Patent Application
20230181721
The present invention relates to a vaccine against a Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and its use.
In 2020, the COVID-19 pandemic due to the rapid worldwide spread of SARS Cov-2 virus (novel coronavirus outbreak 2019-nCoV) is becoming a serious global health threat. It was declared by the WHO as Public Health Emergency of International Concern (PHEIC). A vaccine is urgently needed trying to stop the global spread and the COVID-19 mortality seen in ICU.
The precedent emergent Coronaviruses (RNA viruses which usually cause mild upper respiratory illnesses) are also causing SARS (severe acute respiratory Syndrome) or MERS (Middle east respiratory syndrome) were causing global attention on the clinical significance of coronaviruses. SARS, which is caused by the SARS coronavirus (SARS-CoV), emerged from China and caused nearly 8500 cases and 916 deaths during the outbreak in 2002 and 2003. Overall fatality of SARS-CoV was about 10% in the general population, but >50% in patients aged 65 years and older (Shibo J et al; Future Virology 2013; “Development of SARS vaccines and therapeutics is still needed”).
A number of inactivated and live-attenuated SARS vaccines, as well as those based on vectors encoding the full-length S protein of SARS-CoV, showed high immunogenicity in inducing neutralizing antibody responses and protection against SARS-CoV challenge. However, most of these vaccine candidates may also induce immunopathology or other harmful immune responses such as antibody-dependent enhancement (ADE) phenomenon (Iwasaki et al. Nature Review Immunology 2020), raising concerns about their safety (Weingartl H, et al J. Virol.-2004. Immunization with modified vaccinia virus Ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets).
Virus-like-particle vaccine (whole virus vaccine and an rDNA-produced S protein) induced protection against infection but challenged animals exhibited an immunopathologic-type lung disease (Tseng C T, Sbrana E, Iwata-Yoshikawa N, Newman P C, Garron T, et al. PLOS ONE 2012, Immunization with SARS Coronavirus Vaccines Leads to Pulmonary Immunopathology on Challenge with the SARS Virus). Various approaches (recombinant S protein-based, DNA-based or RNA-based, Viral vector-based, Recombinant RBD protein-based, siRNA, peptides) were explored as candidate vaccines (Du L, He Y, Zhou Y et al. Nat. Rev. Microbiol 2009—The spike protein of SARS-CoV: a target for vaccine and therapeutic development.).
Today, various vaccine technologies from the new SARS-Cov-2 virus are also prepared and will be explored in clinical trials (Le T T et al Nature review 2020—The COVID-19 vaccine development landscape). The most advanced candidates have recently moved into clinical development, including mRNA-1273 (LNP-encapsulated mRNA vaccine encoding S protein—Moderna), Ad5-nCoV—(Adenovirus type 5 vector that expresses S protein—CanSino Biologicals), INO-4800 (DNA plasmid encoding S protein delivered by electroporation—Inovio), LV-SM ENP-DC (Dentritic Cells modified with lentiviral vector expressing synthetic minigene based on domains of selected viral proteins; administered with antigen-specific CTLs—Shenzhen Geno-Immune Medical Institute), specific aAPC (Pathogen-specific aAPC modified with lentiviral vector expressing synthetic minigene based on domains of selected viral proteins—Shenzhen Geno-Immune Medical Institute).
Peptides are also vaccines candidates. Even they are considered as a lower immunogenic strategy, various peptides approaches were studied from the previous SARS-Cov virus spread and are today explored for the new pandemic related to the SARS-COv2 virus (Zheng B J et al; Antiviral Therapy 2005, Synthetic peptides outside the spike protein heptad repeat regions as potent inhibitors of SARS-associated coronavirus). The main research on peptides (fragments of proteins or protein shells that mimic the coronavirus's outer coat) focuses on viral protein subunits—mainly on the virus's spike protein or a key part of it called the receptor binding domain RBD (Callaway E; Nature Vol 580, 30 Apr. 2020, the race for coronavirus vaccine).
As a whole, most candidates presented for which information is available aim to induce neutralizing antibodies against the viral spike (S) protein, preventing uptake via the human ACE2 receptor. However, vaccines based on antibodies produced by B lymphocytes cells are not sufficiently efficient against coronavirus yet. Their action is for the short-term prophylactic response (Wu et al. Emerg Infect Dis. 2007; Ho et al. Emerg Infect Dis. 2005). Besides in some cases and patients, such antibodies may raise a risk of tolerance linked to a problem of increased endocytosis of the virus by cells of the host and/or of excessive inflammatory processes (Iwasaki et al. Nature Review Immunology 2020). Furthermore, previous SARS-COV failed to induce long-term memory B cells (Tang et al. J. Immunol 2011) while memory T cells have been observed up to 11 years after infection (Ng O W Vaccine 2016).
According to another and less known approach, vaccines focusing the activation of T lymphocytes have also been studied: Channappanavar et al. (Journal of Virology 2014 Virus-specific memory CD8 T cells provide substantial protection from lethal SARS-CoV infection); and Tan A C L et al. (Immunol Cell Biol 2013 —The design and proof of concept for a CD8+ T cell-based vaccine inducing cross-subtype protection against influenza A virus). Both teams have demonstrated for other viruses that a peptide vaccine based on two natural CD8 peptides against the SARS-CoV 2003 virus or the Flu Virus induce lasting immunization in mice with in particular the induction of T Resident Memory cells (TRM) in the pulmonary parenchyma and the pulmonary alveoli. Channappanavar was using intravenous peptide-pulsed Dendritic Cells followed by intranasal boosting with recombinant vaccinia virus (rVV) encoding peptides. Tan and collaborators were using three of the natural HLA-A2-restricted influenza epitopes into lipopeptides by intranasal administration.
These peptide vaccines are based on the use of natural, wild-type, naïve epitopes. There is however a need of a better efficiency and in particular of an increased immunogenicity of the vaccine allowing a stronger activation of the immune T cells and preferably CD8 T cells able to destroy cells that are infected by the virus. Furthermore, this prior art used intranasal immunization and/or boost to stimulate the generation of long-lasting antigen specific TRM. However, the intravenous road of administration and preparation of fresh dendritic cells is not an appropriate method for world-wide vaccination strategy. The intranasal road of administration is of interest to stimulate mucosal immunity in the long term but has been also reproducibly describe to induce allergic reaction (Vasu et al. Ther Adv Respir Dis. 2008).
Finally, the research of vaccine epitopes is a very big challenge due to the complexity of coronavirus and the huge number of epitopes that are not useful for a vaccine composition. With 30,000 genetic bases, coronaviruses have the largest genomes of all RNA viruses. Their genomes are more than three times as big as those of HIV and hepatitis C, and more than twice influenza's. 35 000 unique T-cell epitopes are of potential interest, but the final and practical clinical use will be limited to a small number of epitopes.
The present invention provides a vaccine composition against a Severe acute respiratory syndrome-related coronavirus based on a multi-target CD8 T cell peptide composition designed for targeting several structural SARS-Cov-2 proteins such as Spike glycoprotein (S), Nucleocapsid protein (N), and Membrane glycoprotein (M) but also non-structural SARS-Cov-2 proteins, the epitopes being selected in conserved regions on the SARS-Cov-2 genome.
The vaccine composition has the following advantages:
The inventors observed that a single subcutaneous injection of peptides induces a robust immunogenicity in vivo and series of epitopes induce a strong proportion of virus-specific tissue-resident memory T lymphocytes (Trm). They observed high cellular responses upon restimulation with structural and non-structural protein-derived epitopes using blood T cells isolated from convalescent asymptomatic, moderate and severe COVID-19 patients. Finally, the combination of selected CTL epitopes is suitable for use in vaccination of a broad worldwide population, even if the design was based on HLA-A2 subjects.
Accordingly, the present invention relates to a vaccine composition comprising CTL (neo)epitopes of SEQ ID NOs: 70 and/or 146; 23 and 66, and at least 2, 3, 4, 5, 6, 7 or 8 CTL (neo)epitopes selected from SEQ ID NOs: 8, 22, 31, 32, 42, 52, 77 and 97.
In one aspect, the composition further comprises at least 1 HTL peptide/epitope or a T helper peptide, especially PADRE (aKXVAAWTLKAAa with X and a respectively indicating cyclohexylalanine and d-alanine).
In a particular aspect, the vaccine composition comprises CTL (neo)epitopes of SEQ ID NOs: 8, 22, 23, 31, 32, 42, 52, 66, 70, 77, 97 and 146.
In a more specific aspect, the vaccine composition comprises CTL (neo)epitopes of SEQ ID NOs: 8, 22, 23, 31, 32, 42, 52, 66, 70, 77, 97 and 146 and a T helper peptide, especially PADRE (aKXVAAWTLKAAa with X and a respectively indicating cyclohexylalanine and d-alanine).
The vaccine composition may further comprise an adjuvant, in particular a mixture of mineral oil and mannide mono-oleate, especially Montanide® ISA 51.
In a particular aspect, the vaccine composition comprises the CTL (neo)epitopes are each at a dose of between 1 and 100 μg, preferably between 5 and 50 μg. It may comprise the T helper peptide, especially PADRE are at a dose of between 1 and 100 μg, preferably between 5 and 50 μg.
The present invention relates to a vaccine composition as disclosed herein for use for preventing or treating an infection by a severe acute respiratory syndrome-related coronavirus (SARS-CoV).
More particularly, the SARS-CoV is selected from the group consisting of SARS-CoV1, SARS-CoV2 or MERS-CoV virus, preferably SARS-CoV2.
Optionally, the subject to be treated is a subject aged 65 years or older, a subject having a cancer or having had a cancer, a subject being obese (In particular with severe obesity (body mass index [BMI] of 40 or higher [CDC-HCSP BMI>30]), a subject being diabetic, a subject having a hypertension, a subject having a sarcoidosis, a subject being immunocompromised, a subject who lives in a nursing home or long-term care facility, a subject with chronic lung disease or moderate to severe asthma, lung fibrosis, a subject who has serious heart conditions, a subject with chronic kidney disease undergoing dialysis and/or a subject with liver diseases; and/or a subject being HLA-A2.
(A) WT and mutated peptides were incubated with HLA-A*02:01 monomer, exposed to UV for peptide exchange and then HLA-peptide complexes stability at 37° C. was measured by ELISA. Data are mean+/−SEM (n=4) expressed as percentage of binding relative to an internal MEMOPI® positive control neoepitope.
(B) WT and mutated peptides (25 μM) binding to TAP-deficient human cell line (T2) expressing HLA-A2. Data are expressed as percentage of binding relative to an internal MEMOPI® positive control neoepitope.
(A) IFNγ Elispot responses of pooled spleen and draining lymph nodes cells were restimulated for 24 hours with each of the isolated peptides. Data are mean+/−SEM (n=6).
(B) Frequency of Tetramer+CD8 T cells (gating strategy in
(A) Flow cytometry gating strategy to define Tetramer+CD8 T cells responses.
(B) Representative example and gating strategy of Trm phenotype based on CD44, CD103, CXCR3, CD49a and CD69 expression in Tetramer+CD8 T cells as compared to Tetramer−CD8 T cells.
(C)
Left. Same level of responses observed in HLA-A2 positive patients (green color (n=48)) versus HLA-A2 negative patients (orange color (n=40)), that patients being asymptomatic or hospitalized.
Right. The immunogenicity was measured in the IFN gamma responses after stimulation with the peptides using blood T cells isolated from HLA-A2 positive COVID-19 patients (n=48) and HLA-A2 negative COVID-19 patients (n=40).
The vaccine presented in the invention assembles (neo)epitopes combination assuring the involvement of the full repertoire of cells involved in the immune responses to this Specific SARS Coy infection.
The immune cells activated by the vaccine could be in particular:
In a preferred aspect, the selection of the epitopes/neoepitopes of the vaccine composition allows to provide early B cell & HTL response and/or long T cell memory CTL and HTL responses.
This strategy provides advantageously a vaccine composition activating:
The activation of T cells response is of major interest for coronavirus which particularly induces a major problem of T cells response. Further, since some T cells in particular memory T cells are specifically localized in pulmonary tissue, their activation is very helpful against coronavirus which has severe consequence on pulmonary tissue.
In preferred aspect, the vaccine comprises epitopes of the coronavirus that will be recognized by CD8 T cells through the interaction with MHCI system. The activated CD8 T cells convert into Lymphocytes cytotoxic T cells (effector CTL) by the help of helper T Lymphocytes (HTL). The activated T cells are notably CD8 memory T cells allowing the long-term action of the vaccine.
The process of selection of epitopes and their modifications into neoepitopes is a key element of the invention. A second element is the epitopes/neoepitopes combination able to produce a synergy of the immune responses. A third element is the generation of (neo)epitopes with high homology between coronavirus such SARS-CoV (2003), MERS-CoV (2012) and SARS-CoV-2 (2019) allowing vaccination against several and future emergent coronavirus.
As reminded above, a very high number of ˜35 000 unique T-cell epitopes in SARS-CoV-2 genome could be identified. About 200 epitopes could be selected based on the binding capacity by HLA-A2 and their immunogenicity.
Thanks to further complex studies, the inventors further refined the selection by the identification of 55 very promising CTL epitopes of interest (as disclosed in Table 1). The selected epitopes are issued from 4 different parts of the coronavirus (Spike protein (S) (including RBM as epitope B), Membrane protein (M), Nucleocapside (N), and several non-structural viral proteins from viral RNA).
The selected epitopes have the advantages to be well-conserved among SARS-CoV coronaviruses, in particular SARS-CoV-2, SARS-CoV1 and MERS-CoV genomes.
In addition, from the 55 very promising CTL epitopes, the inventors further designed neo-epitopes with an increased binding and/or immunogenicity. These neo-epitopes of interest are disclosed in Tables 2 and 3.
Among these CTL epitopes, the inventors selected a group of 46 preferred CTL (neo)epitopes which induce an immune response in vivo (see Table 7); a group of 27 preferred CTL (neo)epitopes which induce an immune response in vivo and induce cellular responses upon restimulation with these CTL (neo)epitopes using blood T cells isolated from convalescent asymptomatic, moderate and severe COVID-19 patients (see Table 8). Among these peptides, some induce a strong proportion of virus-specific tissue-resident memory T lymphocytes (Trm) (see Tables 7 and 8). Based on these data, the inventors provide a list of preferred CTL peptides (see Table 9). Based on these 12 CTL peptides, it is possible to induce a vaccination of a broad worldwide population (see Table 10). Peptides are able to bind several HLA and show a worldwide population coverage in the range of 40-99%. As shown in Table 10, it is possible to obtain at least one immune response in everybody and to reach herd immunity (65%) in the worldwide population with at least one combination of 5-7 CTL peptides.
Finally, the inventors designed a group of epitopes suitable for inducing an immune response by B Lymphocytes (BCL) that produce neutralizing antibodies, therefore called herein BCL epitopes. B epitopes are selected in the precise 420-500 region of the Spike protein in order to create or generate antibodies blocking the entry of the virus, through an antagonist action of the antibody blocking the recognition between the virus epitopes and the cells of the host. B cell epitopes were rationally design selectively in the receptor-binding domain (RBD), more particularly within the receptor-binding motif (RBM), of the protein Spike. In contrast to other vaccinal strategies with live, attenuated or inactivated virus expressing the whole Spike proteins or vaccination with DNA or RNA coding for Spike or RBD, or vaccination with the total Spike proteins, here by designing selectively B-cell epitopes within the RBM motifs, there is not risk to generate non-neutralizing antibodies which have been associated with antibody-dependent enhancement phenomenon (Iwasaki et al. Nature Review Immunology 2020).
These BCL epitopes of interest are disclosed in Table 4. In addition, these BCL epitopes can be fused to HTL epitopes. For instance, HTL epitopes can be PADRE and such fused epitopes are disclosed in Table 5. The BCL epitopes could be directly coupled or covalently linked to an HTL epitope with an adaptor or through a linker.
The vaccine according to the present invention is a multi-(neo)epitopes combination (wildtype and neoepitopes) with HTL, BCL or CTL purpose so as to induce a synergistic immune response.
The synergy provided by the final combination is a strong element of the invention supporting the original concept of this versatile strategy adapted to the SARS-Cov vaccination in order to obtain an adequate robust response and to limit doses of vaccine.
The final selection for the vaccine composition is based on clinical needs (early responses, long term responses) depending of the state of the patients, immediate risk or long-term risks, evolution of the pandemic and the general condition of the patients (fragile or aged, immunocompromised, debilitating conditions patients).
Although neutralizing antibody production by B cells and cytotoxic activity of CD8+ T cells are well-accepted components of the adaptive immune response to various viruses, the role of the two segments of immunity is rarely covered in one specific vaccine strategy. To consider the COVID-19 pandemic situation and the resurgence of the virus in seasonal condition, it will be important to anticipate specific clinical situation with the ability to adapt the combination to the situation.
In the previous infection to the first SARS-Cov, the frequency of CD4+ memory T-cells to virus structural proteins and anti-SARS coronavirus IgG levels were low by 12 months after infection (Libraty H T et al; Science direct Virology 2007—Human CD4+ memory T-lymphocyte responses to SARS coronavirus infection).
Favorizing early B cell responses and IgG neutralizing antibody for pandemic acute periods is a clinical need for a wide population considered at risks of infection.
Favorizing T cells long term responses for specific populations with a serious risk of mortality is another clinical need covered by the invention.
The vaccine composition has also been designed in order to be effective, not only on the known SARS-Cov viruses but also against SRAS-Cov may emerge in the future.
This whole work leads to a selection of combinations of for instance 10 to 20 epitopes/neoepitopes, fighting against the heterogeneity of the previous, current and future coronaviruses. The selection of the epitopes/neoepitopes can be based on various possible modalities, notably:
In summary the invention describes a (neo)epitopes—based vaccine selected on high binding capability or by specific chemical modification increasing binding property, addressing early (HTL and B cell specific immune response) and/or long-term immunogenicity (HTL and T cells specific immune response) in particular for “fragile” patients.
According to a first aspect, the vaccine composition comprises:
Then, the CTL epitopes and the BCL epitopes of the vaccine are mixed in a common vaccine composition that is administered in one injection, repeated if appropriate.
According to a second aspect, the vaccine composition comprises:
According to a third aspect, the vaccine composition comprises:
According to a fourth aspect, the invention relates to a combination of two vaccine compositions, the first composition comprising:
And the second composition comprising:
Then, the CTL epitopes and the BCL epitopes of the vaccine are in two separate compositions, that are administered sequentially, firstly a CTL epitopes composition then BCL epitopes composition, or firstly BCL epitopes composition then CTL epitopes composition, repeated if appropriate.
Optionally, the BCL epitopes can be fused to the HTL epitopes.
In a preferred aspect, the total number of epitope peptides in the composition can be from 5 to 40, from 7 to 30 or from 10 to 20 peptides.
In a preferred aspect, the CTL epitopes are a mixture of naïve T epitopes (CTL epitopes) and of neo-epitopes (CTL neo-epitopes), advantageously 1 to 15 CTL epitopes and 1 to 15 CTL neo-epitopes. The CTL epitopes are selected from the CTL epitopes of Table 1 and the CTL neo-epitopes are selected from the CTL neo-epitopes of Tables 2 and 3, of Table 2 or of Table 3.
In another preferred aspect, the CTL epitopes are a mixture of naïve T epitopes (CTL epitopes) and of neo-epitopes (CTL neo-epitopes), advantageously 1 to 15 CTL epitopes and 1 to 15 CTL neo-epitopes. The CTL epitopes are selected from the CTL epitopes of SEQ ID NOs: 3, 8, 20, 22, 23, 30, 31, 32, 33, 34, 36, 42, 48, 49 and 52 and the CTL neo-epitopes are selected from the CTL neo-epitopes of SEQ ID NOs: 56, 59, 60, 66, 67, 70, 74, 75, 76, 77, 78, 79, 83, 84, 85, 86, 90, 91, 92, 95, 97, 101, 104, 105, 113, 120, 125, 135, 139, 140, 146 and 153.
In another preferred aspect, the CTL epitopes are a mixture of naïve T epitopes (CTL epitopes) and of neo-epitopes (CTL neo-epitopes), advantageously 1 to 15 CTL epitopes and 1 to 15 CTL neo-epitopes. The CTL epitopes are selected from the CTL epitopes of SEQ ID NOs: 3, 8, 22, 23, 30, 31, 32, 36, 42, 48 and 52 and the CTL neo-epitopes are selected from the CTL neo-epitopes of SEQ ID NOs: 56, 59, 60, 66, 70, 76, 77, 78, 79, 83, 91, 92, 125, 135, 139, 140 and 146.
In another preferred aspect, the CTL epitopes are a mixture of naïve T epitopes (CTL epitopes) and of neo-epitopes (CTL neo-epitopes), advantageously 1 to 7 CTL epitopes and 1 to 5 CTL neo-epitopes. The CTL epitopes are selected from the CTL epitopes of SEQ ID NOs: 8, 22, 23, 31, 32, 42 and 52 and the CTL neo-epitopes are selected from the CTL neo-epitopes of SEQ ID NOs: 66, 70, 77, 97 and 146.
The CTL epitopes or neo-epitopes of the vaccine composition target one or several proteins of SARS-CoV, especially selected in the group consisting of Spike glycoprotein (S), Nucleocapsid protein (N), Membrane glycoprotein (M) and ORfs, more particularly Protein 3a, nsp3, nsp4, nsp6, nsp12, nsp13, nsp14 and nsp16.
Accordingly, the CTL epitopes or neo-epitopes of the vaccine composition are selected for targeting 1, 2, 3, 4, 5, 6, 7 or 8 of Spike glycoprotein (S), Nucleocapsid protein (N), Membrane glycoprotein (M), Protein 3a, nsp3, nsp4, nsp6, nsp12, nsp13, nsp14 and nsp16, preferably at least 5, 6, 7, 8, 9, 10, or 11 proteins of SARS-CoV.
Optionally, for each protein, the CTL (neo)epitopes are selected in the following groups:
Optionally, the CTL epitopes or neo-epitopes of the vaccine composition are selected for targeting at least:
Spike glycoprotein (S) and Nucleocapsid protein (N); Spike glycoprotein (S) and Membrane glycoprotein (M); Spike glycoprotein (S) and Protein 3a; Spike glycoprotein (S) and nsp4; Spike glycoprotein (S) and nsp3; Spike glycoprotein (S) and nsp6; Spike glycoprotein (S) and nsp12; Spike glycoprotein (S) and nsp13; Spike glycoprotein (S) and nsp14; Spike glycoprotein (S) and nsp16; Nucleocapsid protein (N) and Membrane glycoprotein (M); Nucleocapsid protein (N) and Protein 3a; Nucleocapsid protein (N) and nsp4; Nucleocapsid protein (N) and nsp3; Nucleocapsid protein (N) and nsp6; Nucleocapsid protein (N) and nsp12; Nucleocapsid protein (N) and nsp13; Nucleocapsid protein (N) and nsp14; Nucleocapsid protein (N) and nsp16; Spike glycoprotein (S), Nucleocapsid protein (N) and Membrane glycoprotein (M); Spike glycoprotein (S), Nucleocapsid protein (N) and Protein 3a; Spike glycoprotein (S), Nucleocapsid protein (N) and nsp4; Spike glycoprotein (S), Nucleocapsid protein (N) and nsp3; Spike glycoprotein (S), Spike glycoprotein (S), Nucleocapsid protein (N) and nsp6; Spike glycoprotein (S), Nucleocapsid protein (N) and nsp12; Spike glycoprotein (S), Nucleocapsid protein (N) and nsp13; Spike glycoprotein (S), Nucleocapsid protein (N) and nsp14; Spike glycoprotein (S), Nucleocapsid protein (N) and nsp16; Membrane glycoprotein (M) and Protein 3a; Membrane glycoprotein (M) and nsp4; Membrane glycoprotein (M) and nsp3; Membrane glycoprotein (M) and nsp6; Membrane glycoprotein (M) and nsp12; Membrane glycoprotein (M) and nsp13; Membrane glycoprotein (M) and nsp14; Membrane glycoprotein (M) and nsp16; Spike glycoprotein (S), Membrane glycoprotein (M) and Protein 3a; Spike glycoprotein (S), Membrane glycoprotein (M) and nsp4; Spike glycoprotein (S), Membrane glycoprotein (M) and nsp3; Spike glycoprotein (S), Membrane glycoprotein (M) and nsp6; Spike glycoprotein (S), Membrane glycoprotein (M) and nsp12; Spike glycoprotein (S), Membrane glycoprotein (M) and nsp13; Spike glycoprotein (S), Membrane glycoprotein (M) and nsp14; Spike glycoprotein (S), Membrane glycoprotein (M) and nsp16; Nucleocapsid protein (N), Membrane glycoprotein (M) and Protein 3a; Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp4; Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp3; Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp6; Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp12; Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp13; Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp14; Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp16; Spike glycoprotein (S), Nucleocapsid protein (N), Membrane glycoprotein (M) and Protein 3a; Spike glycoprotein (S), Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp4; Spike glycoprotein (S), Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp3; Spike glycoprotein (S), Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp6; Spike glycoprotein (S), Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp12; Spike glycoprotein (S), Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp13; Spike glycoprotein (S), Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp14; or Spike glycoprotein (S), Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp16.
In a particular aspect, the CTL epitopes or neo-epitopes of the vaccine composition are selected for targeting one or several of the following groups
Spike glycoprotein (S) and Membrane glycoprotein (M) and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; and the CTL (neo)epitope targeting M is SEQ ID NO: 66;
Spike glycoprotein (S) and Protein 3a and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; and the CTL (neo)epitopes targeting Protein 3a are selected from one of the groups consisting of (i) SEQ ID NOs: 3, 97 and 101; and (ii) SEQ ID NO: 97;
Spike glycoprotein (5) and nsp3 and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; and the CTL (neo)epitopes targeting nsp3 are selected from one of the groups consisting of (i) SEQ ID NOs: 30, 36, 49, 59, 77, 78, 90, 95 and 125; (ii) SEQ ID NOs: 30, 36, 59, 77, 78 and 125; and (iii) SEQ ID NO: 77;
Spike glycoprotein (5) and nsp4 and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; and the CTL (neo)epitopes targeting nsp4 are selected from one of the groups consisting of (i) SEQ ID NOs: 8, 105 and 139; (ii) SEQ ID NOs: 8 and 139; and (iii) SEQ ID NO: 8;
Spike glycoprotein (5) and nsp6 and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; and the CTL (neo)epitopes targeting nsp6 are selected from one of the groups consisting of (i) SEQ ID NOs: 20, 42, 76, 83, 135 and 140; (ii) SEQ ID NOs: 42, 76, 83, 135 and 140; and (iii) SEQ ID NO: 42;
Spike glycoprotein (5) and nsp12 and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; and the CTL (neo)epitopes targeting nsp12 are selected from one of the groups consisting of (i) SEQ ID NOs: 32, 33 and 153; and (ii) SEQ ID NO: 32;
Spike glycoprotein (5) and nsp13, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; and the CTL (neo)epitopes targeting nsp13 are selected from one of the groups consisting of (i) SEQ ID NOs: 22 and 120; and (ii) SEQ ID NO: 22;
Spike glycoprotein (5) and nsp14 and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; and the CTL (neo)epitope targeting nsp14 is SEQ ID NO: 31;
Spike glycoprotein (5) and nsp16 and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; and the CTL (neo)epitope targeting nsp16 is SEQ ID NO: 52;
Nucleocapsid protein (N) and Membrane glycoprotein (M) and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL (neo)epitope targeting M is SEQ ID NO: 66;
Nucleocapsid protein (N) and Protein 3a and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL (neo)epitopes targeting Protein 3a are selected from one of the groups consisting of (i) SEQ ID NOs: 3, 97 and 101; and (ii) SEQ ID NO: 97;
Nucleocapsid protein (N) and nsp3 and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL (neo)epitopes targeting nsp3 are selected from one of the groups consisting of (i) SEQ ID NOs: 30, 36, 49, 59, 77, 78, 90, 95 and 125; (ii) SEQ ID NOs: 30, 36, 59, 77, 78 and 125; and (iii) SEQ ID NO: 77;
Nucleocapsid protein (N) and nsp4 and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL (neo)epitopes targeting nsp4 are selected from one of the groups consisting of (i) SEQ ID NOs: 8, 105 and 139; (ii) SEQ ID NOs: 8 and 139; and (iii) SEQ ID NO: 8;
Nucleocapsid protein (N) and nsp6 and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL (neo)epitopes targeting nsp6 are selected from one of the groups consisting of (i) SEQ ID NOs: 20, 42, 76, 83, 135 and 140; (ii) SEQ ID NOs: 42, 76, 83, 135 and 140; and (iii) SEQ ID NO: 42;
Nucleocapsid protein (N) and nsp12 and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL (neo)epitopes targeting nsp12 are selected from one of the groups consisting of (i) SEQ ID NOs: 32, 33 and 153; and (ii) SEQ ID NO: 32;
Nucleocapsid protein (N) and nsp13 and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL (neo)epitopes targeting nsp13 are selected from one of the groups consisting of (i) SEQ ID NOs: 22 and 120; and (ii) SEQ ID NO: 22;
Nucleocapsid protein (N) and nsp14 and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL epitope targeting nsp14 is SEQ ID NO: 31;
Nucleocapsid protein (N) and nsp16 and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL epitope targeting nsp16 is SEQ ID NO: 52;
Spike glycoprotein (5), Nucleocapsid protein (N) and Membrane glycoprotein (M), and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL (neo)epitope targeting M is SEQ ID NO: 66;
Spike glycoprotein (5), Nucleocapsid protein (N) and Protein 3a and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL (neo)epitopes targeting Protein 3a are selected from one of the groups consisting of (i) SEQ ID NOs: 3, 97 and 101; and (ii) SEQ ID NO: 97;
Spike glycoprotein (5), Nucleocapsid protein (N) and nsp3 and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL (neo)epitopes targeting nsp3 are selected from one of the groups consisting of (i) SEQ ID NOs: 30, 36, 49, 59, 77, 78, 90, 95 and 125; (ii) SEQ ID NOs: 30, 36, 59, 77, 78 and 125; and (iii) SEQ ID NO: 77;
Spike glycoprotein (5), Nucleocapsid protein (N) and nsp4 and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL (neo)epitopes targeting nsp4 are selected from one of the groups consisting of (i) SEQ ID NOs: 8, 105 and 139; (ii) SEQ ID NOs: 8 and 139; and (iii) SEQ ID NO: 8;
Spike glycoprotein (5), Nucleocapsid protein (N) and nsp6 and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL (neo)epitopes targeting nsp6 are selected from one of the groups consisting of (i) SEQ ID NOs: 20, 42, 76, 83, 135 and 140; (ii) SEQ ID NOs: 42, 76, 83, 135 and 140; and (iii) SEQ ID NO: 42;
Spike glycoprotein (5), Nucleocapsid protein (N) and nsp12 and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL (neo)epitopes targeting nsp12 are selected from one of the groups consisting of (i) SEQ ID NOs: 32, 33 and 153; and (ii) SEQ ID NO: 32;
Spike glycoprotein (5), Nucleocapsid protein (N) and nsp13 and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70, and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL (neo)epitopes targeting nsp13 are selected from one of the groups consisting of (i) SEQ ID NOs: 22 and 120; and (ii) SEQ ID NO: 22;
Spike glycoprotein (5), Nucleocapsid protein (N) and nsp14 and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL (neo)epitope targeting nsp14 is SEQ ID NO: 31;
Spike glycoprotein (5), Nucleocapsid protein (N) and nsp16 and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; and the CTL epitope targeting nsp16 is SEQ ID NO: 52;
Membrane glycoprotein (M) and Protein 3a and wherein the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting Protein 3a are selected from one of the groups consisting of (i) SEQ ID NOs: 3, 97 and 101; and (ii) SEQ ID NO: 97;
Membrane glycoprotein (M) and nsp3, and wherein the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp3 are selected from one of the groups consisting of (i) SEQ ID NOs: 30, 36, 49, 59, 77, 78, 90, 95 and 125; (ii) SEQ ID NOs: 30, 36, 59, 77, 78 and 125; and (iii) SEQ ID NO: 77;
Membrane glycoprotein (M) and nsp4 and wherein the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp4 are selected from one of the groups consisting of (i) SEQ ID NOs: 8, 105 and 139; (ii) SEQ ID NOs: 8 and 139; and (iii) SEQ ID NO: 8;
Membrane glycoprotein (M) and nsp6 and wherein the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp6 are selected from one of the groups consisting of (i) SEQ ID NOs: 20, 42, 76, 83, 135 and 140; (ii) SEQ ID NOs: 42, 76, 83, 135 and 140; and (iii) SEQ ID NO: 42;
Membrane glycoprotein (M) and nsp12 and wherein the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp12 are selected from one of the groups consisting of (i) SEQ ID NOs: 32, 33 and 153; and (ii) SEQ ID NO: 32;
Membrane glycoprotein (M) and nsp13 and wherein the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp13 are selected from one of the groups consisting of (i) SEQ ID NOs: 22 and 120; and (ii) SEQ ID NO: 22;
Membrane glycoprotein (M) and nsp14 and wherein the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitope targeting nsp14 is SEQ ID NO: 31;
Membrane glycoprotein (M) and nsp16 and wherein the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL epitope targeting nsp16 is SEQ ID NO: 52;
Spike glycoprotein (5), Membrane glycoprotein (M) and Protein 3a, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting Protein 3a are selected from one of the groups consisting of (i) SEQ ID NOs: 3, 97 and 101; and (ii) SEQ ID NO: 97;
Spike glycoprotein (5), Membrane glycoprotein (M) and nsp3, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp3 are selected from one of the groups consisting of (i) SEQ ID NOs: 30, 36, 49, 59, 77, 78, 90, 95 and 125; (ii) SEQ ID NOs: 30, 36, 59, 77, 78 and 125; and (iii) SEQ ID NO: 77;
Spike glycoprotein (5), Membrane glycoprotein (M) and nsp4, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp4 are selected from one of the groups consisting of (i) SEQ ID NOs: 8, 105 and 139; (ii) SEQ ID NOs: 8 and 139; and (iii) SEQ ID NO: 8;
Spike glycoprotein (5), Membrane glycoprotein (M) and nsp6, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp6 are selected from one of the groups consisting of (i) SEQ ID NOs: 20, 42, 76, 83, 135 and 140; (ii) SEQ ID NOs: 42, 76, 83, 135 and 140; and (iii) SEQ ID NO: 42;
Spike glycoprotein (5), Membrane glycoprotein (M) and nsp12, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp12 are selected from one of the groups consisting of (i) SEQ ID NOs: 32, 33 and 153; and (ii) SEQ ID NO: 32;
Spike glycoprotein (5), Membrane glycoprotein (M) and nsp13, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp13 are selected from one of the groups consisting of (i) SEQ ID NOs: 22 and 120; and (ii) SEQ ID NO: 22;
Spike glycoprotein (5), Membrane glycoprotein (M) and nsp14, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL epitope targeting nsp14 is SEQ ID NO: 31;
Spike glycoprotein (5), Membrane glycoprotein (M) and nsp16, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL epitope targeting nsp16 is SEQ ID NO: 52;
Nucleocapsid protein (N), Membrane glycoprotein (M) and Protein 3a, and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting Protein 3a are selected from one of the groups consisting of (i) SEQ ID NOs: 3, 97 and 101; and (ii) SEQ ID NO: 97;
Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp3, and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp3 are selected from one of the groups consisting of (i) SEQ ID NOs: 30, 36, 49, 59, 77, 78, 90, 95 and 125; (ii) SEQ ID NOs: 30, 36, 59, 77, 78 and 125; and (iii) SEQ ID NO: 77;
Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp4, and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp4 are selected from one of the groups consisting of (i) SEQ ID NOs: 8, 105 and 139; (ii) SEQ ID NOs: 8 and 139; and (iii) SEQ ID NO: 8;
Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp6, and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp6 are selected from one of the groups consisting of (i) SEQ ID NOs: 20, 42, 76, 83, 135 and 140; (ii) SEQ ID NOs: 42, 76, 83, 135 and 140; and (iii) SEQ ID NO: 42;
Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp12, and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp12 are selected from one of the groups consisting of (i) SEQ ID NOs: 32, 33 and 153; and (ii) SEQ ID NO: 32;
Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp13, and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp13 are selected from one of the groups consisting of (i) SEQ ID NOs: 22 and 120; and (ii) SEQ ID NO: 22;
Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp14, and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL epitope targeting nsp14 is SEQ ID NO: 31;
Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp16, and wherein the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL epitope targeting nsp16 is SEQ ID NO: 52;
Spike glycoprotein (5), Nucleocapsid protein (N), Membrane glycoprotein (M) and Protein 3a, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting Protein 3a are selected from one of the groups consisting of (i) SEQ ID NOs: 3, 97 and 101; and (ii) SEQ ID NO: 97;
Spike glycoprotein (5), Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp3, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp3 are selected from one of the groups consisting of (i) SEQ ID NOs: 30, 36, 49, 59, 77, 78, 90, 95 and 125; (ii) SEQ ID NOs: 30, 36, 59, 77, 78 and 125; and (iii) SEQ ID NO: 77;
Spike glycoprotein (5), Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp4, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp4 are selected from one of the groups consisting of (i) SEQ ID NOs: 8, 105 and 139; (ii) SEQ ID NOs: 8 and 139; and (iii) SEQ ID NO: 8;
Spike glycoprotein (5), Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp6, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp6 are selected from one of the groups consisting of (i) SEQ ID NOs: 20, 42, 76, 83, 135 and 140; (ii) SEQ ID NOs: 42, 76, 83, 135 and 140; and (iii) SEQ ID NO: 42;
Spike glycoprotein (5), Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp12, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp12 are selected from one of the groups consisting of (i) SEQ ID NOs: 32, 33 and 153; and (ii) SEQ ID NO: 32;
Spike glycoprotein (5), Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp13, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting nsp13 are selected from one of the groups consisting of (i) SEQ ID NOs: 22 and 120; and (ii) SEQ ID NO: 22;
Spike glycoprotein (5), Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp14, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL epitope targeting nsp14 is SEQ ID NO: 31; and
Spike glycoprotein (5), Nucleocapsid protein (N), Membrane glycoprotein (M) and nsp16, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL (neo)epitope targeting M is SEQ ID NO: 66; and the CTL epitope targeting nsp16 is SEQ ID NO: 52.
In a particular aspect, the vaccine composition comprises CTL (neo)epitopes targeting at least 5, 6, 7, 8, 9, 10, or 11 proteins of SARS-CoV selected in the group consisting of Spike glycoprotein (5), Nucleocapsid protein (N), Membrane glycoprotein (M), Protein 3a, nsp3, nsp4, nsp6, nsp12, nsp13, nsp14 and nsp16; and for each proteins the CTL epitopes are selected in the following groups:
Optionally, for each targeted protein, the vaccine composition comprises at least one CTL epitope and at least one CTL neoepitope. Optionally, for each targeted protein, the vaccine composition comprises at least one CTL epitope and at least two CTL neoepitopes. Optionally, for each targeted protein, the vaccine composition comprises at least two CTL neoepitopes. Optionally, for each targeted protein, the vaccine composition independently for the different targeted proteins comprises:
Optionally, for each targeted protein, the vaccine composition independently for the different targeted proteins comprises:
Optionally, for each targeted protein, the vaccine composition independently for the different targeted proteins comprises:
Optionally, for each targeted protein, the vaccine composition independently for the different targeted proteins comprises:
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting Spike glycoprotein (S) and the vaccine composition comprises:
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting Spike glycoprotein (S) and the vaccine composition comprises:
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting Spike glycoprotein (S) and the vaccine composition comprises:
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting Spike glycoprotein (S) and the vaccine composition comprises:
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting Nucleocapsid protein (N) and the vaccine composition comprises:
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting Nucleocapsid protein (N) and the vaccine composition comprises:
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting Nucleocapsid protein (N) and the vaccine composition comprises one CTL epitope targeting Nucleocapsid protein (N) of SEQ ID NO: 23 and one CTL neoepitope targeting Nucleocapsid protein (N) of SEQ ID NO:79.
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting Membrane glycoprotein (M) and the vaccine composition comprises:
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting Membrane glycoprotein (M) and the vaccine composition comprises one CTL neoepitope targeting Membrane glycoprotein (M) of SEQ ID NO: 66.
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting at least one ORF selected from the group consisting of Protein 3a, nsp4, nsp3, nsp6, nsp12, nsp13, nsp14 and nsp16, and the vaccine composition comprises:
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting at least one ORF selected from the group consisting of Protein 3a, nsp4, nsp3, nsp6, nsp12, nsp13, nsp14 and nsp16, and the vaccine composition comprises:
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting at least one ORF selected from the group consisting of Protein 3a, nsp4, nsp3, nsp6, nsp12, nsp13, nsp14 and nsp16, and the vaccine composition comprises:
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting at least one ORF selected from the group consisting of Protein 3a, nsp4, nsp3, nsp6, nsp12, nsp13, nsp14 and nsp16, and the vaccine composition comprises:
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting Spike glycoprotein (S) and Nucleocapsid protein (N) and the vaccine composition comprises:
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting Spike glycoprotein (S) and Nucleocapsid protein (N) and the vaccine composition comprises:
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting Spike glycoprotein (S) and Membrane glycoprotein (M) and the vaccine composition comprises:
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting Spike glycoprotein (S) and Membrane glycoprotein (M) and the vaccine composition comprises:
Optionally, the vaccine composition comprises CTL epitopes/neoepitopes targeting Nucleocapsid protein (N) and Membrane glycoprotein (M) and the vaccine composition comprises:
Optionally, the CTL neoepitope targeting Membrane glycoprotein (M) of SEQ ID NO: 66. Optionally, the CTL epitope targeting Nucleocapsid protein (N) consists of SEQ ID NO: 23 and the CTL neoepitope targeting Nucleocapsid protein (N) are selected from the group consisting of SEQ ID NOs: 67, 75, 79, 85, and 113.
In a particular aspect, the vaccine composition comprises at least 1, 2, 3, 4, 5 or 6 CTL (neo)epitopes selected in one of the groups consisting of (i) SEQ ID NOs: 20, 23, 32, 36, 42, 56, 59, 60, 76, 79, 85, 91, 95, 97, 125, 140 and 146; (ii) SEQ ID NOs: 23, 32, 36, 42, 56, 59, 60, 76, 79, 91, 97, 125, 140 and 146; and (iii) SEQ ID NOs: 23, 32, 42, 97 and 146.
In a very particular aspect, the vaccine composition comprises at least 5, 6, 7, 8, 9, 10, 11, or 12 CTL (neo)epitopes selected from SEQ ID NOs: 8, 22, 23, 31, 32, 42, 52, 66, 70, 77, 97 and 146.
In another very particular aspect, the vaccine composition comprises CTL (neo)epitopes of SEQ ID NOs: 70 and/or 146; 23, and 66, and at least 2, 3, 4, 5, 6, 7 or 8 CTL (neo)epitopes selected from SEQ ID NOs: 8, 22, 31, 32, 42, 52, 77 and 97.
In a very specific aspect, the vaccine composition comprises CTL (neo)epitopes of SEQ ID NOs: 8, 22, 23, 31, 32, 42, 52, 66, 70, 77, 97 and 146. More particularly, the vaccine composition may comprise CTL (neo)epitopes of SEQ ID NOs: 8, 22, 23, 31, 32, 42, 52, 66, 70, 77, 97 and 146 and a T helper peptide, especially PADRE (aKXVAAWTLKAAa with X and a respectively indicating cyclohexylalanine and d-alanine).
For instance, the vaccine composition comprises or consists of one of the following CTL (neo)epitopes:
In a particular aspect, the vaccine composition comprises at least one (neo)epitope inducing a virus-specific tissue-resident memory T lymphocytes (Trm). Accordingly, the vaccine composition preferably comprises at least 1, 2, 3, 4, 5 or 6 CTL (neo)epitopes selected in one of the groups consisting of (i) SEQ ID NOs: 20, 23, 32, 36, 42, 56, 59, 60, 76, 79, 85, 91, 95, 97, 125, 140 and 146; (ii) SEQ ID NOs: 23, 32, 36, 42, 56, 59, 60, 76, 79, 91, 97, 125, 140 and 146; and (iii) SEQ ID NOs: 23, 32, 42, 97 and 146.
The addition of a specific HTL epitope as a provider of CD4+ T Helper Lymphocytes cells is an important factor in the combination of (neo)epitopes with T Helper support for the global immune response, both in the B cell early responses and in the T cell Long term specific memory responses.
Considering COVID 19, it has been reported that CD4+ T cell responses correlated with positive outcomes (Braun et al, medRxiv 2020 Presence of SARS-CoV-2-reactive T cells in COVID-19 patients and healthy donors).
The HTL epitopes are either natural or synthetic T cells helper peptides know in the art. Natural helper peptides are for instance a Natural Tetanus sequence alone or linked to another epitope, or a Plasmodium falciparum sequence alone or linked to another epitope.
In a particular aspect, the HTL peptide may comprise a synthetic peptide such as a Pan-DR-binding epitope (e.g., a PADRE® peptide, Epimmune Inc., San Diego, Calif., described, for example, in U.S. Pat. No. 5,736,142), for example, having the formula aKXVAAZTLKAAa, where “X” is either cyclohexylalanine, phenylalanine, or tyrosine; “Z” is either tryptophan, tyrosine, histidine or asparagine; and “a” is either D-alanine or L-alanine (SEQ ID NO: 746). Certain pan-DR binding epitopes comprise all “L” natural amino acid residues; these molecules can be provided as peptides or in the form of nucleic acids that encode the peptide. See also, U.S. Pat. Nos. 5,679,640 and 6,413,935.
The vaccine composition may comprise adjuvants. The adjuvant is preferably an oily adjuvant, which comprises both a hydrocarbon oil and a water-in-oil emulsifier. Such adjuvants act by the so-called “deposition effect”. The hydrocarbon oil may be paraffin oil, a vegetable oil, squalene, squalane or mineral oil, for instance. Suitable W/O emulsifiers may be selected from mannide mono-oleate and sorbitan mono-oleate, for instance. Examples of appropriate oily adjuvants are a mixture of 5-20% mannide mono-oleate with 80-95% mineral oil (Montanide® ISA 51 sold by SEPPIC) or squalene (Montanide® ISA 720 sold by SEPPIC) and similar mixtures. In a specific embodiment, the adjuvant is a mixture of mineral oil and mannide mono-oleate, especially Montanide® ISA 51.
In a particular aspect, the vaccine composition is an emulsion with a mineral oil adjuvant.
The adjuvant used in this invention may alternatively, or in addition to the above oily adjuvants, be selected from micro- and nanoparticles, such as liposomes and microspheres, of PLG, PLA, PLGA or other natural polymers such as gelatin, collagen and chitosan. Other adjuvants may comprise TLR ligands, Toll-like receptor ligands (TLR3 and TLR9), stimulators of IFN genes (STING) agonists, cytokines such as GM-CSF and IL2, carbohydrates, bacterial derivatives, mineral salts and immune stimulating complexes (ISCOM).
Optionally, the vaccine composition may comprise aluminum salts, such as aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate.
The vaccine compositions are intended for parenteral, topical, oral, intrathecal, or local administration. Preferably, the vaccine compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. More preferably, the vaccine composition is intended for subcutaneous administration or intramuscular administration. Optionally, the vaccine composition is intended for nasal administration.
Optionally, each peptide of the composition is present at a concentration of 0.01 mg/ml to 1 g/ml, 0.1 mg/ml to 10 mg/ml. For instance, each peptide can be present at a concentration of 0.5 mg/ml.
In a particular aspect, the vaccine composition is to be administered once, twice or more. For instance, two administrations can be carried out. A prime administration followed by a boost administration. For instance, the injections can be spaced by 3 weeks or 2 weeks and will be adapted to the Immune response requested and to the medical conditions of the subject to be treated.
The present invention relates to a composition of the present invention for use for preventing or treating an infection by a severe acute respiratory syndrome-related coronavirus (SARS-CoV), the use of a composition of the present invention for the manufacture of a vaccine for preventing or treating an infection by a severe acute respiratory syndrome-related coronavirus (SARS-CoV), and to a method for preventing or treating an infection by a severe acute respiratory syndrome-related coronavirus (SARS-CoV) in a subject, comprising the administration of an effective amount of a composition of the present invention.
Optionally, the SARS-CoV is selected from the group consisting of SARS-CoV1, SARS-CoV2 or MERS-CoV virus. Optionally, the SARS-CoV is SARS-CoV1. Optionally, the SARS-CoV is SARS-CoV2. Optionally, the SARS-CoV is MERS-CoV virus.
The present invention relates to a composition of the present invention for use for preventing or treating Covid-19, the use of a composition of the present invention for the manufacture of a vaccine for preventing or treating Covid-19, and to a method for preventing or treating an infection by Covid-19 in a subject, comprising the administration of an effective amount of a composition of the present invention.
Optionally, the subject to be treated is a subject aged 65 years or older, a subject having a cancer or having had a cancer, a subject being obese (In particular with severe obesity (body mass index [BMI] of 40 or higher [CDC-HCSP BMI>30]), a subject being diabetic, a subject having a hypertension, a subject having a sarcoidosis, a subject being immunocompromised, a subject who lives in a nursing home or long-term care facility, a subject with chronic lung disease or moderate to severe asthma, lung fibrosis, a subject who has serious heart conditions, a subject with chronic kidney disease undergoing dialysis and/or a subject with liver diseases. Optionally, the subject can be a subject with a stable comorbidity factor, for instance, stable cancer patients, chronic obstructive pulmonary disease (COPD) patients, stable patients with comorbidity as Obesity or renal dialysis (10 volunteers planned by group of comorbidity).
Many conditions can cause a person to be immunocompromised (including cancer treatment, smoking, bone marrow or organ transplantation, immune deficiencies, poorly controlled HIV or AIDS, prolonged use of corticosteroids and other immune weakening medications).
The patient could be selected on HLA typing. In one aspect, the subject to be treated has one of the HLA typing disclosed in any of the Tables 1-3. In a very specific aspect, the subject is HLA-A2.
However, the vaccine composition comprises a combination of peptides allowing the treatment of subjects having a broad diversity of HLA, then being suitable for the treatment of the worldwide population.
A “diluent” includes sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred diluent for pharmaceutical compositions. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as diluents, particularly for injectable solutions.
An “epitope” is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, T cell receptor or HLA molecule. Alternatively, an epitope can be defined as a set of amino acid. residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor proteins and/or Major Histocompatibility Complex (MHC) receptors. Epitopes are present in nature, and can be isolated, purified or otherwise prepared or derived by humans. For example, epitopes can be prepared by isolation from a natural source, or they can be synthesized in accordance with standard protocols in the art. Synthetic epitopes can comprise artificial amino acid residues, “amino acid mimetics,” such as D isomers of naturally-occurring L amino acid residues or non-naturally-occurring amino acid residues such as cyclohexylalanine. Throughout this disclosure, epitopes may be referred to in some cases as peptides or peptide epitopes.
“Human Leukocyte Antigen” or “HLA” is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al., IMMUNOLOGY, 8TH ED., Lange Publishing, Los Altos, Calif. (1994).
An “HLA supertype or HLA family”, as used herein, describes sets of HLA molecules grouped on the basis of shared peptide-binding specificities. HLA class I molecules that share somewhat similar binding affinity for peptides bearing certain amino acid motifs are grouped into such HLA supertypes. The terms HLA superfamily, HLA supertype family, HLA family, and HLA xx-like molecules (where “xx” denotes a particular HLA type), are synonyms.
“Major Histocompatibility Complex” or “MHC” is a cluster of genes that plays a role in control of the cellular interactions responsible for physiologic immune responses. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. For a detailed description of the MHC and HLA complexes, see, Paul, FUNDAMENTAL IMMUNOLOGY, 3RD ED., Raven Press, New York (1993).
A “native” or a “wild type” sequence refers to a sequence found in nature. Such a sequence may comprise a longer sequence in nature.
The terms “peptide”, “epitope” and “peptide epitope” are used interchangeably with “oligopeptide” in the present specification to designate a series of residues, typically L-amino acid residues, connected one to the other, typically by peptide bonds between the α-amino and carboxyl groups of adjacent amino acid residues.
Optionally, a “peptide”, “epitope” and “peptide epitope” defined by a SEQ ID NO can consist in the particular SEQ ID NO and can also refer to a peptide consisting in the particular SEQ ID NO but including 1 or 2 additional amino acids at the N and/or C terminal end of the SEQ ID NO.
Optionally, one or several “peptide”, “epitope” and “peptide epitope” can be fused together in a same polypeptide.
A “PanDR binding” peptide, a “PanDR binding epitope,” or “PADRE®” peptide (Epimmune, San Diego, Calif.) is a member of a family of molecules that binds more than one HLA class II DR molecule. The pattern that defines the PADRE® family of molecules can be referred to as an HLA Class II supermotif. A PADRE® molecule binds to HLA-DR molecules and stimulates in vitro and in vivo human helper T lymphocyte (HTL) responses. For a further definition of the PADRE® family, see copending application U.S. Ser. No. 09/709,774, filed Nov. 11, 2000; and Ser. No. 09/707,738, filed Nov. 6, 2000; PCT publication Nos WO 95/07707, and WO 97/26784; U.S. Pat. No. 5,736,142 issued Apr. 7, 1998; U.S. Pat. No. 5,679,640, issued Oct. 21, 1997; and U.S. Pat. No. 6,413,935, issued Jul. 2, 2002.
“Pharmaceutically acceptable” refers to a generally non-toxic, inert, and/or physiologically compatible composition or component of a composition.
A “pharmaceutical excipient” or “excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like. A “pharmaceutical excipient” is an excipient which is pharmaceutically acceptable.
A “protective immune response” or “therapeutic immune response” refers to a BCL, CTL and/or an HTL response to an antigen derived from a pathogenic antigen (e.g., an antigen from an infectious agent or a tumor antigen), which in some way prevents or at least partially arrests disease symptoms, side effects or progression. The immune response may also include an antibody response which has been facilitated by the stimulation of helper T cells.
As used herein, a “vaccine” is a composition used for vaccination, e.g., for prophylaxis or therapy, that comprises one or more peptides of the invention. There are numerous embodiments of vaccines in accordance with the invention, such as by a cocktail of one or more peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide. The “one or more peptides” can include any whole unit integer from 1-50, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 peptides of the invention. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class I-binding peptides of the invention can be linked or to otherwise be combined with HLA class II-binding peptides, e.g., a PADRE® universal HTL-binding peptide, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. Vaccines can comprise peptide pulsed antigen presenting cells, e.g., dendritic cells.
The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) which enters the body principally through the nasal and larynx mucosa and progress to the lungs through the respiratory tract. SARS-CoV-2 replicates efficiently in respiratory epithelial cells motivating the development of alternative and rapidly scalable vaccine inducing mucosal protective and long-lasting immunity. Here, the inventors present a multi-target CD8 T cell peptide COVID-19 vaccine design targeting several structural (S, M, N) and non-structural (NSPs) SARS-CoV-2 proteins with selected epitopes in conserved regions on the SARS-CoV-2 genome. They observe that a single subcutaneous injection of several peptides induces robust immunogenicity measured by IFNγ ELIspot. Upon tetramer characterization, they found that a series of epitopes induce a strong proportion of virus-specific CD8 T cells expressing CD103, CD44, CXCR3 and CD49a, the specific phenotype of tissue-resident memory T lymphocytes (Trm). Finally, they observe broad cellular responses, as characterized by IFNγ production, upon restimulation with structural and non-structural protein-derived epitopes using blood T cells isolated from convalescent asymptomatic, moderate and severe COVID-19 patients.
Humoral and cellular adaptive immunity are different and complementary immune defenses engaged by the body to clear viral infection. While neutralizing antibodies have the capacity to block virus binding to its entry receptor expressed on human cells, memory T lymphocytes have the capacity to eliminate infected cells and are required for viral clearance. However, viruses evolve quickly, and their antigens are prone to mutations to avoid recognition by the antibodies (phenomenon named ‘antigenic drift’). This limitation of the antibody-mediated immunity could be addressed by the T-cell mediated immunity, which is able to recognize conserved viral peptides from any viral proteins presented by virus-infected cells. Thus, by targeting several proteins and conserved regions on the genome of a virus, T-cell epitope-based vaccines are less subjected to mutations and may work effectively on different strains of the virus. We design a multi-target T cell-based vaccine containing epitope regions optimized for CD8+ T cell stimulation that would drive long-lasting cellular immunity with high specificity, avoiding undesired effects such as antibody-dependent enhancement (ADE) and antibody-induced macrophages hyperinflammation observed in the COVID-19. The present results showed that a single injection of selected CD8 T cell epitopes induces memory viral-specific T-cell responses with a phenotype of tissue-resident memory T cells (Trm). Trm has attracted a growing interest for developing vaccination strategies since they act as immune sentinels in barrier tissue such as the respiratory tract and the lung. Because of their localization in tissues, they are able to immediately recognize infected cells and, because of their memory phenotype, to rapidly respond to viral infection by orchestrating local protective immune responses to eliminate pathogens. Lastly, such multiepitope-based vaccination platform uses robust and well-validated synthetic peptide production technologies that can be rapidly manufactured in a distributed manner.
Previous research in SARS-CoV-1 suggests that the structural Spike (S) protein is one of the main antigenic components responsible for inducing the host immune responses. However recent evaluation of asymptomatic, moderate and severe convalescent COVID-19 patients showed broad and robust T-cell responses not only on Spike but also on membrane (M), nucleocapsid (N) proteins and several ORFs non-structural proteins (nsp). Deep sequencing data of tens of thousands SARS-CoV-2 genomes from all over the world identified regions that have remained largely invariant to date, and others that have already accumulated significant diversity with several hundreds of point mutations (SNPs) in some key viral proteins, such as the Spike glycoprotein which also displayed a large number of recurrent mutations and several homoplasic site highlighting a possible convergent evolution and adaptation of SARS-CoV-2 to the human host. COVID-19 vaccines reliance on a single antigen (Spike) bearing recurrent mutations and homoplasic sites, as occurring with Spike monovalent vaccines under development, is not without risk of antigen drift, selection pressure and immune evasion.
Using bioinformatics approaches of immune deconvolution to identify T cell epitopes intersected with predicted SARS-CoV-2 T-cell epitopes reported in early manuscripts, population HLA diversity, protein sequence similarity and immunogenicity observed with previous CoVs homologues and the number of copies per virion of SARS-COV-1 (estimated to approximatively 100 copies for S, 1 000 for M, 2 000 for N and only 20 for E proteins), the inventors identified and selected 55 HLA-A2-restricted T-cell epitopes (9-10 mer peptides) derived from 11 of the 29 SARS-CoV-2 proteins in human cells: 3 out of the 4 structural proteins (S, M, N), the largest accessory proteins (ORF3a) and 7 out of the 16 ORF1a/b non-structural proteins (nsp3, nsp4, nsp6, nsp12, nsp13, nsp14, nsp16) (
CD8 T-Cell Epitopes Elicit Tissue-Resident Memory (Trm) Viral-Specific T Cells In-Vivo 134 WT and mutated peptides (neo-epitopes A and B) were produced using synthetic peptide synthesis (Proteogenix, France). HLA-A2 binding property characterization at 37° C., using UV peptide exchange assay on H LA-A*0201 monomer, showed that the majority of selected WT epitopes binds to HLA-A2 with good efficacy (
60 peptides (at least one WT or mutated peptides for each T-cell epitopes outside homoplasic site) has been selected based on HLA-A2 binding, peptide stability and SARS-CoV-2 genome stability for further in-vivo immunogenicity evaluation in HLA-A2.1 transgenic mice. Mice received a single subcutaneous injection of each peptide combined with the universal PADRE helper T-cell epitope and emulsified in Montanide ISA-51 adjuvant. Immunogenicity was assessed in the spleen and draining lymph nodes 11 days after vaccination by ex-vivo restimulation and tetramer phenotypic characterization with the corresponding WT peptide to evaluate cross-reactivity of elicited T cell response towards WT epitopes. CD8 T cells IFNγ ELIspot restimulation showed large immunogenicity in-vivo responses elicited by single vaccination with 14 out of 60 (23%) positive CD8 T cells epitopes derived from 8 out of 11 selected proteins (
In order to identify and select naturally SARS-CoV-2 CD8 T cell immunodominant epitopes, peripheral blood mononuclear cells (PBMC) from asymptomatic and moderate or severe COVID-19 patients with a previously confirmed (at least one month before sampling) and recovered SARS-CoV-2 infection were restimulated ex-vivo for one week with each of the isolated selected 60 peptides derived from 11 proteins. IFNγ responses over 48 hours of restimulation with HLA-A2+ Tap-deficient (T2) human cells were analyzed and compare to PBMC of unexposed healthy donors. The serology of all unexposed healthy donors was negative while all asymptomatic, moderate and severe COVID-19 individuals were IgG+ for anti-Spike antibodies. As recently reported for CD4+ T cells response, the inventors first observed some positive IFNγ response in unexposed donors to few CD8 T cells epitopes, in particular for those derived from structural proteins (Spike and N proteins but no response to the Spike_RBD or M protein) while CD8 T cell responses to non-structural proteins were limited to nsp12 and nsp13 (
Altogether, the inventors identified in previously infected SARS-CoV-2 individuals 22 significantly different CD8 T cell immunodominant epitopes against 3 structural proteins (S, M, N), 1 accessory factor (ORF3a) and 7 non-structural proteins (nsp3, nsp4, nsp6, nsp12, nsp13, nsp14, nsp16) as compared to unexposed healthy donors. 18 of these epitopes are of particular interest for vaccination since able to elicit also in-vivo CD8 T cell immunogenicity against all 11 structural and non-structural SARS-CoV-2 proteins after a single peptide injection in HLA-A2 expressing mice. The inventors selected a combination of 12 CD8 T cells epitopes based on manufacturing, HLA-I coverage, previous CoVs homology and SARS-CoV-2 proteins diversity considerations (Table 9). These 12 epitopes covered the 11 selected proteins, 1 epitope/protein excepting Spike for which 2 epitopes (including 1 RBD epitope) have been selected. Bioinformatic analyses illustrate these 12 epitopes are not restricted to HLA-A*0201 allele, hence are predict (netMHC score <1) to bind efficiently to different HLA-I (A, B, C) alleles with high genetic coverage in all geographical region of the world. Despite HLA polymorphism and different worldwide HLA-I distribution, the combination of these 12 T cell epitopes should induce at least 1 to 3 positive peptides responses in all individuals globally and achieve the 60-70% ‘herd immunity’ threshold with at least 3 to 7 positive peptides responses in in each geographical region (Table 10).
Animal housing and procedures have been conducted according to the guidelines of the French Agriculture Ministry and were approved by the regional ethical committee (APAFIS 25256.) as well as according to the guidelines of Jackson Laboratory (Bar Harbor, USA) and approved by the Institutional Animal Care and Use Committee (IACUC #20031). Human studies were performed under the clinical protocol COVEPIT-1 approved by French Central Ethic Committee (CPP) and registered by the French Regulatory Authority (ANSM) under the ID-RCB no 2020-A01654-35. Written informed consent has been obtained from each of the participating subject.
T-cell WT and mutated peptides binding property on HLA-A2 has been evaluated using the Flex-T HLA-A*02:01 monomer ultraviolet (UV) exchange assay according to the manufacturer recommendation (Biolegend, San Diego, USA). HLA-A*02:01 monomer (200 μg/ml) were exposed to a 366-nm UV lamp in the presence or absence of 400 μM of peptide. After UV-exposure, HLA-peptide complexes were incubated at 37° C. for 30 min to promote unfolding of peptide-free HLA molecule. HLA-peptide complexes stability was detected by ELISA with 132-microglobulin coated antibodies and incubation of 3 ng/ml of complexes for 1 h at room temperature under shaking condition. Avidin-HRP were used to reveal stable biotinylated HLA-peptide complexes and absorbance was monitored at 450 nm. Data are expressed as percentage of binding relative to an MEMOPI® internal positive control neoepitope. MEMOPI® internal positive control neoepitope is a mixture of MPS-216 (SEQ ID NO: 171) and MPS-102 (SEQ ID NO: 172).
A visualization tool was used to determine T-cell and B-cell epitope location in SARS-CoV-2 genomes according to single nucleotide polymorphism (SNPs) and homoplasic site (https://macman123.shinyapps.io/ugi-scov2-alignment-screen/). 23,085 SARS-CoV-2 genomes isolated from patients worldwide were aligned against the Wuhan-Hu-1 reference genome NC_045512.2. A total of 8,667 SNPs has been identified corresponding to 308 homoplasic sites with recurrent mutations. Peptides have been blasted with tblastn algorithm against the Wuhan-Hu-1 reference genome NC_045512.2 to determine the nucleotide coordinates for each peptide. The online tool was then used to identify the peptides corresponding to a homoplasic site.
B6.Cg-Immp2ITg(HLA-A/H2-D)2Enge/J (HLA-A2.1) transgenic mice (The Jackson Laboratory, Bar Harbor, USA) received a single subcutaneous injection of 6 SARS-CoV-2 peptides (50 μg each, WT and mutated peptide of a same epitope have not been evaluated in same mice) plus the universal PADRE helper T-cell epitope emulsified in Montanide ISA-51 adjuvant. Immunization was measured 11 days after injection. 3 males and 3 females have been evaluated per group. Freshly harvested spleen and draining lymph nodes have been pooled by sex per group and analyzed by flow cytometry analyses. CD8+ T cells have been isolated using MACS microbeads and restimulated individually with each evaluated peptide. The frequency of IFNγ-secreting CD8+ T cells was measured by ELIspot in parallel of tetramer staining for each peptide evaluated by flow cytometry. Control Memopi® peptides are a mixture of MPS-216 (SEQ ID NO: 171), MPS-102 (SEQ ID NO: 172), MPS-112 (SEQ ID NO: 173), MPS-106 (SEQ ID NO: 174), MPS-213 (SEQ ID NO: 175), and MPS-103 (SEQ ID NO: 176) plus the universal PADRE helper T-cell epitope emulsified in Montanide ISA-51 adjuvant.
All subjects were enrolled in the COVEPIT-1 clinical trial, an open-label, multicentric and prospective study with minimal risk and constraints designed to assess the memory T cell in subjects who recovered from COVID-19. Subjects were enrolled at the Groupement Santé du Bataillon des Marins-Pompiers (Marseille-France) and GHR MSA—Hôpital Emile Muller (Mulhouse, France). The main objective was to test the subject' memory T cells reactivity to a selection of SARS-CoV-2 antigens. Enrolled subjects must have a proven COVID-19 infection which recovered 1 to 6 months before study entry. Eligible subjects are male and female of 18 to 70 years old diagnosed for COVID-19 using a PCR test from a nasal and/or oropharyngeal swab, and/or a serological test, and/or a chest CT with lesions suggestive of COVID-19. Subjects were excluded if pregnant or breastfeeding, unable to fulfill the protocol requirement, with an history of cancer 5 years prior to study entry (except for localized or in situ cancer), history of head injury or sepsis 1 year prior to study entry, chronic infections (e.g. HIV infection, chronic hepatitis B, active viral hepatitis C or bacterial or fungal infection) requiring a systemic treatment in the month prior to COVID-19, disease (auto immune or inflammatory disease, transplant recipients . . . ) requiring a immunosuppressive or immunomodulator treatment and/or, corticosteroids at an equivalent dose of prednisone >10 mg/d for more than 15 days or >40 mg/d for the last 15 days prior to COVID-19; corticosteroid during COVID-19 were not considered as an exclusion criterion.
Ex-Vivo PBMC Restimulation with SARS-CoV-2 Peptide
PBMC were isolated after a Ficoll density-gradient centrifugation and a red blood cell lysis. HLA-A2 phenotyping was performed by flow cytometry (clone BB7.2, BD Bioscience). Ex-vivo stimulation protocol was adapted from a previously described protocol (Mitra, A. et al. Nature Communications 11, 1839 (2020)). HLA-A2+ positive PBMC (106/well) were incubated in RPMI 1640 containing 10 mM HEPES, 2 mM L-glutamine, 1 mM Sodium Pyruvate, 2% human AB serum, 10% bovine serum and non-essential amino acids in 48-well plates. During the first week of culture, PBMC were cultured with 3 μg/mL of each isolated peptides and IL-21 (30 ng/mL; Miltenyi, Paris France). Fresh medium containing IL-21 (30 ng/mL), IL-7 (5 ng/mL; BioRad, Paris France), and IL-2 (10 ng/mL; Miltenyi, Paris France) and peptide-loaded HLA-A2+ Tap-deficient (T2) cells were added to the culture for the next two days. Ex vivo T-cell viral stimulation was evaluated by IFNΥ supernatant quantification (BD Biosciences, US). The percentage of background IFNΥ secretion was determined by the response of PBMC co-cultured with non-loaded T2 cells and negative control peptide, then fold change was calculated over the IFNΥ secretion background for each donor.
Continuous variables were expressed as the mean±SEM, unless otherwise indicated, and raw data were compared with nonparametric tests: Mann-Whitney for 2 groups or Kruskall-Wallis with Dunn's comparison when the number of groups was >2. P values of <0.05 were considered statistically significant. All statistical analyses were performed on GraphPad Software (GraphPad Software, San Diego, Calif.).
CoVepit is the combination of 13 following peptides
aKXVAAWTLKAAa, a Pan DR HTL epitope (SEQ ID NO: 170)
12 CTL epitope HLA restricted epitopes:
A) CTL Immunogenicity Evaluation after CoVepiT Immunization in Different HLA-A2+ Transgenic Mouse Strains
CoVepiT immunogenicity was evaluated in different strains of HLA transgenic mice (
Different HLA-A2 Strain were Tested:
(Group 1) B6.Cg-Immp2ITg (HLA-A2/H2D) 2Enge/J mice mice. (n=16, age 6-7 weeks); 8 mice were immunized (4 males, 4 females) and 8 naïve mice were used as negative control (4 males, 4 females).
(Group 2) HLA-A2/HLA-DR1+ double transgenic mice (strain from Pasteur institute, Pascolo S, Lemonnier FA, JEM 1997) (n=39 mice age 10-48 weeks), 27 mice were immunized and 12 naïve mice were used as negative control. 3 mice were pooled for each IFN-γ analysis.
(Group 3) HLA-A2/H2kB transgenic mice (CB6F1-Tg(HLA-A*0201/H2-Kb)A*0201; Taconic stock #9659) (n=7 mice age 5-6 weeks, males), 4 mice were immunized and 3 naïve mice were used as negative control).
T cell effector responses was determined by measuring IFN-γ production by CD8+ T cells compared to naïve mice (non-vaccinated mice) after in vitro restimulation for 24 hours with the pool of 12 corresponding wild-type peptides. As shown on
Because PADRE binds with high affinity to human HLA-DR types, and with moderate affinity to mouse I-Ab/d MHC haplotypes, the use of HLA-A2 and HLA-DR1 double transgenic mice allow to study the impact of PADRE in a more relevant model for CD4+ T cell responses which indirectly impact the quality of CD8+ T cell responses. Data show that CoVepiT immunization in HLA-A2/DR1 mice is highly effective in this specific HLA-A2/DR1+ double transgenic model mimicking the human situation (HLA ABC and HLA DR).
Cytotoxic T lymphocytes (CTLs) are controlling intracellular pathogens by recognizing and clearing infected viral target cells. Experimental methods were implemented, to estimate the CTL's efficacy in detecting granzyme B protease inducing target cell death and direct killing assay.
To demonstrate that specific CD8 T cells generated after immunization by CoVepiT achieve cytotoxic activity against SARS-COV2 infected cells, Granzyme B secretion and cytolytic activity of CD8+ T cells were measured after in vitro restimulation with SARS-COV2 peptides-presenting HLA-A2+ human cells.
Granzyme B is established as a caspase-like serine protease that is released by cytotoxic lymphocytes to kill virus-infected cells.
For this study, HLA-A2/H2 KB transgenic mice (n=7, 7-28 weeks old males) were immunized with 12 CTL peptides of CoVepiT+pan-HLA-DR PADRE peptide emulsified in Montanide ISA 51 (50 μg of each peptide+25 μg of HTL peptide). On Day 10, spleen and draining lymph nodes were collected. CD8+ T cells were sorted. To measure secretion of granzyme B, CD8+ T cells were in vitro restimulated with wild-peptide 14/23/48/19 (SEQ ID NOs: 14, 23, 48 and 19, respectively) encoding for Spike/Receptor Binding Domain/Membrane/Nucleocapsid (S/RBD/M/N) SARS COV-2 proteins (10 μg/mL each) plus CD8− T cells (ratio 1:1) (0.1×106CD8+ T cells) to present peptides. As demonstrated in
Cytotoxic activity of CD8+ T Cells obtained after CoVepiT vaccination, was also measured by Chromium 51 release assay in HLA-A2 T2 human cells presenting 4 viral proteins. This second method was employed to confirm direct cytolytic capacity of CD8+ T cells against SARS-COV2 infected target cells using chromium 51 release assay. To mimic SARS-COV2 infection and viral peptides presentation by HLA-A2 cells, HLA-A2+T2 human cells were pulsed overnight with the same wild-type peptides from viral proteins Spike/Receptor Binding Domain/Membrane/Nucleocapsid (48/19/14/23 S/RBD/M/N, SEQ ID NOs 48, 19, 14 and 23 respectively) and used as target cells to measure cytotoxic functions of elicited CD8+ T cells after vaccination. Prior the assay, CD8 T cells isolated from immunized mice were expanded in vitro with peptide vaccine 14/23/48/19 (SEQ ID NOs 48, 19, 14 and 23 respectively) (2 μg/mL each) plus cytokine (IL-7, IL-21 and IL-2) to increase the pool of viral-specific T cells. T2 cells were labeled with chromium 51 then cocultured with CD8 T cells at ratio 40:1 or 15:1 for 4 hours. Chromium released by target T2 cells was counted in the supernatant using a gamma counter to quantify specific cytolysis.
Altogether, these immunogenicity and cytotoxicity data indicate that CoVepiT elicits SARS-COV2 specific Th1-biased CTL cells, producing IFNg and Granzyme B cytolytic granules. These immunogenicity results were more pronounced where the bi-transgenic model was expressing both HLA A2 and HLA DR. Finally, the CTL cells induced by the CoVepiT vaccine are capable to recognize and kill SARS-COV2 peptides-presenting human cells in a direct manner.
This experiment was conducted using 56 transgenic mice (male, age 6-7 weeks) HLA-A/H2-D. Experimental design is detailed in the table below. Nine different groups were studied for pharmacology and immunogenicity analysis with 3 escalating doses and one or two injections schedule studied.
One or 2 administrations of CoVepiT vaccine were also compared (group 1 versus group 2). The product was prepared with 12 CTL peptides plus Pan DR HTL epitope emulsified with the adjuvant Montanide ISA 51 (1/1 m/m) to be injected by subcutaneous route (100 uL). In parallel, groups of Naïve mice (group 3A) or mice injected with adjuvant only (group 4A and 4B) were used as negative control for immunization.
For pharmacology measurement, on Day 14 necropsy (one injection at DO) or Day 21 necropsy (2 injections D0-D14), spleen, Inguinal, axillary, and brachial lymph nodes were harvested and processed for single suspension to isolate CD8+ T cells from each individual mouse. CD8 T cells were sorted using negative sorting Macs Miltenyi microbeads and restimulated in vitro (0.3×106 cells) with of the pool of 12 corresponding wild-type peptides (10 μg/mL each) to evaluate cross-reactivity of elicited T cell response towards SARS-COV2 virus antigens.
The naïve mice group elicits few immunogenicity and serve as negative control whereas all treated animals treated with CoVepiT showed strong immunogenicity response.
In this experiment in HLA-A/H2-D transgenic mice, it was not observed significative impact, or correlation between 1 or 2 administrations related to immunogenicity, as shown in
Considering the different dose tested, the 5 μg dose (5 μg/each CTL peptide) induced better immunogenicity, the maximal effect was observed after a single injection. However, no difference was observed between 1, 5 or 50 μg after a second injection providing the same good level of immunogenicity for the 3 doses groups. Altogether, the intermediate dose (5 μg/each CTL peptide) is likely to be efficient to induce strong CD8 T cell immunogenicity. The highest dose (50 μg/each CTL peptide) gives also a strong immunogenicity validating the dose for the pharmaco-toxicological model.
Increasing doses or two injections are not providing increased of immunogenicity in this Transgenic model HLA-A/H2-D.
HLA-A2/HLA DR1 double transgenic mice were used for the study n=30 mice (aged 10-12 weeks).
The HLA-A2.1/HLA-DR1 double transgenic model was selected to explore at the highest dose (50 μg/each CTL peptide), the schedule of one injection versus two injections following protocol described in
A higher immunogenicity response was observed after the second administration of the vaccine versus the One injection group in the HLA A2-DR1 model.
A significant higher immunogenicity response was observed after the second administration of the vaccine versus the One injection group in the HLA A2−/HLADR1 model.
Serum of immunized mice for this part of the study corresponds to the group 1C, 2C and 3A N=24 mice (12 females and 12 males, age 6-7 weeks).
SARS-COV2 specific antibody was also quantified in the sera of immunized mice to determine whether the vaccine can promote humoral B cell response. HLA-A/H2-D transgenic mice were subcutaneous injected with CoVepiT (12 peptides 50 μg plus HTL 25 μg emulsified in Montanide) (Group 1C and 2C detailed in the table). Sera from Day 14 (one injection) or Day 21 (2 injection) were collected and antibody specific for spike was quantified in the sera by ELISA. For this test, Spike or RBD recombinant proteins were immobilized on the plate (10 μg/mL) then sera were added at serial dilutions. Revelation of Mouse IgG and IgA performed using a polyclonal peroxidase labeled antibody and Tetramethylbenzidine substrate. A positive serum of mice containing specific anti spike and RBD antibodies was used as positive control for the detection. Data in
Altogether these data in HLA A2-DR+ transgenic model demonstrate that CoVepiT vaccine elicits strong Th1-biased (IFNg+) CD8 T cell responses against SARS-CoV-2 antigens and with a significant higher immunogenicity response observed after the second administration of the vaccine.
CoVepiT as T multiepitope vaccine (epitopes selected from 11 proteins of SARS-CoV 2 including Spike and RBD protein) induced a strong T cellular response and this T cellular vaccine is not eliciting humoral response versus the Spike or RBD proteins.
HLA-A2/HLADR1 double transgenic model was used for this experiment (30 young animals (10-12 weeks)-30 aged animals (10-12 months). N=24 young mice and N=36 aged mice were immunized with CoVepiT; n=6 young and n=6 aged mice were naïve (no vaccination).
SARS-Cov2 multiepitope vaccine (CoVepiT) was evaluated in vivo in HLA-A2/DR1 double transgenic in young mice (12 weeks) versus aged mice (10-12 months) in order to evaluated CoVepiT response in immunosenescent situations. The age of 10-12 months is the oldest age that could be tested for HLA-A2/DR1 transgenic mice since this transgenic strain has a short-life expectancy (1 year).
As detailed in the
T cell immunogenicity in young and aged mice was evaluated after in vitro stimulation with SARS-COV2 wild type peptides for 24 hours. IFNγ response was quantified by ELISPOT on Day 14 or Day 21 following vaccination; CD8+ T cells mixed with CD8− cells pulsed with peptides (10 μg/mL each). No peptide stimulation (Medium) was used as basal IFN-γ aspecific secretion. Similar IFNγ response was observed after CoVepiT immunization of young and aged mice illustrating the efficacy of this vaccine in immunosenescent situation (
Altogether, the data demonstrate that CoVepiT vaccine elicits CTL immunogenicity in periphery as well as in the lung and respiratory tract with similar magnitude response in young and aged immunosenescent mice and confirm the immunogenicity all SARS-COV2 peptides without immunodominance of a peptide.
Tetramer analysis by Flow cytometry were further performed as one of the available research tools to help to further characterize elicited T cell phenotype. Parenchyma resident T cells and circulating T cells into the lung were discriminated in this test with the CD8α/CD8β staining. Anti-CD8α APCe670 antibody was injected intravenously few minutes prior sacrifice of the mice, this method allows the staining of only circulating CD8 T cells with the Anti-CD8α APCe670 antibody. Tissue resident lung T cell were characterized by CD8α−/6+ phenotype (
The data demonstrate that CoVepiT vaccine elicits Tissue-resident viral specific CD8 T cells in the lung and respiratory tract of both young and aged animals constituting hence a local barrier provided by sentinel memory T cells with cytotoxic function.
Immunization experiments were conducted with the highest dose of 50 μg CTL/epitope. 40 transgenic mice were studied in 5 groups with 4 Males and 4 Females in each group (age 6-7 weeks)—the transgenic model was mice HLA-A/H2-D 2Enge/J.
Groups used in this pharmaco-toxicological study:
Mice were injected with 1 injection (D0, group 1C) or 2 injections (D0-D14, group 2C) of CoVepiT vaccine (50 μg of each CTL peptide+HTL peptide 25 μg). The peptides were emulsified with the adjuvant Montanide ISA 51 (1/1 m/m) and injected by subcutaneous route (100 uL) as the subcutaneous route is also intended for the phase I clinical study. In parallel, groups of Naïve mice not treated (Group 3A) and a group receiving the adjuvant Montanide ISA 51 (One or two injections, Group 4A and 4B) served as negative controls of the immunization.
Daily clinical observations were performed by a veterinarian. Each mouse was assessed for any visible abnormalities in the fur, skin with focus at the injection site, eyes, respiration, behavior, etc. The cage was also observed for signs of food and water intake, and urination and defecation. Mice were also weighted weekly using a countertop scale.
No evidence of local irritation at the injection sites, behavior, morbidity, or mortality was observed following injection of CoVepiT vaccine regardless of the doses (1 μg, 5 μg and 50 μg) tested.
After one injection, the follow up of clinical observation was done every day (from D0 to D14) and was qualified as normal in all groups treated.
After two injections, the follow up every day (from D11 to D21) reported the same clinical observation for all mice. Only a hair loss was observed in 2 mice treated with CoVepiT (one hair loss observation at 1 μg and at 50 μg), one hair loss observation was also observed in the group treated with the adjuvant Montanide alone. Such observations were considered common in mice and are frequently correlated with stress and environmental modifications.
No difference in the weight (
For this study, the highest dose of 50 μg/peptide was selected to evaluate the potential toxicity in transgenic mice model as this model was established to elicit expected immunogenicity responses to the vaccine. Covepit was administered one or 2 times as detailed in the Table (Group 1C and 2 C).
Blood was collected on Day 2 and Day 10 for the one injection groups or Day 14 and Day 21 for the two injections groups. In parallel, naïve mice receiving no treatment and mice treated with adjuvant (emulsion only with Montanide ISA51) one or 2 injections were used as control. Mice were sacrificed on Day 0 (naïve mice), Day 14 (one injection) or Day 21 (2 injections)
Clinical chemistry parameters were including Albumine, Alkaline phosphatase, Creatine kinase, Lactate deshydrogenase, Alanine aminotransferase, Aspartate Aminotransferase, Sodium, Potassium, Chloride analysed in (
No specific biological change was observed in the treated groups with the test item CoVepiT at 50μg versus the control group (naïve not treated mice) or the adjuvant. It was not observed notable elements with one or two injections of CoVepiT compared to the adjuvant group (receiving one or two injections) considering albumin, Alkaline phosphatase, CK, LDH, AST, ALT, Sodium, Potassium, Chloride.
In parallel, White blood cell count, Red blood cell count, Hemoglobin, hematocrites, platelets, were also counted (
Hematological parameters were not substantially modified in the treated groups with the test item CoVepiT at 50μg versus the control group (naïve not treated mice). It was not observed notable elements with one injection of CoVepiT compared to the adjuvant group (receiving one injection) considering the blood cells counts: white blood cell count, Red Blood cell count, Hemoglobulin, hematocrit, platelets; lymphocytes, neutrophiles, monocytes and eosinophiles.
With two injections, no significant changes were observed, except a slight increase of monocytes and a slight decrease of lymphocytes population after the two CoVepiT vaccination versus the naïve group and the Montanide group (also receiving two injections) on Day 21.
The strong immunogenicity of the selected peptides and the recognition by memory T Cells was confirmed in convalescent COVID-19 Patients (Erreur ! Source du renvoi introuvable.) with the same responses (quantity and intensity) observed between HLA-A2+ and HLA-A2− donors.
The ex vivo clinical update in 88 COVID 19 convalescent patients confirms the immunogenicity of the 12 CTL selected peptides on T cells both in asymptomatic and in hospitalized (moderate to severe) COVID-19 convalescent patients, whatever their HLA-A2 status (negative or positive).
CTL long term Immunogenicity was evaluated after one or two administrations of CoVepiT vaccine in HLA-A2 transgenic mice, a total of 24 mice were vaccinated (n=12 one injection, and n=12 two injection) and 6 naïve mice (non-immunized) were used as negative control for the experiment. A dose of 50 ug of each peptide+25 ug HTL emulsified in Montanide ISA51 (1/1 m/m) was subcutaneously injected in the mice, the schedule of injections is described in
Long-term T cell immunogenicity was assessed on Day 60 (1 administration) or on Day 74 (2 administrations) after ex vivo restimulation. CD8+ T cells were isolated from the spleen and T cells were isolated from the lung or the Bronchoalveaolar lavage (BAL) of the immunized and naïve mice. In each group, spleen, BAL, and lung of 3 mice were pooled for analysis. IFN-γ response was quantified by ELISPOT after ex vivo restimulation with 12 wild-type corresponding peptides (10 ug/mL peptide) or without peptide (Medium) to measure basal IFN-γ aspecific secretion.
CoVepiT elicits a high long-term CTL immunogenicity in the spleen 2 months after a single or double vaccination
Item 1—A vaccine composition comprising one or several peptides (CTL peptide) inducing a CTL response against a SARS-CoV protein and optionally one or several peptides (BCL peptide) inducing a B cell response against SARS-CoV protein and optionally one or several peptides (HTL peptide) inducing a T helper response.
Item 2—The vaccine composition of item 1, wherein the composition comprises
Item 1—A vaccine composition comprising one or several peptides selected from one or several peptides (CTL peptide) inducing a CTL response against a SARS-CoV protein, and optionally one or several peptides (HTL peptide) inducing a T helper response, wherein the composition comprises:
Spike glycoprotein (5), Nucleocapsid protein (N), Membrane glycoprotein (M) and Protein 3a, and wherein the CTL (neo)epitopes targeting S are selected from one of the groups consisting of (i) SEQ ID NOs: 34, 48, 56, 60, 70, 74, 84, 86, 91, 92, 104, and 146; (ii) SEQ ID NOs: 48, 56, 60, 70, 91, 92 and 146; and (iii) SEQ ID NOs: 70 and 146; the CTL (neo)epitopes targeting N are selected from one of the groups consisting of (i) SEQ ID NOs: 23, 67, 75, 79, 85 and 113; (ii) SEQ ID NOs: 23 and 79; and (iii) SEQ ID NO: 23; the CTL epitope targeting M is SEQ ID NO: 66; and the CTL (neo)epitopes targeting Protein 3a are selected from one of the groups consisting of (i) SEQ ID NOs: 3, 97 and 101; and (ii) SEQ ID NO: 97;
| Number | Date | Country | Kind |
|---|---|---|---|
| 20305469.7 | May 2020 | EP | regional |
| 20305930.8 | Aug 2020 | EP | regional |
| 21305071.9 | Jan 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/062481 | 5/11/2021 | WO |
