared to young donors, but a proportion of elderly donors exhibit significant, high levels of M-reactive T cell responses. These data provide new insights into age-related alteration of pre-existing SARS-CoV-2-specific T cells.

The disclosure may provide the invention as follows.
(1) A method of inducing antigen-specific immune response (preferably, T cell immune response), against coronavirus, in a subject, comprising:
administering a vaccine or an immunogenic composition comprising an effective amount of (i) M protein of coronavirus or a fragment thereof, or (ii) an mRNA comprising an open reading frame encoding M protein of coronavirus or fragment thereof to the patient, wherein the subject is over 50 years old, or preferably over 60 years old.
(2) The method according to (1) above, wherein the subject is over 70 years old.
(3) The method according to (1) or (2) above, wherein the fragment of M protein comprises position 145 to 160 of the amino acid sequence registered under GenBank Accession No.: QII57163.1, or has an amino acid sequence comprising an amino acid sequence corresponding to the position 145 to 160 of the amino acid sequence.
(4) The method according to any one of (1) to (3) above, wherein the M protein or the fragment thereof is in form of a fusion protein with another protein.
(5) The method according to (4) above, wherein said another protein is S protein of the corona virus.
(6) A coronavirus vaccine, comprising (i) M protein of coronavirus or a fragment thereof or (ii) an mRNA comprising an open reading frame encoding a coronavirus M protein of coronavirus or a fragment thereof, wherein the mRNA is encapsulated or formulated in a nanoparticle.
(7) The coronavirus vaccine according to (6) above, wherein the nanoparticle is a lipid nanoparticle.
(8) The coronavirus vaccine according to (6) or (7) above, which is to be administered to a subject who is over 50 years old, or preferably over 60 or 70 years old.
(9) An mRNA comprising an open reading frame encoding a coronavirus M protein or a fragment thereof for use in a method of inducing antigen-specific immune response (preferably, T cell immune response) in a subject, comprising: administering a vaccine or an immunogenic composition comprising an effective amount of M protein of coronavirus or a fragment thereof of coronavirus to the patient, wherein the subject is over 50 years old, or preferably over 60 or 70 years old.
(10) M protein of coronavirus or a fragment thereof for use in a method of inducing antigen-specific immune response (preferably, T cell immune response) in a subject, comprising: administering a vaccine or an immunogenic composition comprising an effective amount of M protein of coronavirus or a fragment thereof to the patient, wherein the subject is over 50 years old, or preferably over 60 or 70 years old.

The disclosure may further provide the invention as follows.
(11) The coronavirus vaccine according to (6) or (7) above, wherein the fragment of M protein comprises an amino acid sequence at position 145 to 160 of the amino acid sequence registered under GenBank Accession No.: QII57163.1, or has an amino acid sequence comprising an amino acid sequence corresponding to the position 145 to 160 of the amino acid sequence.
(12) The coronavirus vaccine according to any one of (6), (7), and (11) above, wherein the M protein or the fragment thereof is in form of a fusion protein with another protein, preferably an antigen protein (e.g., a component of coronavirus).
(13) The coronavirus vaccine according to (12) above, wherein said another protein is S protein of the corona virus.
(14) The coronavirus vaccine according to any one of (6), (7), (11), and (12) above, for use in a method of inducing antigen-specific immune response (preferably, T cell immune response, including CD4 positive memory T cell response) in a subject.
(15) The coronavirus vaccine according to (14) above, wherein the antigen-specific immune response contains an antigen-specific T cell response.
(16) The coronavirus vaccine according to (14) or (15) above, wherein the antigen-specific immune response comprises an immune memory in T cells.
(17) The coronavirus vaccine according to any one of previous items, wherein the coronavirus is a beta-coronavirus.
(18) The coronavirus vaccine according to any one of previous items, wherein the coronavirus is SARS-CoV-2.
(19) The coronavirus vaccine according to any one of previous items, wherein the coronavirus vaccine is to prevent or treating SARS-CoV-2 infection, and M protein is derived from coronavirus, preferably beta-coronavirus (e.g., common cold coronavirus), other than SARS-CoV-2.
(20) The coronavirus vaccine according to any one of previous items, wherein the coronavirus vaccine is to prevent or treating SARS-CoV-2 infection, and M protein is derived from SARS-CoV-2.

Fig. 1 shows altered pre-existing T cell responses to SARS-CoV-2 structural proteins in elderly donors. PBMCs isolated from seronegative (A) and seropositive (B) young (20-50 years of age) and elderly (>70 years of age) donors were stimulated with peptide pools for SARS-CoV-2 S, N, and M proteins and subjected to IFN-γ ELISpot analysis. Spot-forming units representing the frequency of IFN-γ-secreting cells in seronegative young (n=45), seronegative elderly (n=41), seropositive young (n=19), and seropositive elderly (n=11) are shown. The sum of spots formed by cells stimulated with S, N, and M (SNM) is also shown. Statistical comparisons between age groups utilized the Mann-Whitney test. *P<0.05, ns: not significant.

Fig. 2 shows M-specific pre-existing T cell responses predominate in elderly donors. Ratios of spots formed by cells stimulated with SARS-CoV-2 S, N, and M peptide pools in ELISpot data (in Fig. 1.) were analyzed in seronegative (A) and seropositive (B) donors who had >40 spots/10⁶ PBMCs in the sum of spots formed by cells stimulated with S, N, and M.

Fig. 3 shows pre-existing and SARS-CoV-2-induced M-specific T cells exhibit similar phenotypes. PBMCs isolated from seronegative (n=5) and seropositive (n=5) M responders were stimulated with an M peptide pool for 7 hours and analyzed by flow cytometry. (A) Dot plots represent data of subject #31 (seronegative, >70 years of age). (B) Percentages of IFN-g-expressing cells among CD4 and CD8 T cells were analyzed. (C) Percentages of naive (Tn), central memory (Tcm), and effector memory (Tem) among IFN-γ-expressing cells stimulated with M. (D, E) Percentages of TNF-α- and IL-2-expressing cells among total CD4 T cells (D) and IFN-γ-expressing CD4 T cells (E) stimulated with M. (B-E) Data shown as mean ± SD. Each dot represents an individual donor. Blue and orange dots indicate results of young and elderly donors, respectively. Statistical analysis utilized unpaired two-tailed Student’s t tests. *P<0.05, ns: not significant.

Fig. 4 shows SARS-CoV-2 M epitopes recognized by T cells. PBMCs isolated from M responders in seropositive (A) and seronegative (B) groups were stimulated with SARS-CoV-2 M matrix pools (15 pools) for 16 h and subjected to IFN-γ ELISpot analysis. Spot-forming units representing the frequency of IFN-γ-secreting cells. Blue and orange bars indicate results of young and elderly donors, respectively.

DETAILED DESCRIPTION

The term “subject” is a mammal including primate such as human. A human can be 50 years old or older, 60 years old or older, 70 years old or older, 80 years old or older, 90 years old or older, or 100 years old or older. A human can be a man or a woman.

The term “comprise” or “comprising” means containing at least specified technical matter(s) as an object of the term in a sentence, and the term does not exclude containing a matter not specified in the sentence. The term “consist of” or “consisting of” means containing substantially only specified technical matter(s) as an object of the term in a sentence. The term “comprise” or “comprising” encompasses “including” as well as “consisting”. For example, a composition “comprising” A may consist exclusively of A or may include something additional, for example, A and B.

The term “coronavirus” refers to viruses belonging to the family Coronaviridae of the order Nidoviridae. Coronaviruses are named coronaviruses because they have multiple projections by spike proteins (S proteins) on the surface envelope structure of viral particles and their images observed under an electron microscope resemble the corona of the sun. In humans, it is known to cause respiratory infections such as the common cold. However, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), MERS coronavirus (MERS-CoV), and 2019 novel coronavirus (SARS-CoV-2) are lethal. Coronaviruses include, for example, coronaviruses of the coronavirus subfamily, alpha coronaviruses (e.g., canine coronavirus, alpha coronavirus 1, human coronavirus 229E, human coronavirus NL63, porcine epidemic diarrhea virus), beta coronaviruses (e.g., embekovirus subgenus, salbekovirus subgenus, and Melbekovirus subgenus, Novecovirus subgenus, e.g., human enteric coronavirus 4408, human coronavirus OC43, mouse coronavirus, human coronavirus HKU1, and SARS-associated coronaviruses (e.g., SARS-CoV, SARS-CoV2, MERS coronavirus, and equine coronaviruses), gamma coronaviruses (e.g., avian coronavirus, white-lipped coronavirus SW1), and delta coronaviruses (e.g., Helicobacter coronavirus HKU11, munia coronavirus HKU13, and thrush coronavirus HKU12). In addition, coronaviruses also include their mutant virus strains.

Coronaviruses comprises nucleocapsid containing an RNA genome and Nucleocapsid protein (N protein), which is encapsulated by an envelope. The envelope comprises envelope (E) protein, spike (S) protein, and membrane (M) protein, which are exposed to the surface of the envelope. An example of M protein is M protein having the amino acid sequence registered under GenBank Accession No.: QII57163.1, which is an amino acid sequence set forth in SEQ ID NO: 1. These proteins may be mutated by repeated infections in hosts. In M protein, the regions at positions 2 to 19 and at positions 72 to 79 are exposed to virion surface (ectodomains); and the regions at positions 41 to 50 and at positions 101 to 222 are intravirion domains.

According to multiple sequence alignment analysis of beta coronavirus, M protein has a significant homology (e.g., 98% identity) among strains obtained from humans, bats, and pangolin. Therefore, M protein has an amino acid sequence with 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to the amino acid sequence registered under GenBank Accession No.: QII57163.1 or the amino acid sequence set forth in SEQ ID NO: 1. M protein plays an important role in the morphogenesis and assembly of coronavirus particles, and the functions of M protein include, for example, but not limited to, facilitating the morphogenesis and assembly of coronavirus particles. M protein may have one or more mutation (e.g., 1 to 10, 1 to 5, 1 to 3, or 1 or 2 mutations) in its amino acid sequence and have a function of M protein. Mutations can be selected from the group consisting of insertion, deletion, addition, or substitution. Substitutions can be amino acid substitutions including conservative amino acid substitution where an amino acid is replaced with another amino acid having the same physical property (e.g., aromatic amino acids such as tyrosine, tryptophan, and phenylalanine; positively charged amino acids such as arginine, histidine, and lysin; negatively charged amino acids such as aspartic acid and glutamic acid; non-charged hydrophilic amino acids such as asparagine, glutamine, serine, threonine; hydrophobic amino acids such as alanine, glycine, isoleucine, leucine, proline, and valine; and Sulphur-containing amino acids such as methionine and cysteine). M protein have an amino acid sequence with 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to the amino acid sequence registered under GenBank Accession No.: QII57163.1 or the amino acid sequence set forth in SEQ ID NO: 1; and has a function of M protein. M protein may be a naturally occurring M protein or a wild type M protein. M protein may be a naturally occurring or wild type M protein having an amino acid sequence with 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% identity to the amino acid sequence registered under GenBank Accession No.: QII57163.1 or the amino acid sequence set forth in SEQ ID NO: 1.

The term “immunogenic” as used herein means that an agent is capable of eliciting a humoral or preferably cellular immune response. An immunogenic composition can elicit a humoral or preferably cellular immune response in a subject who has received an effective amount of the immunogenic composition.

The term “epitope” refers to a portion of an antigen recognized by B cells or preferably by T cells. T cells can recognize a peptide comprising an epitope when the peptide is presented by antigen-presenting cells (APCs) via major histocompatibility complex (MHC). In an APC, a protein is digested to produce small peptide fragments of the protein, and then, the peptide fragment (also simply referred to as “peptide”) is presented by the APC via MHC. Thus, T cells can recognize such fragmented peptide of a protein regardless of whether or not the protein or peptide is derived from intracellular protein or intracellular domain.

The term “vaccine” or “vaccine composition” as used herein refers to biologics that can induce an immunity in a body of a subject against a specific infection including coronavirus (e.g., beta-coronavirus). Vaccine can be administered to a subject in order to obtain a prophylactic effect and/or a therapeutic effect. Vaccine or vaccine composition may comprise an active ingredient such as a part of an invader and a pharmaceutically acceptable additive. Vaccine or vaccine composition may further comprise an adjuvant that can enhance the induction of an immunity in a body of a subject.

The term “effective amount” or “therapeutically effective amount” as used herein refers to an amount of an active ingredient that can provide a detectable level of a desired effect on a biological activity in a treated subject. In the present invention, the term “effective amount” may mean an amount that is sufficient to prevent an infection, and/or inhibit a worthening of an infection.

In an embodiment, the present invention provides a method of inducing antigen-specific immune response (preferably, T cell immune response) in a subject. The antigen-specific immune response may involve an immune memory. Thus, an elderly people (e.g., a human subject over 50 years old, 60 years old, 70 years old, or 80 years old) can prevent an infection with coronavirus by inducing an antigen specific immune response including an immune memory.

In an embodiment, the present method may comprise administering a vaccine or an immunogenic composition comprising an effective amount of (i) M protein of coronavirus or a fragment thereof, or (ii) an mRNA comprising an open reading frame encoding M protein of coronavirus or fragment thereof to a subject, wherein the subject is over 50 years old, 60 years old, 70 years old, or 80 years old. As shown below, M protein can strongly induce an antigen-specific immunity in an elderly people. The induced immunity involves a memory immunity against the antigen (i.e., M protein or fragment thereof). Thus, M protein is particularly suitable for induction of an antigen-specific immunity among the other protein components of coronavirus, including S protein or the like. An epitope that can strongly induce an immune response in an elderly is located at positions 145 to 160 of an amino acid sequence registered under GenBank Accession No.: QII57163.1. A peptide comprising the epitope can be incorporated into an antigen-presenting cell such as a dendritic cell and then be digested into a smaller oligo peptide with 9 to 15 amino acids in length. The digested smaller oligo peptides are presented with major histocompatibility complex (MHC) or human leukocyte antigen to induce an antigen-specific immunity (e.g., antigen-specific acquired immunity). Thus, the peptide having an amino acid sequence at positions 145 to 160 of an amino acid sequence registered under GenBank Accession No.: QII57163.1 can be useful in or suitable for induction of the antigen-specific immunity.

In an embodiment, a vaccine or an immunogenic composition may comprise a peptide having an amino acid sequence at positions 145 to 160 of an amino acid sequence registered under GenBank Accession No.: QII57163.1. In a preferable embodiment, a vaccine or an immunogenic composition may comprise a partial peptide of M protein having an amino acid sequence registered under GenBank Accession No.: QII57163.1, wherein the partial peptide at least comprises an amino acid sequence at positions 145 to 160 of the M protein. The peptide may have, for example, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, 200 or more, or 210 or more amino acids in length. The peptide may have, for example, 210 or less, 200 or less, 190 or less, 180 or less, 170 or less, 160 or less, 150 or less, 140 or less, 130 or less, 120 or less, 110 or less, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 19 or less, 18 or less, 17 or less, or 16 or less amino acids in length. The peptide may have, for example, 16 to 210, 16 to 200, 16 to 190, 16 to 180, 16 to 170, 16 to 160, 16 to 150, 16 to 140, 16 to 130, 16 to 120, 16 to 110, 16 to 100, 16 to 90, 16 to 80, 16 to 70, 16 to 60, 16 to 50, 16 to 40, 16 to 30, 16 to 20 amino acids in length.

In an embodiment, a vaccine or an immunogenic composition may comprise a peptide having an amino acid sequence corresponding to an amino acid sequence at positions 145 to 160 of an amino acid sequence registered under GenBank Accession No.: QII57163.1. In an embodiment, a vaccine or an immunogenic composition may comprise a partial peptide of M protein wherein the partial peptide has an amino acid sequence comprising an amino acid sequence corresponding to an amino acid sequence at positions 145 to 160 of an amino acid sequence registered under GenBank Accession No.: QII57163.1.

In an embodiment, a vaccine or an immunogenic composition may comprise a polynucleotide or gene encoding M protein or a fragment thereof such as the peptide as defined above. In a preferable embodiment, the polynucleotide or gene is an mRNA. In a preferable embodiment, the mRNA is encapsulated or formulated in a nanoparticle, preferably a lipid nanoparticle.

In an embodiment, (i) M protein of coronavirus or a fragment thereof, or (ii) an mRNA comprising an open reading frame encoding M protein of coronavirus or fragment thereof is isolated, purified, or synthesized.

An mRNA may contain a modified nucleic acid. Examples of modified nucleic acids include, but are not limited to, pseudouridine, which is known as a modified nucleoside for in vivo expression of mRNA. Modified nucleosides can replace unmodified nucleosides. Pseudouridine includes, for example, 1-methyl-3-(amino-5- carboxypropyl) pseudouridine (m1acp3Ψ), 1- methyl pseudouridine (m1Ψ), 2'-O-methylpseudouridine (Ψ m), 5-methyldihydriuridine (m5D), 3- methyl pseudouridine (m3Ψ), and others can substitute for uridine. The modified mRNA preferably contains pseudouridine and can preferably further contain 5-methylcytidine. In a preferable embodiment, all of uracils in an mRNA are replaced with m1Ψ pseudouridine. An mRNA usually contains a 5' cap structure, a 5' untranslated region (UTR), a coding region, a 3' UTR, and a polyadenine sequence. The 5' cap structure is, for example, a cap containing N7-methylguanosine (m7G) containing caps (e.g., m7GpppG cap, 3 '-O-methyl-m7 GpppG cap). 5' UTR and 3' UTR, for example. facilitate translation of proteins from mRNA.

The lipid nanoparticle is suitable for mRNA delivery. Examples of lipid nanoparticles include, but are not limited to, the nucleic acid-lipid particles described in US8,058,069B, which is incorporated herein by reference in its entirety. Amphiphilic lipids can form lipidic nanoparticles in aqueous solution. In a preferable lipid nanoparticle, 50 mol% to 60 mol% of total lipid is cationic lipid such as DLin-MC3-DMA, ALC-0315, and SM-102; 4 mol% to 10 mol% of total lipid is phospholipid such as DSPC; 30 mol% to 40 mol% of total lipid is cholesterol or a derivative thereof; and 0.5 mol% to 2 mol% of total lipid is a conjugated lipid such as PEGylated lipid such as PEG2000-DMG and ALC-0159, which can inhibit aggregation of particles. In a preferable embodiment, an mRNA comprises m1Ψ pseudouridine and is encapsulated in a lipid nanoparticle, preferably, wherein 50 mol% to 60 mol% of total lipid is cationic lipid, 4 mol% to 10 mol% of total lipid is phospholipid such as DSPC, 30 mol% to 40 mol% of total lipid is cholesterol or a derivative thereof, and 0.5 mol% to 2 mol% of total lipid is a conjugated lipid such as PEGylated lipid.

In an embodiment, an induced antigen-specific immunity is an antigen-specific T cell immunity (particularly by a CD4 positive or CD4 single positive T cell, preferably a CD4 single positive memory T cell), which may involve an immune memory. In an embodiment, an induced antigen-specific immunity is an antigen-specific B cell immunity, which may involve an immune memory. In an embodiment, an induced antigen-specific immunity is an antigen-specific T cell immunity (particularly by a CD4 positive or CD4 single positive T cell, preferably a CD4 single positive memory T cell) and B cell immunity, which may involve an immune memory.

In an embodiment, M protein or fragment thereof may be isolated, purified, or synthesized. In an embodiment, a gene encoding M protein of fragment thereof is provided.

In an embodiment, a vaccine or an immunogenic composition may further comprises a pharmaceutically acceptable additive. Examples of pharmaceutically acceptable additive include, but are not limited to, diluent, solubilizer, buffering agent, tonicity agent, emulsifier, viscosity-increasing agent, antioxidant, and soothing agent.

In a preferable embodiment, a vaccine or an immunogenic composition may further comprises an adjuvant. An adjuvant is a substance that can enhance an immune response against an antigen non-specifically. Examples of adjuvants include, but are not limited to, alum, aluminum salts, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, aluminum hydroxyphosphate sulfate, on which antigen can be adsorbed; Freund’s incomplete adjuvant, and Freund’s complete adjuvant.

In an embodiment, a vaccine or an immunogenic composition may comprise physiological sarin and M protein or a fragment thereof.

In an aspect, the present invention provides a method of inducing an antigen-specific immunity against coronavirus in a subject. The method may comprise administering to the subject a vaccine or an immunogenic composition comprising an effective amount of (i) M protein of coronavirus or a fragment thereof, or (ii) an mRNA comprising an open reading frame encoding M protein of coronavirus or fragment thereof. In a preferable embodiment, the subject is preferably an elder human subject as defined above.

In an aspect, the present invention provides a method of preventing or treating a coronavirus infection in a subject. The method may comprise administering to the subject a vaccine or an immunogenic composition comprising an effective amount of (i) M protein of coronavirus or a fragment thereof, or (ii) an mRNA comprising an open reading frame encoding M protein of coronavirus or fragment thereof. In a preferable embodiment, the subject is preferably an elder human subject as defined above.

In an aspect, the present invention provides (i) M protein of coronavirus or a fragment thereof, or (ii) an mRNA comprising an open reading frame encoding M protein of coronavirus or fragment thereof for use in a method of inducing an antigen-specific immunity against coronavirus in a subject.

In an aspect, the present invention provides use of (i) M protein of coronavirus or a fragment thereof, or (ii) an mRNA comprising an open reading frame encoding M protein of coronavirus or fragment thereof in the manufacture of a medicament for use in a method of inducing an antigen-specific immunity against coronavirus in a subject.

Examples

Methods

Subjects
The study design was approved by the OIST human subjects ethics committee (applications HSR-2020-024, HSR-2020-028). All donors provided informed written consent. Young (20 to 50 years of age, n=66) and elderly volunteers (over 70 years of age, n=52) were recruited in Okinawa, Japan, between October, 2020 and April, 2021. 90 donors (48 young and 42 elderly) had no history of COVID-19, while 28 donors (18 young and 10 elderly) who recovered from COVID-19 had positive COVID-19 PCR test results 1-3 months before blood collection. Plasma from each donor was tested for SARS-CoV-2-specific antibodies using SARS-Cov-2 Antibody Detection Kits (KURABO RF-NC001, RF-NC002) or Cellex qSARS-Cov-2 IgG/IgM Cassette Rapid Tests (Cellex 5513C). Four (3 young and 1 elderly) of 90 donors who had no history of COVID-19 and 26 (16 young and 10 elderly) of 28 donors who had recovered from COVID-19 were seropositive for SARS-CoV-2 S antigen. Based on antibody test results, donors were grouped into seronegative young (n=45, 40 % male, 60% female; mean age 38 years, age rage 23-49 years), seronegative elderly (n=41, 17 % male, 83% female; mean age 81 years, age rage 70-93 years), seropositive young (n=19, 63 % male, 37% female; mean age 41 years, age rage 20-50 years), and seropositive elderly (n=11, 45 % male, 55% female; mean age 78 years, age rage 70-91 years).

Peripheral blood mononuclear cells (PBMCs) and plasma isolation.

Blood samples were collected in heparin-coated tubes (TERUMO; VP-H100K). PBMCs and plasma were separated using Leucosep tupes pre-filled with Ficoll-Paque Plus (Greiner; 163288). After adding 5 mL of blood and 3 mL of AIM-V medium (Thermo; 12055091), Leucosep tubes were centrifuged at 1,000 g at room temperature for 10 min. The white layer containing PBMCs was collected, washed with 10 mL AIM-V medium and centrifuged for 7 min at 600 g, followed by a second washing with centrifugation for 7 min at 400 g. PBMC pellets were resuspended in 500 mL CTL test medium (Cellular Technology Limited (CTL); CTLT-010). Fresh PBMCs were used for IFN-g ELISpot assays. PBMCs used for flow cytometry analysis and epitope mapping analysis were stored with CTL-cryo ABC media (CTL; CTLC-ABC) in liquid nitrogen.

IFN-g ELISpot assay

Peptide pools for SARS-CoV-2 S (JPT; PM-WCPV-S-1), N (Miltenyi;130-126-698), and M (Miltenyi;130-126-702) proteins dissolved in DMSO (500 mg/mL for S) or water (50 mg/mL for N and M) were used for cell stimulation. IFN-g ELISpot assays were performed using Human IFN-g Single-Color Enzymatic ELISpot kits (CTL; hIFNgp-2M), according to the manufacturer’s instructions. Briefly, freshly isolated PBMCs (1-4 x 10⁵ cells per well) were stimulated with 1 μg/mL peptide solutions for each SARS-CoV-2 protein for 18-20 h. For each sample analysis, negative controls (cells treated with equimolar amounts of DMSO) and positive controls (cells treated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 mg/mL ionomycin) were included. After incubation, plates were washed and developed with detection reagents included in the kits. Spots were counted using a CTL ImmunoSpot S6 Analyzer. Antigen-specific spot counts were determined by subtracting background spot counts in a negative control well from the wells treated with peptide pools. If >30 spots/10⁶ PBMCs in the negative control well or <30 spots/10⁶ PBMCs in the positive control well were detected, sample data were excluded from analysis.

Flow cytometry

Frozen PBMCs were thawed, washed with CTL wash supplement (CTL; CTL-W-010), and rested in CTL test medium overnight. Then, cells were resuspended in RPMI1640 (Gibco) medium supplemented with 5% (v/v) human AB-serum (PAN-Biotech; P30-2901), seeded into 96-well, U-bottom culture plates (10⁶ cells per well), and either left unstimulated (cells treated with equimolar amounts of DMSO) or stimulated with 1 μg/mL SARS-CoV-2 M peptide pool for 7 h in the presence of 1μg/mL anti-CD40 (5C3; Biolegend; 334302) and 1 μg/mL anti-CD28 antibodies (CD28.2; Biolegend; 302934). Brefeldin A (1 μg/mL) (Biolegend; 420601) was added for the last 2 h. After stimulation, cells were incubated with anti-Fc receptor-blocking antibody (Biolegend; 422301) and NIR-Zomibie (Biolegend; 423106), and stained with anti-CD3 (OKT3; Biolegend; 1:200), anti-CD4 (clone PPA-T4; Biolegend; 1:200), anti-CD8 (SK1; Biolegend; 1:200), anti-CD45RA (HI100; Biolegend; 1:100), and anti-CCR7 (G043H7; Biolegend; 1:100) antibodies. For intracellular cytokine analysis, cells were subsequently fixed and permeabilized using Foxp3 Staining Buffer Sets (eBioscience; 00-5253-00) and stained with anti-IFN-g (B27; BD; 1:100), anti-TNF-a (MAb11; Biolegend; 1:20) and anti-IL-2 (MQ1-17H12; Biolegend; 1:20) antibodies. Samples were analyzed on a Fortessa X-20 (BD), and data were analyzed with FlowJo software version 10.7.1 (FlowJo LLC).

Matrix peptide pools of SARS-CoV-2 M

Matrix peptide pools included in Epitope Mapping Peptide Set SARS-CoV-2 (VME1) (JPT EMPS-WCPV-VME-1) were used to analyze M epitopes recognized by T cells. PBMCs (0.5-1.5 x 10⁵) were stimulated with 1 mg/mL of each M matrix peptide pool (15 pools of 6-8 peptides) for 18 h and subjected to IFN-g ELISpot assays.

Statistical analysis

Unpaired t tests or Mann-Whitney U tests were performed using GraphPad Prism 9.1.0 software. Statistical details are provided in figure legends.

Results

Age-related differences in SARS-CoV-2-specific T cell responses
To assess whether there are age-related differences in SARS-CoV-2-specific T cell responses, we collected peripheral blood from young (20 to 50 years of age) and elderly (>70 years of age) donors in Okinawa between October 2020 and April 2021. These included 18 young and 10 elderly donors who had recovered from mild COVID-19 1-3 months prior to blood collection. Antibody tests using freshly purified sera showed that 93% of donors with previously diagnosed COVID-19 and 4% of those without, were seropositive for SARS-CoV-2 Spike (S). Then we divided donors into 4 groups: young seronegative (n=45), elderly seronegative (n=41), young seropositive (n=19), and elderly seropositive (n=11).

To compare SARS-CoV-2-specific T cell responses between age groups we performed Interferon-γ (IFN-γ) ELISpot assays using freshly purified peripheral blood mononuclear cells (PBMCs) stimulated with each of 4 peptide pools covering the major viral structural proteins [N-terminal S (S1), C-terminal S (S2), Membrane (M), or Nucleoprotein (N)]. First, we analyzed the sum of spots formed by IFN-γ-expressing cells reactive to S1, S2, N, or M antigens (hereafter referred to as spots of SNM-reactive T cells), indicating the magnitude of SARS-CoV-2-specific T cell responses in each donor. Almost all seropositive donors exhibited strong T cell responses to SARS-CoV-2 antigens regardless of age; the frequency of SNM-reactive spots was >40 per 10⁶ PBMCs in 92% of seropositive donors. The frequency of SNM-reactive T cells in seronegative donors was more variable than in seropositive donors, but a substantial proportion of seronegative donors exhibit T cell responses comparable to those of seropositive donors (53% of young donors and 42% of elderly donors had >40 spots per 10⁶ PBMCs). In both seronegative and seropositive populations, there were no significant differences in the frequency of SNM-reactive T cells between young (seronegative; median:53, IQR:21-96, seropositive; meidan:312, IQR:75-1509) and elderly (seronegative; median:32, IQR:7-331, seropositive; meidan:778, IQR:351-2392) (Fig. 1A, 1B).

Next, we compared frequencies of T cells reactive to individual viral antigens between young and elderly donors. Among seronegative donors, the frequencies of S-2- and N-reactive T cells were significantly lower in elderly than in young persons (Fig. 1B). However, there were no significant differences in the frequencies of S-1- and M-reactive T cells between seronegative young and elderly donors (Fig. 1B). Consistent with previous reports²², T cell responses to S-2 were higher than S-1 in the seronegative population (Fig. 1B). Frequencies of T cells specific to S1, S2, N, and M were comparable between seropositive young and elderly donors (Fig. 1B).

We also analyzed intraindividual immunodominance of each SARS-CoV-2 antigen in donors who had >40 spots per 10⁶ PBMCs. Remarkably, M-specific responses were dominant in 6/23 young and 12/14 elderly seronegative donors (Fig. 2A). In contrast, T cell responses specific to S-1 and S-2 were more prominent than M in seropositive donors (Fig. 2B). Taken together, these data suggest that although SARS-CoV-2 infection can induce comparable T cell responses to various viral antigens in young and elderly individuals, pre-existing memory T responses specific to SARS-CoV-2 S and N antigens decrease in elderly people, while M-specific T cell responses are maintained.

Phenotypes of M-reactive T cells

Given that a proportion of seronegative donors exhibited M-specific responses comparable to those of seropositive donors, we compared phenotypes of M-reactive T cells by flow cytometry analysis. We analyzed 5 seronegative (1 young and 4 elderly) and 5 seropositive (2 young and 3 elderly) donors who had high responses to the M peptide pool (M responders). We stimulated PBMCs with the M peptide pool and stained cells with anti-CD4 or anti-CD8 antibodies, followed by intracellular cytokine staining with anti-IFN-g antibody. Upon stimulation with the M peptide pool, CD4 T cells but not CD8 T cells significantly increased expression of IFN-g in both seronegative and seropositive M responders and at comparable levels (Fig. 3A, 3B).

We next analyzed CD45RA and CCR7 expression to determine proportions of naive and memory cells among M-reactive CD4 T cells. Although there was significant variation between individuals in proportions of M-reactive CD4 T cells exhibiting naive (CD45RA⁺CCR7⁺), effector memory (CD45RA⁺ CCR7^-), and central memory (CD45RA^- CCR7⁺) phenotypes, all M responders had M-reactive memory T cells (Fig. 3C). There was no obvious difference in the proportion of naive, effector memory, and central memory CD4 T cells between seronegative and seropositive M responders (Fig. 3C).

We also analyzed frequencies of cells expressing IL-2 and TNF-a among M-reactive CD4 T cells. Although several donors showed increased IL-2 and TNF-a expression upon stimulation with M in both seronegative and seropositive M responders, in seropositive donors, only IL-2 reached statistical significance (Fig. 3D). Seronegative and seropositive M-responders showed no detectable difference in IL-2 and TFN-a expression in T cells stimulated with M (Fig. 3D). Expression of TNF-a and IL-2 in IFN-g-expressing CD4 T cells was comparable between seronegative and seropositive M responders (Fig. 3E). These data indicate that M-reactive T cell responses are mediated by CD4 T cells expressing IFN-g in both seronegative and seropositive M responders, suggesting that pre-existing M-reactive T cells and SARS-CoV-2-induced memory M-specific T cells might serve similar functions in SARS-CoV-2 infection.

Epitopes of SARS-CoV-2 M protein

To compare epitopes recognized by M-reactive T cells of seronegative and seropositive donors, we stimulated PBMCs isolated from M responders with SARS-CoV-2 M matrix pools (15 pools of 6-8 peptides) where each of 56 M-derived peptides (15-mers) is allocated to 2 different pools. In seropositive M responders, stimulation with

pools

5 and 13 induced high levels of IFN-g responses (Fig. 4A). These matrix pools shared a single peptide, M_145-160, suggesting that the M_145-160, which was previously identified as an immunodominant viral epitope in COVID-19 convalescent patents²³, is the epitope recognized by T cells of seropositive M responders. In contrast, in seronegative M responders, stimulation of PBMCs with many M matrix pools induced comparable IFN-g responses (Fig. 4B), suggesting that various M epitopes are recognized by pre-existing T cells.

Discussion

Our data indicate that a fraction of elderly donors possesses significantly high levels of pre-existing SARS-CoV-2 M-specific T cell responses, though the frequency of pre-existing SARS-CoV-2 S- and N-specific T cells in this population is lower. Other recent studies have also reported an age-related decline of S-specific pre-existing T cells [19, 24]. These data suggest that pre-existing T cells specific to SARS-CoV-2 are heterogeneously affected by age in a target antigen-dependent manner. There was no obvious difference in the sum of IFN-γ-expressing cells specific to S, N, and M antigens, suggesting that abundant M-specific T cells can compensate for the loss of S- and N-specific T cells, at least in the magnitude of T cell-mediated IFN-γ production, in elderly individuals. Taken together, these data suggest that multi-specificity of pre-existing T cells may decline with age, but the magnitude of pre-existing T cell responses can be maintained with T cells specific to certain viral proteins such as M.

As the frequency of pre-existing T cells specific to viral structural proteins S, N, and M is associated with protection from SARS-CoV-2 infection¹⁶, focused M responses might be particularly important for protection of elderly individuals who have lower responses to S and N. Flow cytometry analyses revealed that CD4 T cells mainly mediate M-specific T cell responses, and their naive/memory phenotypes and their capacity to produce IFN-γ, IL-2, and TNF-α cytokines were comparable between seronegative and seropositive groups. The phenotypic similarity suggests that pre-existing M-reactive T cells may serve similar functions to SARS-CoV-2-induced M-specific memory T cells. We speculate that pre-existing M-specific CD4 T cells play a protective role in SARS-CoV-2 infection by promoting cellular immunity through IFN-g production and humoral immunity by providing T cell help to S- and N-specific B cells via linked recognition.

However, we cannot exclude the possibility that pre-existing M-specific T cells are harmful for some elderly individuals. Several studies suggest that pre-existing SARS-CoV-2-specific T cells are detrimental in COVID-19⁸. In particular, the frequency of M-specific T cells in COVID-19 patients is thought to be a risk factor, as it is correlated with age and severity of disease⁸, although how pre-existing M-specific T cells affect magnitude, kinetics and functions of M-specific T cell responses in COVID-19 patients remains unclear. Thus, both protective and pathogenic functions of pre-existing M-specific T cells can be speculated. A longitudinal comparison of susceptibility and symptom severity of COVID-19 between individuals with and without high pre-existing M-specific T cell responses might provide insights into this issue.

Despite defects in pre-existing T cell responses to S and N, most elderly donors who recovered from mild COVID-19 had abundant T cells specific to SARS-CoV-2 antigens at levels comparable to those of young donors, suggesting that elderly individuals can induce SARS-CoV-2-specific T cell responses upon SARS-CoV-2 infection. However, our analysis is limited to only a few patients who had recovered from mild COVID-19. Therefore, the relationship between age-related alteration of pre-existing T cells and T cell responses during infection in patients with diverse clinical outcomes of COVID-19 should be investigated in a larger, statistically valid test population.

Whether diverse SARS-CoV-2-induced T cell clones can mediate long-lasting memory responses should be addressed in future studies. It has recently been shown that SARS-CoV-2-induced memory T cells persist at least 6 months after infection²⁵. Interestingly, SARS-CoV-1 infection induces long-lasting (>11 years) CD8 memory T cells specific to M_141-155 peptide²⁶. Our data show that the overlapping peptide (M_145-160) is immunodominant in SARS-CoV-2 infection, which is consistent with a recent study showing CD4 T cell responses to the M_145-160 peptide in convalescent COVID-19 patients²³ The amino acid sequence of M_145-160peptide from SARS-CoV-2 shows high homology with SARS-CoV-1 and other coronaviruses (SARS-CoV-1: 81.3%, NL63: 33.0% , OC43: 47.0%, 229E: 22.7%, HKU1: 47.0%). This short M peptide is likely a potent inducer of SARS-CoV-2 M-specific memory T cells. In contrast, pre-existing T cells likely recognize various M peptides, possibly including M_145-160, rather than focusing on this single M peptide, as we observed in epitope mapping analysis.

What induces pre-existing M-specific T cells? Common cold coronaviruses may induce pre-existing SARS-CoV-2-specific T cell^{27, 28}. Amino acid sequence homology between SARS-CoV-2 and other common cold coronaviruses is relatively high for M (NL63: 25.2%, OC43: 36.9%, 229E: 26.7%, HKU1: 32.4%), S1 (NL63: 11.0%, OC43: 15.4%, 229E: 12.8%, HKU1: 15.2%), S2 (NL63: 27.3%, OC43: 36.9%, 229E: 28.0%, HKU1: 35.3%), and N (NL63: 22.3%, OC43: 26.5%, 229E: 16.2%, HKU1: 26.5%). These coronaviruses may induce polyclonal M-specific T cells. Age-related loss of memory T cells specific to common cold coronavirus S protein²⁴ supports the hypothesis that pre-existing M-focused T cell responses are induced by common cold coronavirus infection in elderly people. Our data showing higher frequency of pre-existing T cells specific to S-2 than S-1 are also consistent with the fact that S-2 shows higher homology between SARS-CoV-2 and other coronaviruses. However, some young donors, as well as elderly donors, had abundant pre-existing T cells specific to M, but not to S and N, suggesting that focused T cell responses to M are not necessarily due to age-related loss of pre-existing T cells specific for S and N antigens. Interestingly, a recent study reported that T cells specific to commensal bacteria can cross-react with SARS-CoV-2 S antigen²⁹. Similarly, there may be specific microbes that induce pre-existing M-specific T cells.

It is worth considering the potential of novel COVID-19 vaccines to induce M-specific immunity. Current vaccine strategies are to induce S-specific antibody and T cell responses^{30, 31}. Recent studies reported a correlation between the frequency of pre-existing S-specific T cells and vaccine-induced S-specific T cell responses¹⁹, which suggests a role of pre-existing S-specific T cells in cognate T cell help. However, elderly individuals likely would not benefit fully from pre-existing S-specific T cells. To enhance vaccine efficacy among the elderly, it might be reasonable to consider a strategy to induce not only S-specific, but also M-specific immunity, using vaccines based on inactivated viruses or M-fused S antigens. Linked recognition of M-specific T helper cells by S-specific B cells can promote S-specific antibody production by overcoming the defect of cognate T cell help in elderly individuals. Further characterization of M-specific T cells in young and elderly may provide new insights into vaccine-induced immunity that is less affected by age.

References

1. Oran DP, Topol EJ: Prevalence of Asymptomatic SARS-CoV-2 Infection. Annals of Internal Medicine 2020, 173(5):362-367.
2. O’Driscoll M, Ribeiro Dos Santos G, Wang L, Cummings DAT, Azman AS, Paireau J, Fontanet A, Cauchemez S, Salje H: Age-specific mortality and immunity patterns of SARS-CoV-2. Nature 2021, 590(7844):140-145.
3. Takahashi T, Ellingson MK, Wong P, Israelow B, Lucas C, Klein J, Silva J, Mao T, Oh JE, Tokuyama M et al: Sex differences in immune responses that underlie COVID-19 disease outcomes. Nature 2020, 588(7837):315-320.
4. Wolff D, Nee S, Hickey NS, Marschollek M: Risk factors for Covid-19 severity and fatality: a structured literature review. Infection 2021, 49(1):15-28.
5. Li H, Liu S-M, Yu X-H, Tang S-L, Tang C-K: Coronavirus disease 2019 (COVID-19): current status and future perspectives. International Journal of Antimicrobial Agents 2020, 55(5):105951.
6. Bertoletti A, Tan AT, Le Bert N: The T-cell response to SARS-CoV-2: kinetic and quantitative aspects and the case for their protective role. Oxford Open Immunology 2021, 2(1).
7. Song P, Li W, Xie J, Hou Y, You C: Cytokine storm induced by SARS-CoV-2. Clinica Chimica Acta 2020, 509:280-287.
8. Sattler A, Angermair S, Stockmann H, Heim KM, Khadzhynov D, Treskatsch S, Halleck F, Kreis ME, Kotsch K: SARS-CoV-2-specific T cell responses and correlations with COVID-19 patient predisposition. Journal of Clinical Investigation 2020, 130(12):6477-6489.
9. Rydyznski Moderbacher C, Ramirez SI, Dan JM, Grifoni A, Hastie KM, Weiskopf D, Belanger S, Abbott RK, Kim C, Choi J et al: Antigen-Specific Adaptive Immunity to SARS-CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity. Cell 2020, 183(4):996-1012.e1019.
10. Chen G, Wu D, Guo W, Cao Y, Huang D, Wang H, Wang T, Zhang X, Chen H, Yu H et al: Clinical and immunological features of severe and moderate coronavirus disease 2019. Journal of Clinical Investigation 2020, 130(5):2620-2629.
11. Le Bert N, Clapham HE, Tan AT, Chia WN, Tham CYL, Lim JM, Kunasegaran K, Tan LWL, Dutertre C-A, Shankar N et al: Highly functional virus-specific cellular immune response in asymptomatic SARS-CoV-2 infection. Journal of Experimental Medicine 2021, 218(5).
12. Braun J, Loyal L, Frentsch M, Wendisch D, Georg P, Kurth F, Hippenstiel S, Dingeldey M, Kruse B, Fauchere F et al: SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19. Nature 2020, 587(7833):270-274.
13. Le Bert N, Tan AT, Kunasegaran K, Tham CYL, Hafezi M, Chia A, Chng MHY, Lin M, Tan N, Linster M et al: SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature 2020, 584(7821):457-462.
14. Woldemeskel BA, Kwaa AK, Garliss CC, Laeyendecker O, Ray SC, Blankson JN: Healthy donor T cell responses to common cold coronaviruses and SARS-CoV-2. Journal of Clinical Investigation 2020, 130(12):6631-6638.
15. Wyllie D, Mulchandani R, Jones HE, Taylor-Phillips S, Brooks T, Charlett A, Ades AE, Makin A, Oliver I, Moore P et al: SARS-CoV-2 responsive T cell numbers are associated with protection from COVID-19: A prospective cohort study in keyworkers. In.: Cold Spring Harbor Laboratory; 2020.
16. Wyllie DH, Mulchandani R, Jones HE, Taylor-Phillips S, Brooks T, Charlett A, Ades A, Makin A, Oliver I: SARS-CoV-2 responsive T cell numbers are associated with protection from COVID-19: A prospective cohort study in keyworkers. In.: Cold Spring Harbor Laboratory; 2020.
17. Bacher P, Rosati E, Esser D, Martini GR, Saggau C, Schiminsky E, Dargvainiene J, Schroder I, Wieters I, Khodamoradi Y et al: Low-Avidity CD4(+) T Cell Responses to SARS-CoV-2 in Unexposed Individuals and Humans with Severe COVID-19. Immunity 2020, 53(6):1258-1271 e1255.
18. Chen Z, John Wherry E: T cell responses in patients with COVID-19. Nature Reviews Immunology 2020, 20(9):529-536.
19. Loyal L, Braun J, Henze L, Kruse B, Dingeldey M, Reimer U, Kern F, Schwarz T, Mangold M, Unger C et al: Cross-reactive CD4+ T cells enhance SARS-CoV-2 immune responses upon infection and vaccination. In.: Cold Spring Harbor Laboratory; 2021.
20. Sahin U, Muik A, Derhovanessian E, Vogler I, Kranz LM, Vormehr M, Baum A, Pascal K, Quandt J, Maurus D et al: COVID-19 vaccine BNT162b1 elicits human antibody and TH1 T cell responses. Nature 2020, 586(7830):594-599.
21. Cohen CA, Li AP, Hachim A, Hui DS, Kwan MY, Tsang OT, Chiu SS, Chan WH, Yau YS, Kavian N et al: SARS-CoV-2 specific T cell responses are lower in children and increase with age and time after infection. In.: Cold Spring Harbor Laboratory; 2021.
22. Braun J, Loyal L, Frentsch M, Wendisch D, Georg P, Kurth F, Hippenstiel S, Dingeldey M, Kruse B, Fauchere F et al: SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19. Nature 2020, 587(7833):270-274.
23. Keller MD, Harris KM, Jensen-Wachspress MA, Kankate VV, Lang H, Lazarski CA, Durkee-Shock J, Lee P-H, Chaudhry K, Webber K et al: SARS-CoV-2-specific T cells are rapidly expanded for therapeutic use and target conserved regions of the membrane protein. Blood 2020, 136(25):2905-2917.
24. Saletti G, Gerlach T, Jansen JM, Molle A, Elbahesh H, Ludlow M, Li W, Bosch B-J, Osterhaus ADME, Rimmelzwaan GF: Older adults lack SARS CoV-2 cross-reactive T lymphocytes directed to human coronaviruses OC43 and NL63. Scientific Reports 2020, 10(1).
25. Jiang X-L, Wang G-L, Zhao X-N, Yan F-H, Yao L, Kou Z-Q, Ji S-X, Zhang X-L, Li C-B, Duan L-J et al: Lasting antibody and T cell responses to SARS-CoV-2 in COVID-19 patients three months after infection. Nature Communications 2021, 12(1).
26. Ng O-W, Chia A, Tan AT, Jadi RS, Leong HN, Bertoletti A, Tan Y-J: Memory T cell responses targeting the SARS coronavirus persist up to 11 years post-infection. Vaccine 2016, 34(17):2008-2014.
27. Sagar M, Reifler K, Rossi M, Miller NS, Sinha P, White LF, Mizgerd JP: Recent endemic coronavirus infection is associated with less-severe COVID-19. Journal of Clinical Investigation 2021, 131(1).
28. Mateus J, Grifoni A, Tarke A, Sidney J, Ramirez SI, Dan JM, Burger ZC, Rawlings SA, Smith DM, Phillips E et al: Selective and cross-reactive SARS-CoV-2 T cell epitopes in unexposed humans. Science 2020, 370(6512):89-94.
29. Lu X, Hosono Y, Ishizuka S, Nagae M, Ishikawa E, Motooka D, Ozaki Y, Sax N, Shinnakasu R, Inoue T et al: Identification, crystallization and epitope determination of public TCR shared and expanded in COVID-19 patients. In.: Cold Spring Harbor Laboratory; 2021.
30. Corbett KS, Edwards DK, Leist SR, Abiona OM, Boyoglu-Barnum S, Gillespie RA, Himansu S, Schafer A, Ziwawo CT, Dipiazza AT et al: SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness. Nature 2020, 586(7830):567-571.
31. Polack FP, Thomas SJ, Kitchin N, Absalon J, Gurtman A, Lockhart S, Perez JL, Perez Marc G, Moreira ED, Zerbini C et al: Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. New England Journal of Medicine 2020, 383(27):2603-2615.

Claims

A method of inducing antigen-specific immune response (preferably, T cell immune response) in a subject, comprising:
administering a vaccine or an immunogenic composition comprising an effective amount of (i) the M protein of a coronavirus or a fragment thereof, or (ii) an mRNA comprising an open reading frame encoding the M protein of the coronavirus or a fragment thereof to the subject, wherein the subject is over 50 years old, or preferably over 60 years old.
The method according to claim 1, wherein the subject is over 70 years old.
The method according to claim 1 or 2, wherein the fragment of the M protein comprises positions 145 to 160 of the amino acid sequence registered under GenBank Accession No.: QII57163.1, or has an amino acid sequence comprising an amino acid sequence corresponding to the positions 145 to 160 of the amino acid sequence.
The method according to any one of claims 1 to 3, wherein the M protein or the fragment thereof is in a form of a fusion protein with another protein.
The method according to claim 4, wherein said another protein is the S protein of the coronavirus.
A coronavirus vaccine, comprising (i) the M protein of a coronavirus or a fragment thereof or (ii) an mRNA comprising an open reading frame encoding the M protein of the coronavirus or a fragment thereof, wherein the mRNA is encapsulated or formulated in a nanoparticle.
The coronavirus vaccine according to claim 6, wherein the nanoparticle is a lipid nanoparticle.
The coronavirus vaccine according to claim 6 or 7, which is to be administered to a subject who is over 50 years old, or preferably over 60 years old.
An mRNA comprising an open reading frame encoding the M protein of a coronavirus or a fragment thereof for use in a method of inducing antigen-specific immune response in a subject, comprising: administering a vaccine or an immunogenic composition comprising an effective amount of the M protein of the coronavirus or a fragment thereof to the subject, wherein the subject is over 50 years old, or preferably over 60 years old.
An M protein of a coronavirus or a fragment thereof for use in a method of inducing antigen-specific immune response in a subject, comprising: administering a vaccine or an immunogenic composition comprising an effective amount of the M protein of a coronavirus or a fragment thereof to the subject, wherein the subject is over 50 years old, or preferably over 60 years old.
The coronavirus vaccine according to claim 6 or 7, wherein the fragment of the M protein comprises positions 145 to 160 of the amino acid sequence registered under GenBank Accession No.: QII57163.1, or has an amino acid sequence comprising an amino acid sequence corresponding to the position 145 to 160 of the amino acid sequence.
The coronavirus vaccine according to any one of claim 6, 7, and 11, wherein the M protein or the fragment thereof is in a form of a fusion protein with another protein.
The coronavirus vaccine according to claim 12, wherein said another protein is the S protein of the coronavirus.
The coronavirus vaccine according to any one of claim 6, 7, 11, and 12, for use in a method of inducing antigen-specific immune response (preferably, T cell immune response) in a subject.
The coronavirus vaccine according to claim 14, wherein the antigen-specific immune response contains an antigen-specific T cell response.
The coronavirus vaccine according to claim 14 or 15, wherein the antigen-specific immune response comprises an immune memory in T cells.
The coronavirus vaccine according to any one of claims 6 to 8 and 11 to 16, wherein the coronavirus is a beta-coronavirus.
The coronavirus vaccine according to any one of claims 6 to 8 and 11 to 17, wherein the coronavirus is SARS-CoV-2.
The coronavirus vaccine according to any one of claims 6 to 8 and 11 to 18, wherein the coronavirus vaccine is to prevent or treat SARS-CoV-2 infection, and the M protein is derived from a coronavirus other than SARS-CoV-2.
The coronavirus vaccine according to any one of claims 6 to 8 and 11 to 18, wherein the coronavirus vaccine is to prevent or treat SARS-CoV-2 infection, and the M protein is derived from SARS-CoV-2.