Method for generating a vaccine composition that elicits a human leukocyte antigen class I-restricted CD8 T cell response against virus-derived epitopes composed of non-viral particles

Abstract

A method for providing a vaccine composition capable of effectively inducing a systemic immune response and / or a local immune response upon administration, wherein the composition comprises a human leukocyte antigen class I (HLA I)-restricted epitope selected from viral pathogen non-viral particle constituent proteins (non-VIPs), and thus elicits a CD8 T cell response specific for virus-infected cells.

Description

Technical Field

[0001] The present invention relates to immunology and vaccines. More specifically, the present invention relates to methods for preparing vaccine compositions and vaccine compositions for combating viral pathogens. Specifically, the present invention relates to methods for generating administrable vaccine compositions that can serve as prophylactic or therapeutic agents against viral infections. Background Art

[0002] Viral vaccine composition

[0003] Currently, commercially available vaccine preparations mainly target whole-virus open reading frames (ORFs), and generally seek to elicit neutralizing immunoglobulin (Ig) responses to confer immunity against the target viral pathogen. Vaccination strategies generally do not seek to specifically select HLA-restricted T cell epitopes for inclusion in the vaccine composition. Typically, the characterization of any HLA-restricted epitopes (HREs) is limited to epitopes present in proteins including virions or "virion-integral proteins" (VIPs), because in clinically manifest viral infections, T cell responses to these specific viral ORF products are mainly observed, and thus it is speculated that they can confer productive T cell-mediated cellular immunity. However, the inclusion of VIP-derived HREs in vaccine compositions is usually the result of aiming to elicit B cell-driven Ig responses, generally without considering the life cycle of the target virus replication and spread within the infected host, nor how this life cycle elicits T cell-centered cellular immunity within the host. Therefore, when attempting to utilize or circumvent T cell immunity in vaccine compositions, the viral life cycle, viral immune escape, and the possibility of immunopathology should be carefully considered.

[0004] Adaptive immune response to viral infection

[0005] The clinical manifestations of viral infections are usually associated with viral immune escape and / or virus-induced immunopathology. These immunopathologies are typically attributable to an excessive response to "virally encoded proteins" (VEPs). During natural viral infections with clinical manifestations, the production of Ig against VEPs is usually limited to the VIPs available on the surface of virions, which are used for B cell sampling and maturation against viral Ig epitopes. This is similar to the dominant T cell response in viral infections with clinical manifestations, where patient T cells are usually limited to HREs derived from VIPs. In contrast, due to a combination of viral life cycle factors and the way the host immune system responds during the viral life cycle, the non-virion-integral proteins (non-VIPs) of the virus usually drive subdominant T cell responses. Host immunity usually engages in active immune escape of specific innate and adaptive responses driven by VEPs. In fact, viral non-VIPs usually cause dysregulation of the host innate immune response driven by the virus, thereby altering the process of downstream adaptive immune responses.

[0006] Challenges in defining human leukocyte antigen (HLA)-restricted epitopes of vaccine compositions

[0007] Due to the high diversity of HLA haplotypes in the population, the standardization of incorporating viral HREs into vaccine compositions has been challenging. HLA allele variants represent the most diverse gene family in higher organisms, where the genetic diversity of HLA genes confers a wide range of possibilities for diverse HRE presentation at the population level. That is, the genetic diversity of HLA genes drives diversity at the protein level, which in turn drives differential selection of HRE loading and presentation on the cell surface. There is a general lack of systematic and accurate tools to functionally analyze the highly complex processing and presentation of specific HREs, which limits the systematic and reliable identification of HREs from VEPs. In addition, the complex T cell receptor (TCR)-driven T cell recognition of these HREs drives a diverse array of downstream T cell-centered adaptive immunity and immune tolerance. The lack of systematic biotechnological tools to functionally analyze HRE-specific T cell responses further hinders the directed analysis of T cell responses against VEPs to determine whether to incorporate them into vaccine compositions.

[0008] HLA class I and II and related T cell responses

[0009] Two major classes of HLA, namely class I (HLA I) and class II (HLA II), typically elicit CD8 and CD4 T cell responses, respectively, and are found in all jawed vertebrates. HLA I molecules are presented by all nucleated cells and typically present intracellular proteins that are processed mainly by proteasomal degradation and transport of the degradation products to the endoplasmic reticulum (ER). Within the ER, further proteolytic processing can occur during the enzyme-driven loading of HLA I molecules with HLA I-HRE and subsequent transport to the cell surface for T cell sampling. HLA I molecules are also presented on the surface of professional antigen-presenting cells (APCs). Professional APCs such as dendritic cells (DCs) or macrophages are central mediators of adaptive immunity. Within professional APCs, there are also other processing and transport pathways that enable the delivery of protein antigen degradation products derived from sampling of the extracellular environment to the ER for HLA I cross-presentation. In contrast, HLA II molecules are constitutively presented only on the surface of professional APCs. HLA II molecules are largely restricted to presenting protein degradation products derived from sampling of the extracellular environment in such processing, which involves endocytosis of extracellular payloads within endocytic vesicles to fuse with different vesicular compartments containing HLA II molecules, and a series of processing and chaperone enzymes that direct the loading of HLA II molecules with HRE and then export to the cell surface for T cell sampling.

[0010] HLA class I and CD8 T cell responses

[0011] The major function of the CD8 T cell population is to act as cytotoxic lymphocytes (CTLs), which, upon detection of target cells presenting associated HLA I-HRE epitopes, mediate the execution of multiple cell death mechanisms against the target cells, where the HLA I-HRE epitopes activate the TCR carried by the relevant CD8 CTL clones. Thus, the detection of HLA I-HRE by CD8 T cells involves surveillance of the intracellular proteome of nucleated cells and thus represents a central mechanism by which adaptive cellular immunity responds to non-self proteins expressed intracellularly. This marks the CD8 T cell response detecting HLA I-HRE as a major defense against dysplasia and malignant cell transformation, as well as viral infections, and bacterial, parasitic, or other intracellular microbial infections. HLA I-HRE are relatively short peptides with more stringent physicochemical constraints compared to HLA II-HRE. The relatively stringent nature of HLA I-HRE reflects the requirement to tightly control the CD8 CTL adaptive response to avoid tissue damage caused by false CD8 CTL responses against HLA I-HER derived from self and non-self antigen components.

[0012] HLA class II and CD4 T cell responses

[0013] The HLAII-HRE-directed CD4 T cell response is more complex and diverse than the HLAI-HRE CD8 T cell response. Generally, CD4 T cells are regarded as "helper" cells that coordinate innate and adaptive immune responses by signaling to both leukocytes and non-leukocytes alike. In terms of adaptive immunity, the HLAII-HRE CD4 T cell response is necessary for driving B cell maturation to produce high-affinity Ig responses and also involves shaping CD8 T cells towards, for example, memory cell differentiation. When detecting HLAII-HRE sampled from the extracellular space, CD4 T cells are crucial for coordinating the adaptive immune response to infections, including extracellular microbes, mainly by driving the progression of the humoral Ig response through B cell maturation. For example, regulatory T cells (Tregs), which are responsible for immune tolerance to commensal microbes and food, can also be mainly identified as CD4 T cells sampling HLAII-HRE. These HLAII-HRE CD4 Treg responses also play key quality control and inflammation resolution roles in the innate and adaptive immune responses against pathogenic microbes, such as limiting the likelihood of virus-associated immunopathology.

[0014] Viral life cycle and host CD8 T cell responses

[0015] Viral infection of host cells leads to the delivery of the viral genome and the expression of the encoded ORFs, which can be classified as non-VIPs and VIPs. Generally, non-VIPs are responsible for viral replication and immune escape from host defenses, while VIPs are the proteins that are assembled into de novo synthesized virions within infected cells. Multiple types of immune escape mechanisms in certain viruses can also be attributed to VIPs. Generally speaking, non-VIPs are mainly responsible for the processing and replication of the viral genome, coordinating viral genome packaging, controlling de novo synthesized virion biogenesis, and mediating immune escape.

[0016] Eliciting productive CD8 CTLs against HLAI-HRE expressed on infected cells is central to the adaptive immune response to viral infection. Eliminating virus-infected cells through CD8 CTL action is considered a key aspect of virus clearance and the establishment of acquired immunity. Infected host cells trigger a series of innate immune responses to attract CD8 T cells to sample HLAI-HRE, whether naive CD8 T cells initiate the CD8 CTL adaptive immunity or memory CD8 T cells recall immunity against previous natural infections or vaccinations. The major intracellular innate immune response driving CD8 T cell recruitment and sampling is the type I interferon (IFN-I) pathway.

[0017] Viral non-VIPs are typically widely expressed in cytoplasmic compartments and are thus accessible to the HLA-I-HRE processing and presentation machinery, while VIPs are typically sequestered from these mechanisms. The sequestration of VIPs from HLA-I-HRE processing and presentation is particularly evident in enveloped viruses, where during de novo virion biogenesis, structural VIPs are sequestered to membranes and vesicles. Thus, by eliminating productively infected cells, CD8+ CTL responses against viral non-VIPs can be considered the major driver of virus clearance in many forms of viral infection.

[0018] Professional APCs are strong inducers of HLA-I-HRE CD8+ CTL responses. In fact, especially DCs have mechanisms to promote cell infection, thereby driving beneficial CD8+ CTL responses. However, in the absence of productive professional APC infection, the priming of CD8+ CTL responses can be biased towards VIPs by endocytosis of whole virions and trafficking of VIPs to the ER for HLA-I-HRE cross-presentation by DCs. This may represent a key driver of the immunodominant nature of CD8+ T cell responses against VIP-derived HLA-I-HREs in many clinically manifest viral infections.

[0019] Beneficial CD8 T cell host responses to viral infection and immune escape

[0020] The most productive CD8+ T cell responses to viral infection are those that prime CD8+ CTL responses that can ultimately drive the clearance of virus-infected cells. This is the main reason for the rapid triggering of innate immune responses during viral infection and the recognition of viral factors and genomes, where IFN-I plays an important role in inducing innate immune pathways. The IFN-I pathway plays a key role in promoting the recruitment and activation of CD8+ T cells, thereby facilitating the priming of beneficial CD8+ CTL responses against HLA-I-HREs presented by productively infected cells.

[0021] An important mechanism of viral immune escape is the dysregulation and / or inhibition of the innate IFN-I response, and many viral genomes encode numerous non-VIPs that disrupt IFN-I signaling. This promotes viral immune escape, especially in terms of CD8+ T cell detection of HLA-I-HREs in infected cells. This immune escape mechanism allows virus-infected cells to escape CD8+ CTL-driven clearance while viral replication can continue. This has obvious implications for potentially non-productive or even harmful HLA-I-HRE CD8+ T cell responses during viral infection.

[0022] Non-productive CD8 T cell responses to viral infection

[0023] During viral infection, the escape of virus-infected cells from CD8 T cell responses has a significant impact on driving non-productive or even harmful CD8 T cell responses. Importantly, the escape of virus-infected cells from CD8 T cell responses will selectively limit the CD8 CTL responses against viral non-VIP-derived HLAI-HREs, where the CD8 CTL responses against viral non-VIP-derived HLAI-HREs are most favorable for viral clearance by eliminating infected cells. Crucially, this immune escape enables infected cells to produce and release new viral particles and amplifies the infection. This necessarily leads to an increasing number of viral particles in the extracellular space being sampled by professional APCs (including DCs and macrophages) through pinocytosis, endocytosis, or phagocytosis. However, the currently available HLAI-HRE epitopes must be derived from VIPs, rather than the more productive non-VIP-derived HLAI-HERs, unless the professional APCs themselves are productively virus-infected.

[0024] The results of immune escape of CD8 CTL responses against non-VIP-derived HLAI-HREs presented by productively infected cells, and the emergence of immunodominant responses against VIP-derived HLAI-HERs triggered by professional APCs, have a significant impact on further downstream viral immune escape. First, the predominance of professional APCs triggering CD8 T cell responses shifts the CD8 CTL responses from productive non-VIP-derived HLAI-HREs to VIP-derived HLAI-HERs. Second, the immunodominance of VIP-derived HLAI-HREs will trigger CD8 CTL responses that promote the elimination of professional APCs by CD8 CTLs. In fact, the elimination of professional APCs mediated by CD8 CTLs is considered to be a factor in eliminating the inflammatory response to viral infection. However, during active viral infection, this can be regarded as immune response dysregulation, which drives the depletion of professional APCs at the site of infection and in the draining lymph nodes (DLNs), leading to a reduced ability of professional APCs to coordinate the overall adaptive immune response, and critically, the HLAI-HRE / CD4 T cell response. Downstream of the initial immune escape of CTL action against productively infected cells, this form of cumulative immune escape of adaptive immunity is crucial in viral infections with a limited host cell infection spectrum, especially in cases of productive infection with a lack of or inefficient professional APCs.

[0025] Derived from CD8 CD4 T cell and Ig immune escape with dysregulated CTL responses

[0026] The accumulation of the above-described CD8 CTL responses against VIP-derived HLAI-HRE (which can drive the exhaustion of professional APCs during infection) can lead to a series of downstream effects that are detrimental to an effective adaptive immune response to viral infection. Importantly, professional APCs are crucial for HLAII-HRE-specific CD4 T cell responses, and these responses themselves are crucial for the establishment of humoral Ig immunity. The immunodominant VIP-derived HLAI-HRE CD8 CTL responses that eliminate professional APCs at the site of infection (at the cost of responses against productive non-VIP-derived HLAI-HER) also lead to a reduced availability of the professional APC platform in the DLN that can coordinate CD4 T cell responses. This inevitably results in a reduced ability to effectively coordinate the timely maturation of B cells and the production of high-affinity neutralizing Ig, which would otherwise contribute to viral clearance and sustained immunity. This can also lead to a reduced ability of CD4 T cell responses, where the CD4 T cell responses effectively mediate the shaping of CD8 CTL responses against non-VIP-derived HLAI-HRE to memory differentiation and the effective establishment of CD8-centered sustained immunity.

[0027] Ig-driven immunopathology and vaccine-related diseases

[0028] Most current vaccine strategies aim to provide compositions of substantially intact VIP, usually within whole inactivated or attenuated viruses, with the central goal of eliciting the production of neutralizing antibodies. However, without the support of coordinated HRE T cell responses, these strategies often result in poor neutralizing antibody titers and incomplete immunity to the target viral pathogen. In addition, some vaccine strategies have led to exacerbated immunopathology in animal and human subjects, possibly in part due to dysregulated Ig responses downstream of misdirected HRE-specific T cell responses to VIP. In some cases, immunopathology associated with the production of self-neutralizing antibodies has been found in both developed vaccination strategies and natural infections, particularly in respiratory mucosal infections, where high titers of antibody secretion can lead to mucus hypersecretion and obstructive respiratory distress. Due to the high complexity of the HRE repertoire during viral infection, the high diversity of HLA haplotypes among individuals, and the incomplete understanding of the overall adaptive immune response to viral infection, these vaccine-related disease phenomena are not well understood and are difficult to address.

[0029] Eliciting selected CD8 Perspectives on viral vaccine compositions that elicit T cell responses

[0030] There is substantial evidence to support the crucial role of CD8 CTL responses in preventing the establishment of viral infections and clearing viral infections. In fact, animal models clearly demonstrate the significant protective effect of antigen-specific CD8 T cells against bolus viral challenge. These observations point to a vaccine strategy in which selected non-VIP-derived HLA I-HREs are provided in a vaccine composition to selectively elicit beneficial CD8 CTL responses. To this end, the vaccine composition is designed to provide a front-line defense against invading viruses while further avoiding the potentially non-productive or harmful elicitation of CD4 and Ig responses, which can lead to the exacerbation of immunopathology following subsequent natural viral infection.

[0031] To date, it has been challenging to analyze HRE-specific T cell responses with sufficient depth and precision to determine the precise HREs of an effective vaccine composition aimed at eliciting favorable T cell responses. In addition to T cell responses to these antigens, emerging biotechnological methods now also allow for in-depth, rapid, and accurate functional analysis of HREs (see, for example, WO2018083316, WO2018083317, WO2018083339, and WO2018083318). Summary of the Invention

[0033] The present invention provides a method for generating an administrable vaccine composition for use as a prophylactic or therapeutic agent against viral infections. The present invention particularly provides a vaccine composition capable of effectively inducing a systemic immune response and / or a local immune response upon administration, wherein the composition comprises a human leukocyte antigen class I (HLA I)-restricted epitope (HLA I-HRE) selected from non-virion-integral proteins (non-VIPs) of a viral pathogen, thereby eliciting a CD8 T cell response specific for virus-infected cells.

[0034] The present invention provides a method for selecting non-VIP-derived HLA I-HREs, by which a vaccine composition is generated that selectively elicits CD8 CTL responses favorable for viral immunity and clearance while avoiding non-productive or harmful CD8 CTL responses to VIP-derived HLA I-HERs.

[0035] This method includes selecting HLA I-HREs for inclusion in a vaccine composition that is designed to elicit a selective CD8 CTL response against virus-infected cells while avoiding viral immune escape mechanisms and spurious CD8 CTL responses driven by downstream immunopathology.

[0036] Specifically, the present invention provides a method for generating an administrable vaccine composition as a prophylactic or therapeutic agent against viral infection. The present invention particularly relates to a vaccine composition capable of effectively inducing a systemic immune response and / or a local immune response upon administration, wherein the composition comprises one or more non-VIP-derived HLA-I-HREs selected from the target viral pathogen while avoiding HLA-I-HERs derived from VIP, thereby triggering a restricted and highly defined CD8 T cell response specific for virus-infected cells.

[0037] In one aspect, the present invention provides a method for generating a vaccine composition for combating a viral pathogen, comprising:

[0038] a. Identifying human leukocyte antigen class I-restricted epitopes (non-VIP-derived HLA-I-HREs) derived from non-virion constituent proteins of the viral pathogen, wherein a vaccine composition against the viral pathogen is desired;

[0039] b. Classifying the immunogenicity of the identified non-VIP-derived HLA-I-HREs in a naive CD8 T cell population isolated from donors without prior target viral infection and / or in a memory CD8 T cell population isolated from donors with confirmed active, latent, or resolved target viral infection;

[0040] c. Selecting non-VIP-derived HLA-I-HREs with confirmed immunogenicity in donors without prior target viral infection or non-VIP-derived HLA-I-HREs with observed CD8 T cell responses in donors with confirmed active, latent, or resolved target viral infection;

[0041] d. Incorporating the selected non-VIP-derived HLA-I-HREs into the vaccine composition.

[0042] The immunogenicity of non-VIP-derived HLA-I-HREs in donors without prior infection or donors with confirmed prior infection should be against HLA-I-HERs, which have been confirmed to be processed and presented in a cell model incorporating a productive viral infection to confirm the availability of the epitope in the normal viral life cycle of infected cells. That is, the use of a single-expressed viral ORF, ORF fragment, or the use of a recombinant peptide is not sufficient to confirm the immunogenicity of a given non-VIP-derived HLA-I-HRE. This can be confirmed independently of the directed analysis of T cell responses in donors without prior infection or infected donors, for example, by detecting non-VIP-derived HLA-I-HREs in an ex vivo cell model that has been productively or non-productively infected with the viral pathogen.

[0043] A systematic comparative analysis of non-VIP-derived HLAI-HRE immune responses can be performed in the following categories of human subjects:

[0044] 1. Subjects with asymptomatic SARS-CoV-2 infection

[0045] 2. Subjects with resolved SARS-CoV-2 infection

[0046] 3. Subjects with severe Covid-19 disease

[0047] 4. Subjects known not to be infected with SARS-CoV-2

[0048] The priority order for non-VIP-derived HLAI-HRE identified in each subject category to be preferentially incorporated into the vaccine composition is 1>2>3>4.

[0049] In a second aspect, the present invention provides a method of inoculating a human or veterinary subject to provide immunity against a virus, which comprises administering a vaccine composition prepared by the above method.

[0050] These vaccine preparations can be used for the treatment and prevention of known pathogens, as well as for rapidly generating a response to new pathogens. In addition, knowledge of non-VIP-derived HLAI-HRE enables the development of diagnostic procedures for clinical and epidemiological surveillance. Brief Description of the Drawings

[0052] Figure 1 . Initial SARS-CoV-2 infection results in the escape of innate immunity mediated by virus-encoded proteins and its mediation of the escape of early adaptive CD8 T cell responses.

[0053] Figure 2 . The infection established during initial immune escape results in a delayed recruitment of the immune response.

[0054] Figure 3 . The late initiation of the adaptive immune response results in a bias towards professional APC sampling of accumulated virions and a directed T cell response against HLA-restricted antigens derived from the constituent proteins of the virions. This is the correct directed response of HLAII / CD4 helper cells and B cells. This may be counterproductive for HLAI / CD8 CTL responses, where the HLAI / CD18 CTL response should target non-virion proteins highly expressed in productively infected cells rather than those presented in the virions.

[0055] Figure 4 .The misdirected CD8 CTL response against HLA-I restricted epitopes from VIP cross-presented by professional APCs during cumulative viremia and tissue damage leads to local professional APC depletion, resulting in dysregulated T cell and B cell responses, which can potentiate cytokine storm in severe Covid-19 manifestations and lead to an overall weak neutralizing antibody response to SARS-CoV-2 infection.

[0056] Figure 5 .A) The selected core HLA-A, B, C alleles, B) the proportion of each ethnic group predicted to carry at least n core allele sets, C) the cumulative probability of observing at least n core alleles in a given ethnic group.

[0057] Description of the Invention

[0058] The invention is described in the items listing aspects and embodiments below:

[0059] 1. A method of generating a vaccine composition for combating a viral pathogen, comprising:

[0060] a. Identifying a human leukocyte antigen class I restricted epitope derived from a non-viral particle constituent protein (non-VIP-derived HLAI-HRE) from the viral pathogen, wherein a vaccine composition against the viral pathogen is desired;

[0061] b. Classifying the immunogenicity of the identified non-VIP-derived HLAI-HRE in a naive CD8 T cell population isolated from donors without prior target virus infection and / or in a memory CD8 T cell population isolated from donors with confirmed active, latent, or resolved target virus infection;

[0062] c. Selecting a non-VIP-derived HLAI-HRE that has confirmed immunogenicity in donors without prior target virus infection or a non-VIP-derived HLAI-HRE that has an observed CD8 T cell response in donors with confirmed active, latent, or resolved target virus infection;

[0063] d. Incorporating the selected non-VIP-derived HLAI-HRE into the vaccine composition.

[0064] 2. The method of item 1, wherein multiple non-VIP-derived HLAI-HREs are selected for incorporation into the vaccine composition to represent one or more HLAI-HERs in the selected HLAI alleles, and the selected HLAI alleles represent those alleles carried by at least 60% of the individuals within the target population for which the vaccine composition is designed.

[0065] 3. The method according to item 1, wherein one or more non-VIP-derived HLAI-HREs are selected and incorporated into the vaccine composition to represent one or more HLAI-HERs among the one or more HLAI alleles selected, and the one or more HLAI alleles selected are carried by the individual for whom the vaccine composition is designed.

[0066] 4. The method according to any one of items 1-3, wherein the vaccine composition comprises one or more inoculation vectors selected from the following:

[0067] a. Recombinant non-replicating or replicating viral vectors;

[0068] b. Virus-like particles;

[0069] c. Recombinant RNA constructs with or without modified nucleotides;

[0070] d. Recombinant DNA constructs with or without modified nucleotides;

[0071] e. Recombinant proteins with or without modified amino acids;

[0072] f. Synthetic polypeptides with or without modified amino acids.

[0073] 5. The method according to item 4, wherein the one or more inoculation vectors are selected from a, b or c, and wherein the selected non-VIP-derived HLAI-HREs are incorporated into an expression construct that does not allow the expression of a functional non-VIP protein in host cells during vaccine delivery to avoid the immune escape activity of the viral non-VIP.

[0074] 6. The method according to item 5, wherein the selected non-VIP-derived HLAI-HREs are provided by one or more of the following means:

[0075] a. Introducing point mutations and / or sequence insertions and / or sequence deletions that inactivate protein function within the full-length non-VIP ORF;

[0076] b. Constructing a synthetic nucleic acid sequence containing a non-VIP ORF fragment encoding the selected HLAI-HREs in a tandem construct;

[0077] c. Constructing a synthetic nucleic acid sequence containing a non-VIP ORF fragment encoding the selected HLAI-HREs within the vector protein sequence.

[0078] 7. The method according to item 4, wherein the one or more inoculation vectors are selected from d or e, wherein the recombinant protein or synthetic polypeptide comprises one or more non-VIP-derived HLAI-HREs, and the protein or polypeptide molecule comprises tandem HLAI-HREs or encodes the HLAI-HER within the vector protein or polypeptide.

[0079] 8. The method of any one of items 1-7, wherein the viral pathogen is selected from adenovirus, alphavirus, arbovirus, Borna disease, bunyavirus, calicivirus, condyloma acuminata, coronavirus, coxsackievirus, cytomegalovirus, dengue virus, contagious ecthyma, Epstein-Barr virus, erythema infectiosum, hantavirus, viral hemorrhagic fever, viral hepatitis, herpes simplex virus, varicella-zoster virus, HIV, infectious mononucleosis, influenza, Lassa fever virus, measles, mumps, molluscum contagiosum, paramyxovirus, phlebotomus fever, polyomavirus, poxvirus, retrovirus, Rift Valley fever, rubella, slow virus, smallpox, subacute sclerosing panencephalitis, tumor virus infection, West Nile virus, yellow fever virus, rabies virus, and respiratory syncytial virus.

[0080] 9. The method of item 8, wherein the viral pathogen is a coronavirus, such as SARS-Cov2.

[0081] 10. The method of any one of items 4-9, wherein the one or more vaccine vectors further encode one or more B cell / immunoglobulin epitopes to elicit a neutralizing Ig response.

[0082] 11. The method of any one of items 4-10, wherein the one or more vaccine vectors further encode one or more selected HLAII-HRE epitopes to elicit a CD4 T cell response to support B cell maturation and neutralizing antibody production, and / or promote the differentiation of non-VIP-derived HLAI HRE-specific CD8 T cells into memory.

[0083] 12. The method of item 10, wherein the one or more B cell / immunoglobulin epitopes are selected from the VIP proteins of the target viral pathogen and are expressed on the surface of the virion.

[0084] 13. The method of any one of items 10 and 12, wherein the one or more B cell / immunoglobulin epitopes are modified to remove HLAI-HRE from the VIP to avoid eliciting a CD8 T cell response against the VIP protein during vaccine delivery.

[0085] 14. The method of item 11, wherein the one or more HLAII-HRE may comprise sequences derived from the target viral pathogen, or may be synthetic or naturally occurring HLAII-HE epitopes, which, when included in the vaccine composition, can promote beneficial CD4 T cell responses to support B cell maturation and / or CD8 T cell memory differentiation.

[0086] 15. The method of any one of items 4 to 10, 13, and 14, wherein the one or more vaccine vectors further comprise one or more vaccine adjuvants.

[0087] 16. A method according to any one of items 4 - 15, wherein the vaccine vector is further formulated into a vaccine preparation for administration to a human or veterinary subject, and wherein the vaccine preparation further comprises a pharmaceutically suitable excipient.

[0088] 17. A vaccine composition for combating a viral pathogen, prepared by the method described in any one of items 1 - 16.

[0089] 18. A vaccine preparation comprising the vaccine composition described in item 17 and at least one pharmaceutically acceptable excipient.

[0090] 19. The vaccine preparation of item 18, which further comprises at least one vaccine adjuvant.

[0091] 20. A method of inoculating a human or veterinary subject to provide immunity against a virus, which comprises administering the vaccine composition described in item 17, the vaccine preparation described in any one of items 18 - 19, or a vaccine composition prepared by the method described in any one of items 1 - 16.

[0092] 21. A method of treating a human or veterinary subject suffering from an acute, chronic or latent viral infection, which elicits an immune response by immunizing the human or veterinary subject with the vaccine composition described in item 17, the vaccine preparation described in any one of items 18 - 19, or a vaccine composition prepared by the method described in any one of items 1 - 16.

[0093] 22. A method of eliciting a CD8 T cell response against a viral infection in a human or veterinary subject, which comprises administering the vaccine composition described in item 17, the vaccine preparation described in any one of items 18 - 19, or a vaccine composition prepared by the method described in any one of items 1 - 16.

[0094] 23. A method according to any one of items 20 - 22, wherein the route of administration of the vaccine is selected from intramuscular, intranasal, oral, intraperitoneal, subcutaneous, topical, intradermal and transdermal delivery.

[0095] 24. A method according to any one of items 20 - 22, wherein the vaccine is administered intranasally.

[0096] 25. A method according to any one of items 20 - 22, wherein the vaccine is administered intramuscularly.

[0097] 26. A method according to any one of items 20 - 22, wherein the vaccine is administered intradermally.

[0098] 27. A method according to any one of items 20 - 26, wherein the vaccine is administered on two separate occasions, at least 7 days apart, to represent a prime - boost vaccination strategy.

[0099] 28. The method of any one of items 20 - 27, wherein the priming - boosting vaccine is administered by the same route.

[0100] 29. The method of any one of items 20 - 27, wherein the priming vaccine is delivered intramuscularly and the boosting vaccine is delivered intranasally.

[0101] 30. The method of any one of items 27 - 29, wherein the priming and boosting vaccine compositions, vaccine carriers, and / or vaccine formulations can be the same or different. Examples

[0102] Example 1: A SARS - CoV - 2 infection immunopathogenesis model demonstrating the advantages of non - VIP - derived HLAI - HRE vaccine compositions

[0103] A few years ago, several cases of infections of unknown etiology were discovered. These infection cases were later associated with a novel virus called Severe Acute Respiratory Syndrome Coronavirus - 2 (SARS - CoV - 2). On March 11, 2020, the World Health Organization declared that SARS - CoV - 2 was causing Coronavirus Disease (COVID - 19), which is now considered a pandemic. This disease is characterized by respiratory infections that, depending on the severity, can lead to acute respiratory distress disease. Compared to younger individuals, the prognosis of elderly subjects infected with SARS - CoV - 2 is much worse, indicating that the decline in the ability of the adaptive immune response in elderly subjects plays a role in the severity of SARS - CoV - 2 infection, progression to COVID - 19, and the risk of severe complications and mortality. This example presents a common model of SARS - CoV - 2 immunopathogenesis that has a direct impact on determining the current and past infection status in non - elderly subjects, understanding the severity of COVID - 19 in elderly subjects, and has implications for designing vaccine compositions selectively containing non - VIP - derived HLAI - HRE.

[0104] For a long time, the development of viral vaccines has been limited due to the lack of understanding of antigen - specific T - cell responses to natural viral infections and multiple vaccine modalities. This limited understanding of T - cell responses is due to the lack of systematic and high - precision tools to identify HLA - restricted epitopes presented from viral proteins. In addition, the lack of rapid and definitive tools to detect the responses of human T - cells to these multiple HLA - restricted antigens is a serious limitation.

[0105] Several respiratory virus infections are associated with acute inflammatory responses of the respiratory mucosa, which are generally associated with dysregulated T cell responses. Historically, RSV, SARS-CoV, and even invasive influenza strains have been regarded as the causes of severe inflammatory syndromes of the respiratory mucosa. Now, subjects infected with SARS-CoV-2 who experience the most severe clinical manifestations of Covid-19 suffer cytokine storms that ultimately lead to morbidity. These severe immunopathologies are clearly driven by excessive T cell responses during viremia.

[0106] Historical experiences of vaccine-related disease exacerbation in attempts to vaccinate against respiratory virus infections point to significant risks in current initiatives to deploy vaccines against the novel coronavirus. For decades, it has been recognized that RSV has a high unmet medical need, and attempts to vaccinate against RSV in the early 1960s led to exacerbation of acute respiratory distress in vaccine recipients following subsequent natural infection. In the decades of research on this phenomenon, antigen-specific CD8 and CD4 T cell responses were thought to equally participate in driving virus clearance and immunopathology. Similarly, experimental vaccines against SARS-CoV, the early novel coronavirus that caused the 2003 SARS outbreak, led to severe inflammatory responses in vaccine development studies, which could be partly attributed to anti-spike immunoglobulins.

[0107] To support the significant role of T cell responses in coronavirus infections, studies of SARS-CoV infections have shown that in recovered subjects, B cell responses are weak, but antigen-specific T cell responses are persistent—a trend reflected in the SARS-CoV-2 pandemic. In addition, a prominent feature of SARS during SARS-CoV and SARS-CoV-2 infections is lymphopenia, presumably driven by massive recruitment of T cells to the respiratory mucosa. Combining the obvious correlation between lymphopenia and poor prognosis in Covid-19 patients, and the emerging details of T cell immunopathology in severe Covid-19, there is clearly a need for precise analysis of T cell responses in subjects infected with SARS-CoV-2. This is required to evaluate the safety and efficacy of current candidate vaccines and to directly inform the development of the next generation of vaccines.

[0108] Summary of the SARS-CoV-2 immunopathogenesis model

[0109] Initial immune escape by CD8 CTLs during the early stages of infection leads to the establishment of infection, which is more likely to occur in elderly subjects as the naive CD8 T cell repertoire in elderly subjects is known to be reduced due to decreased thymic output. During this stage, an effective early CD8-mediated response can clear the infection even asymptomatically without the subsequent generation of detectable high-affinity neutralizing antibodies, but can establish memory T cells. Apparently, other healthy non-elderly subjects typically present with asymptomatic SARS-CoV-2 infection, or very mild symptoms that are cleared by low-level and transient Ig responses.

[0110] Initial immune escape results in the evasion of detection of "non-viral particle constituent proteins" (non-VIP) HLA-I restricted epitopes, the detection of which is an ideal requirement for clearing virus-infected cells.

[0111] The accumulation of danger associated molecular patterns (DAMP) and pathogen associated molecular patterns (PAMP) during infection establishment and epithelial cell death leads to the characteristic recruitment of immune cells into the infected respiratory mucosa.

[0112] Initial immune escape allows the accumulation of virions, which are then sampled by professional APCs that themselves may not be productively infected.

[0113] Processing and presentation of virion constituent proteins (VIP) by professional APCs elicits HLA-I / CD8 responses and HLA-II / CD4 responses.

[0114] The HLA-II / CD4 response that detects VIP initiates CD4 helper responses to support the maturation of B cells against the viral particle surface proteins sampled by B cells, leading to a productive adaptive immune response that generates neutralizing antibodies. In many cases, but not all, the infection can thus resolve.

[0115] The HLA-I / CD8 response that detects VIP is not ideally suited to target epithelial cells or other cells with productive viral infection, but these responses are capable of targeting professional APCs that cross-present HLA-I restricted antigens from virus-infected cells.

[0116] Misdirected CD8 CTL responses against VIP can lead to the depletion of professional APCs at the site of infection and in the DLN, resulting in a complete breakdown of the coordination of T cell responses and the loss of support for B cell maturation.

[0117] Poor B cell responses can explain the inconsistent antibody responses in subjects infected with SARS-CoV-2 and those with clinical manifestations of Covid-19. Therefore, using serology alone for policy-making in the clinical management and epidemiology of patients is unreliable as T cell responses should also be considered.

[0118] Vaccination strategies that target a large number of VIPs (such as targeting the Spike protein in SARS-CoV-2) can trigger counterproductive CD8 CTL responses and exacerbate disease upon natural infection, especially in vaccinees carrying HLA alleles that confer sensitivity to respond to such specific HLA-restricted epitopes.

[0119] Vaccination strategies aimed at precisely eliciting HLA-I-restricted CD8 T cell responses against non-VIP-derived epitopes to establish CD8 T cell memory responses would be beneficial, and the memory responses can drive virus clearance as early as possible during virus encounter and infection, without the risk of misdirected ADE or harmful CD8 CTL responses mediated by VIP.

[0120] Detailed model of SARS-CoV-2 immunopathogenesis

[0121] This section outlines a four-step model of immune response dysregulation initiated by initial immune escape caused by inhibition of innate immune signaling in virus-mediated infected cells by CD8 CTL responses early in infection. Innate immune signaling in professional APCs may subsequently be inhibited even in the absence of productive infection of these cells. Collectively, initial immune escape in infected cells redirects CD8 CTL responses against virion constituent proteins (VIP). In addition to the early initiation of CD8 CTL-driven professional APC exhaustion, immune escape results in poor clearance of virus-infected cells, which are normally associated with the resolution of the inflammatory response following significant virus clearance. This model of immunopathology can explain many clinical observations associated with severe Covid-19 clinical manifestations, in addition to asymptomatic or mild disease in young individuals with confirmed SARS-CoV-2 infection. This model has direct implications for understanding the epidemiology of the SARS-CoV-2 pandemic, compared to unreliable serological testing of significantly weak and transient Ig responses in young individuals with SARS-CoV-2 infection, by detecting CD8 T cell effector and memory populations to more reliably detect current mild or past infection. This model supports the sole and selective use of non-VIP-derived HLAI-HRE in vaccine compositions for the prevention or treatment of SARS-CoV-2 infection, where eliciting CD8 CTL responses against non-VIP-derived HLAI-HER is considered necessary and sufficient for protective immunity against SARS-CoV-2 and a range of other viral infections.

[0122] Step 1: Initial SARS-CoV-2 infection and immune escape of CD8 T cells. Figure 1 。

[0123] Figure 1 Illustrate that initial SARS-CoV-2 infection leads to escape of innate immunity mediated by virus-encoded proteins and its mediation of escape of early adaptive CD8 T cell responses.

[0124] A) Virion entry into target epithelial cells occurs through docking of the ACE2 complex and involves furin pre-cleavage in addition to host cell serine protease action, especially TMPRSS2, for spike complex processing and entry into host cells from endocytic compartments.

[0125] B) Entry into host cells leads to complex transcription of the viral genome, producing virus-encoded proteins (VEP).

[0126] C) The virion - associated proteins (VIPs) of known coronaviruses include factors responsible for immune - escape activity that bind to both structural and non - structural proteins. It is thought that the delivery of VIPs conferring immune - escape activity inhibits the innate immune response immediately in infected cells by delivering these VIP factors from the virion envelope to the host membrane. Transcription of the viral genome drives the further expression of accessory proteins to enhance this immune - escape activity.

[0127] D) Non - virion - associated proteins (non - VIPs) produced by viral genome transcription can further participate in the inhibition of the host cell innate immunity.

[0128] E) The main means by which VEP drives innate immune suppression is to block the type I interferon (IFN I) response.

[0129] F) Inhibition of the IFN I response in infected host cells results in reduced recruitment and sampling of naive CD8 T cells, thus reducing the ability to detect and respond to viral HLA - I - restricted epitopes in the early stages of infection. Therefore, the relative availability of naive and active memory CD8 is crucial for clearing early - stage infections at this stage.

[0130] G) Through immune - escape from the front - line CD8 T - cell response, infected cells can freely carry out viral genome replication and de novo virion genesis.

[0131] H) The release of de novo - synthesized virions from infected cells further exacerbates host cell infection.

[0132] Second step: Virus dissemination and immune activation. Figure 2 。

[0133] Figure 2 Illustrate that the infection established during initial immune - escape leads to a delayed recruitment of the immune response.

[0134] A) The accumulation of cellular dysfunction, VEP expression, viral genome replication, and loss of membrane integrity leads to the release of damage - associated molecular patterns (DAMPs) and pathogen - associated molecular patterns (PAMPs).

[0135] B) Innate immune receptors in both local healthy epithelial cells and resident alveolar macrophages recognize DAMPs and PAMPs, inducing the production of pro - inflammatory chemokines and cytokines, including: IL - 6, MCP1, MIP1a, MIP1b, and IP - 10.

[0136] C) De novo - synthesized virions released from infected host cells infect neighboring epithelial cells and initiate the spread of the virus to other host cells.

[0137] Step 3: Late initiation of adaptive immunity and misdirected CD8 CTL responses. Figure 3 。

[0138] Figure 3 Illustrate that the late initiation of the adaptive immune response leads to a bias in the sampling of professional APCs for accumulated virions and the directed T cell response against HLA-restricted antigens derived from the virion constituent proteins. This is a correctly directed response for HLAII / CD4 help and B cell responses. This can productively balance the HLAI / CD8 CTL response, where the HLAI / CD18 CTL response should target non-virion proteins highly expressed in productively infected cells rather than those present in virions.

[0139] A) Cell death of chronically infected epithelial cells leads to mucosal permeability, cellular infiltration into the lung cavity, edema, and the initiation of infection. In particular, alveolar macrophages are important for this process and are responsible for phagocytosing infected cells and virions.

[0140] B) Viral spread increases virion production within the host and raises the extracellular virion titer, thus allowing further spread of the infection and sampling of virions by professional APCs and B cells to initiate the deeper adaptive immune responses typical of any form of infection.

[0141] C) Sampling of accumulated virions by DCs via the endocytic mechanism provides VIPs for the processing and presentation of HLA-restricted epitopes. It is unclear whether DCs are productively infected, and reports in the literature are conflicting. However, it is clear that delivery of virions to DCs can partially inhibit innate signaling, and DCs in contact with virions are significantly less activated. This DC activation typically triggers an increase in the processing and presentation of HLAI- and HLAII-restricted epitopes. In the absence of such activation, the overall detection of HLA-restricted epitopes by CD4 and CD8 T cells is insensitive – biasing the activation of T cell responses towards the most abundant proteins presented by virions (i.e., VIPs).

[0142] D) Transport of VIPs sampled from the extracellular environment for the processing and presentation of HLAI-restricted epitopes.

[0143] E) In the absence of productive infection of DCs and in the presence of inhibition of DC activation, naive CD8 T cells sampling HLAI-restricted epitopes presented by DCs are activated mainly against VIPs rather than non-VIPs. This can be considered incorrect priming, as ideally, the clearance of virus-infected cells mediated by CD8 CTLs would target non-VIPs highly expressed in productively infected cells.

[0144] F) Processing of VIPs sampled from the extracellular environment leads to the presentation of HLAII-restricted epitopes.

[0145] G) Naive CD4 T cells sampling HLA-II restricted epitopes presented by DC are activated mainly against VIP rather than non-VIP. This can be considered as proper immune priming because CD4 helper effector functions against VIP are necessary for B cell maturation into neutralizing antibody production.

[0146] H) B cell sampling of virions via BCR initiates the humoral response with intact CD4 helper effector support, providing the first low-affinity antibodies against VIP, along with the normal progression of clonal selection and isotype switching.

[0147] I) The virus further spreads to cells expressing ACE2, notably which apparently includes alveolar macrophages.

[0148] Step 4: Misdirected CD8 CTL responses lead to overall immune dysregulation. Figure 4 。

[0149] Figure 4 It is illustrated that misdirected CD8 CTL responses against HLA-I restricted epitopes from VIP cross-presented by professional APCs during cumulative viremia and tissue damage lead to local professional APC exhaustion, thereby resulting in T cell and B cell response dysregulation, which can potentiate cytokine storm in severe Covid-19 manifestations and lead to overall weak neutralizing antibody responses against SARS-CoV-2 infection.

[0150] A) Apoptotic and necrotic cells are marked by early antibody responses and undergo macrophage phagocytosis. Further disruption of epithelial cells leads to severe tissue damage of the respiratory mucosa and cumulative mucus production, accompanied by cellular infiltration, edema and worsening infection.

[0151] B) Substances phagocytosed by macrophages can contribute to VIP-derived HLA-restricted epitopes through presentation by these professional APCs. Alveolar macrophages are known to express ACE2 and are thought to be infected. Due to strong innate PAMP receptor expression and generally enhanced innate immune pathways in macrophages, it is uncertain whether this infection is productive under most physiological conditions (i.e., inhibition of viral transcription and replication). In any case, in addition to free virions that are also phagocytosed, these cells can also be sensitive to the already initiated CTL responses against VIP by continuously phagocytosing abundant VIP proteins in infected cells and dead cells.

[0152] C) Cumulative CTL responses against VIP-derived HLA-I restricted epitopes can contribute to macrophage exhaustion via antigen-specific CTL action during immune response dysregulation.

[0153] D) Persistent DC sampling of virions in viremia supports the cross-presentation of VIP-derived HLAI-restricted epitopes by these cells.

[0154] E) The cumulative CTL response against VIP-derived HLAI-restricted epitopes can contribute to the depletion of macrophages by antigen-specific CTL action during immune response dysregulation, resulting in a lack of professional APCs in MALT and DLN, thus contributing to T cell and B cell response dysregulation, leading to cumulative immune dysfunction and cytokine storm.

[0155] F) Depletion of DCs in MALT and DLN results in dysregulated and inefficient CD4 T cell responses, as well as a lack of CD4 helper effector function to drive the maturation of B cells into high-quality and diverse neutralizing antibody responses.

[0156] Epidemiological implications of SARS-CoV-2 infection and other viral infections in humans

[0157] It is recognized that a large proportion of subjects infected with SARS-CoV-2 do not generate high-quality or even detectable antibody responses. The mechanism behind this is unclear, but dysregulation of the B cell maturation response mediated by the depletion of professional APCs caused by misdirected CD8 CTL activity will result in the inability to reliably detect antibody responses even in convalescent Covid-19 patients.

[0158] In addition, just as phagocytic and endocytic sampling by macrophages and DCs can respectively cause partial immunosuppression of antigen presentation activity, B cells can be vulnerable to virus-mediated inhibition during viremia. In fact, BCR-driven B cell endocytosis during viral epitope sampling will lead to the internalization of virions, which include innate immunosuppressive factors expressed in their envelopes. This alone can see the leakage of immunosuppressive VIP through membrane fusion events between host cells and viral envelopes, transmitting innate immunosuppression to B cells and thus weakening overall B cell maturation. Of course, in the absence of productive infection and destruction of macrophages, DCs or B cells, the sampling of virions delivers high doses of VIP-derived HLAI-restricted epitopes, which can render these professional APC populations vulnerable to misdirected CTL action, which is harmful to the overall adaptive immune response by limiting support for antigen-specific CD4 T cell responses and B cell maturation.

[0159] Many studies have linked productive and non-productive infections of professional APC populations to dysregulated IFN-I responses and reduced professional APC maturation, which can be linked to the direct inhibition of innate signaling pathways by VEP. This can contribute to poorer overall immunopathogenesis and exacerbate the impact of cumulative adaptive immune dysfunction in individuals with overt COVID-19.

[0160] Regardless of the exact mechanisms underlying adaptive immune dysregulation, it is clear from emerging reports that serology is unreliable in subjects with asymptomatic or mild Covid-19 presentations. Moreover, it is unclear to what extent subjects with asymptomatic or subclinical SARS-CoV-2 infections mount detectable antibody responses. In contrast, it appears that most subjects who have cleared subclinical SARS-CoV-2 do so by mounting sufficient CD8 CTL responses against VEP and may never mount antibody responses against VIP.

[0161] It is currently unclear whether subjects with mild infections mount CD8 CTL responses against other VIP- and / or non-VIP-derived HLA-restricted epitopes. Due to the historically limited depth of analysis of antigen-specific T cell responses, it is unclear what role CD8 CTL responses against specific VIP- and non-VIP-derived HLA-I-restricted epitopes play in viral clearance during subclinical infections with any virus in human subjects.

[0162] Risks of vaccines encoding VIP

[0163] This article does not purport to explain the general risks of adverse events when deploying VIP-centered vaccines in the population. However, as described above, with respect to the immunopathologic mechanisms of severe Covid-19 presentations, there are specific risks associated with eliciting sub-neutralizing Ig responses and, in fact, transient neutralizing Ig responses in vaccinees using VIP-centered compositions.

[0164] Primarily, incomplete or transient Ig-driven protection in vaccinees against subsequent natural SARS-CoV-2 infections can contribute to vaccine-associated disease worsening through two different mechanisms. First, sub-neutralizing antibody responses can lead to antibody-dependent enhancement of Covid-19 by facilitating the uptake of partially opsonized (i.e., non-neutralizing) viral particles by professional APCs and promoting misdirected CD8 CTL responses against VIP-derived HLA-restricted epitopes. Second, full-length VIP (including the "spike" protein) can equally trigger CD8 T cell responses, which can lead to accelerated immunopathogenesis and reduced ability to clear virus during subsequent natural infections.

[0165] Even in vaccinees with productive Ig-driven protective immunity using VIP-encoding vaccines, which concomitantly elicit potentially harmful CD8 CTL responses against VIP-derived HLA-I-HRE, the decline in Ig titers over time can lead to worsening of vaccine-associated disease because memory CD8 CTL populations remain in circulation for a long time after protective Ig titers wane.

[0166] In fact, despite decades of research, there remains a lack of effective vaccines against similar lower respiratory tract viral infections such as respiratory syncytial virus (RSV), and only partially effective vaccines against, for example, influenza. This can be due to the combination of viral vaccine composition and delivery, but in light of the above observations, this can also be due to the dual priming of beneficial neutralizing antibody responses and harmful CD8 CTL responses in inactivated virus vaccines or recombinant vaccines that deliver VIPs for the purpose of eliciting neutralizing antibody responses. Historical precedents of vaccine-associated disease exacerbation in RSV vaccination highlight the inherent risk of providing VIPs (e.g., via inactivated virus vaccines) as the primary antigen source during vaccination, while VIPs naturally do not provide non-VIP antigen targets.

[0167] Regarding the risks associated with the immunopathogenesis model presented above for VIP-centered vaccination strategies, inactivated virus vaccines, full-length VIPs, and VIP fragment (i.e., gene fragments or protein domains presenting neutralizing antibody epitopes) vaccine compositions inherently have a risk profile that, just in terms of the sequence space represented by each of these compositions, provides VIP-derived HLA-I restricted epitopes to prime potentially harmful CD8 CTL responses that can exacerbate disease during subsequent natural viral infections. In fact, for the emerging SARS-CoV-2 virus that has not been widely exposed to the human immune system and in the absence of long-term selection pressure from the human immune system, there is a high likelihood of the presence of a large number of HLA-I restricted epitopes within any VEP. This implies an enhanced risk of vaccine-associated disease exacerbation in vaccine strategies against SARS-CoV-2 compared to more established human viral pathogens of similar nature.

[0168] In any case, especially in the context of rapid vaccine deployment, a major logistical challenge in detecting vaccine-associated disease exacerbation is that adverse events are not expected to occur at the initial vaccine challenge but rather during random subsequent viral infections. This is not only a technical challenge in the complexity of systematically evaluating T cell responses to HLA-restricted antigens, but also due to temporal and probabilistic factors, potential adverse events may only become apparent in later studies when many individuals have already undergone the vaccine challenge. Additionally, since VIP-derived HLA-I-HRE-specific CD8 CTL memory cells persist for far longer than significant VIP-directed Ig titres, such adverse events can occur months and years after the primary vaccine administration without subsequent booster vaccinations.

[0169] Next-generation vaccine compositions comprising non-VIP-derived HLA-I-HREs

[0170] Regarding the above observations presented, a vaccine composition comprising the selected non-VIP-derived HLAI-HRE is warranted. It is well recognized that a combination of CD8 T cell memory, neutralizing antibody titers, and B cell memory will provide optimal protection following vaccination. However, given the challenges and risks inherent in VIP-centered SARS-CoV-2 (and indeed other lower respiratory tract invasive viral infections) vaccine strategies, highly selective incorporation of non-VIP-derived HLAI-HRE may be the safest and most productively deployable means of vaccinating. That is, non-VIP-derived HLAI-HRE is necessary and sufficient to confer protective immunity through selective CD8 CTL responses against SARS-CoV-2 and a range of other human viral pathogens.

[0171] Vaccines that do not seek to elicit B cell responses against VIPs but rather CD8 T cell responses specific for non-VIP antigens may potentially have enhanced safety. Indeed, due to the complex nature of human HLA-restricted antigen presentation, it is quite reasonable to assume that both vaccine efficacy and vaccine-related adverse events can be closely predicted by the recipient's HLAI haplotype and to a lesser extent by the HLAII haplotype.

[0172] In terms of controlling adverse events, of course, T cell responses against immunodominant HLA-restricted epitopes (potentially derived from VIPs) in patients and vaccine recipients can be quantified by established laboratory techniques (i.e., ELISPOT assays on original samples). However, due to poor sensitivity and resolution, the identification, classification, and characterization of HLAI-restricted antigenic epitopes that elicit subdominant T cell responses in clinically manifest SARS-CoV-2 infections cannot be addressed by such techniques.

[0173] High-resolution and systematic techniques for the precise analysis of the putative HLAI-restricted epitopes (i.e., non-VIP-derived HLAI-HRE) and their immunogenicity lay the foundation for the next generation of vaccines against emerging SARS-CoV-2 and viruses with similar infection life cycles. Given the limited payloads carried by modern recombinant RNA, DNA, and protein vaccine vectors, the compact nature of HLAI-HRE allows for maximized epitope coverage to match the HLA haplotypes of the target population to be vaccinated. Even with the most restrictive recombinant RNA vaccine vectors, at least 60% HLAI allele coverage can be achieved worldwide with a limited set of non-VIP-derived HLAI-HRE, especially in conferring population-wide protection in the rapid deployment of vaccines against emerging viral pathogens.

[0174] In existing studies of similar invasive viral infections of the respiratory mucosa, several themes suggesting possible T cell immunodysregulation have emerged in addition to T cell- and immunoglobulin-related immunopathology.

[0175] From an important perspective, acute respiratory distress syndrome (ARDS) is associated with strong viral immunoglobulin responses in natural infections and in live vaccines. This implies that in persistent infections, robust HLAII / CD4 T cells contribute to driving high-titer antibody production, which can be a hallmark of excessive mucus production and respiratory distress. This may be related to poor control of infection by HLAI / CD8 responses in susceptible individuals early in infection, followed by high viral titers driving strong HLAII / CD4 helper responses and B cell maturation. This is consistent with the observed strong correlation between severe Covid-19 manifestations and age, as well as the near-complete absence of SARS-CoV-2 infection symptoms in adolescents and young adults. It is well known that the size of the naive T cell population decreases with age; however, there is also a significantly faster relative decline in the naive CD8 population compared to the naive CD4 population. Older subjects may develop immunodysregulation, in part due to a naturally declining CD8 T cell response associated with the naive CD8 T cell repertoire, which permits progression of infection and viremia, thus driving B cell maturation and immunoglobulin-related immunopathology.

[0176] To support this view of Covid-19 disease progression, it has been observed that subjects recovering from the SARS-CoV virus associated with the 2003 SARS pandemic had weak and short-lived virus-specific immune responses that lasted only a few months. In contrast, in recovering subjects, a significant CD8 T cell memory population was observed to persist one year after infection.

[0177] Currently, many strategies for live vaccines against SARS-CoV-2 focus on the spike protein as a key antigen payload. Notably, this is likely to be counterproductive if immunoglobulin-related immunopathology contributes to severe Covid-19 inflammatory manifestations. Indeed, this also seems to be the case in similar attempts to vaccinate against other viral infections of the respiratory mucosa.

[0178] In addition, an important aspect of the viral life cycle is that, during natural infection, the spike protein is a poor library of HLA-I restricted epitopes. In fact, the spike protein is a membrane component that is produced and inserted into the ER membrane and then transported to vesicles where viral particle assembly occurs and then buds out. This means that the spike protein is largely isolated from the cytosolic pathway that is a major contributor to antigen processing and cross-presentation of HLA-I restricted epitopes. This not only limits the effective priming potential of CD8-centered immune priming, but the spike protein itself is more likely to drive HLA-II / CD4 priming and the initiation of an immunoglobulin response, leading to exacerbation of vaccine-induced disease upon subsequent natural infection.

[0179] Given the above observations, it can be hypothesized that immunization with HLA-restricted epitopes to promote the priming of a CD8 T cell immune response is a safe and productive way to provide significant protection against SARS-CoV-2 infection. A highly defined payload encoding HLA-I restricted epitopes will help establish a CD8 T cell memory response as the frontline protection against natural infection while avoiding potentially harmful priming of HLA-II / CD4 or immunoglobulin responses. This approach requires the ability to accurately identify and characterize HLA-restricted epitopes, as well as the CD8 T cell responses to these epitopes during natural infection.

[0180] An important feature of coronaviruses and other invasive viruses that infect the respiratory mucosa is the regulatory proteins encoded in their genomes, which disrupt the innate immunity within cells and may lead to T cell immune dysregulation. In fact, the SARS-CoV virus that caused the 2003 SARS pandemic encoded three ORFs that were able to disrupt the innate immune response leading to interferon production, and they are similarly included in the SARS-CoV-2 genome. These cytosolic available proteins represent ideal targets and natural HLA-I restricted epitope libraries during natural infection. The ideal design of a vaccine payload would incorporate tandem ORF fragments from these viral proteins to avoid T cell dysregulation during vaccine delivery while still promoting a strong HLA-I restricted epitope-specific CD8 T cell response.

[0181] Due to the obvious central role of T cell immunity in SARS-Cov-2 immunity and immunopathology, the safe and effective deployment of a vaccine must incorporate targeted immunity against HLA-restricted epitopes. Given the huge diversity of haplotypes between ethnic categories and geographical regions, this is a major challenge. Preparing for the design and deployment of vaccines for such market or population segments is a major challenge, and while this represents a worse-case scenario for future SARS-CoV-2 outbreaks or pandemics, it is a strategic aspect that cannot be ignored.

[0182] One important strategic aspect to consider is to more deeply analyze the HLA-restricted epitopes of selected viral ORFs in the existing HLA allele core set, and / or expand the scope of this analysis to cover different HLA I haplotypes that are of strategic importance in the global SARS-CoV-2 pandemic.

[0183] A key point in controlling the emerging pandemic is the ability to systematically evaluate potentially emerging immune escape virus strains. It should be noted that the novel coronavirus SARS-CoV-2 may have originated from a transfer from other animal species to humans, and thus has not been widely exposed to the human immune system and the selective pressure of the human immune system. It is reasonable to assume that a large number of HLA I-restricted epitopes will be presented by the emerging SARS-CoV-2 strains driving the pandemic that began in 2019, and with widespread infection, new strains may emerge that contain mutations that escape the presentation of specific HLA I-restricted epitopes.

[0184] Example 2: Method for determining immunogenic non-VIP-derived HLA I-HREs to be incorporated into a vaccine composition

[0185] This example outlines a method for selecting non-VIP-derived HLA I-HREs for use in generating a vaccine composition using the SARS-CoV-2 virus infection outlined in Example 1.

[0186] This section outlines methods for rapidly identifying and evaluating the immunogenicity of HLA-restricted epitopes from the SARS-CoV-2 genome. The T cell immunogenicity of these epitopes was analyzed in three different groups: patients with severe Covid-19 manifestations, subjects known to be infected but with mild symptoms, and healthy donors not infected with SARS-CoV-2.

[0187] The purpose of this overall analysis is to identify the HLA-restricted epitopes that most effectively mediate virus clearance and potentially identify HLA-restricted epitopes that drive dysregulation of antigen-specific T cell responses. These analyses enable the next generation of valuable vaccines to provide key data on antigen-specific T cell responses in uninfected subjects, patients with severe Covid-19, and patients in whom the infection has resolved.

[0188] These example methods can be divided into 5 key work packages:

[0189] Work Package 1 (WP1)

[0190] SARS-CoV-2 HLA I-restricted epitope discovery

[0191] Work Package 2a (WP2a)

[0192] Antigen-specific CD8 T cell activation and TCR screening

[0193] Work Package 2b (WP2b)

[0194] Generation of HLAI multimers and TCR reagent libraries

[0195] Work Package 3 (WP3)

[0196] Validation of HLAI antigen-specific T cell assays

[0197] Work Package 4 (WP4)

[0198] Execution of studies in Covid-19 and uninfected subjects

[0199] SARS-CoV-2 HLAI-restricted epitope discovery – WP1

[0200] Purpose:

[0201] To perform the first complete HLAI-restricted epitope scan of all SARS-CoV-2 ORFs to identify functionally processed and cross-presented HLAI-restricted epitopes within the 16 most prevalent alleles in major markets.

[0202] To provide a foundational dataset for the selection of non-VIP-derived HLAi-HREs for use in the studies performed in WP2a, WP2b, WP3, and WP4.

[0203] Techniques and methods:

[0204] The most basic form of an engineered antigen-presenting cell (eAPC) system is a system for the rapid generation of eAPC cell lines expressing the analyte of interest. These analytes represent only the target HLA alleles and target antigen open reading frames (ORFs) in which the target HLA-restricted epitopes will be identified. This is achieved through standardized donor vectors for HLA and antigen ORF constructs that are paired with genomic acceptor sites within a functionally engineered immortal cell line representing the “programmable” eAPC (WO2018083316).

[0205] This eAPC platform provides high-throughput or high-content generation of analyte eAPCs to supply mass spectrometry (MS)-based methods for the identification of HLA-restricted antigens from the integrated analyte ORFs within the context of the native HLAI-restricted repertoire derived from the eAPC protein itself.

[0206] This enables systematic analysis of analyte ORFs in a single HLA background (i.e., "monoallelic") by direct observation of epitopes from functional processing and presentation of the analyte sequence. Optionally, a series of expression constructs incorporating tandem microgene analytes, and optionally proteasome targeting motifs that enhance cytoplasmic processing of the analyte protein in an expression system, can be used. This mass spectrometry readout is achieved by sample preparation with capture of HLA from eAPC cell lysates, liquid chromatography fractionation of the samples, and then MS identification of HLA-restricted epitopes.

[0207] The set of 16 most common alleles forms a core working set for HLA-restricted antigen discovery. As Figure 5 shown, these are selected to capture optimal HLA coverage of the major markets.

[0208] Deliverables:

[0209] A database of SARS-CoV-2 HLA-restricted epitopes presented by the selected 16 core HLA alleles.

[0210] Metadata includes relative intensity scores (z-scores) for the eAPC intrinsic HLA-restricted epitope repertoire, and scores for the HLA allele epitope concordance model.

[0211] Antigen-specific CD8 T cell activation and TCR screening – WP2a

[0212] Objectives:

[0213] Establish validated immunoresponsive HLA-restricted epitopes identified in WP1 by unbiased screening of uninfected and SARS-Cov-2-infected subjects.

[0214] Evaluate the sensitivity of the identified HLA-restricted SARS-CoV-2 epitopes based on CD8 T cell activation and tetramer-based assays.

[0215] Provide materials for TCR validation and analysis standards in WP2b.

[0216] Techniques and methods:

[0217] The eAPC platform described in WP1 enables reliable and highly defined HLA-restricted antigens to be presented to T cells from uninfected donors ( Primary CD8 T cell isolates from donors or patients. This is a key component for measuring HLA-restricted epitope responses in cell-based assays, which further enhances TCR discovery capabilities. In fact, "monoallelic" eAPCs are a reliable and reproducible model for stimulating and testing HLA-restricted epitope responses in T cell subsets, without the need for complex isolation and culture of multiple primary cell types from each sample. This is particularly important for identifying immunogenic HLA-restricted epitopes during natural viral infections.

[0218] This work package effectively deploys a set of technologies and methods;

[0219] 1. eAPC-based HLA allele-restricted stimulation of naive and memory CD8 T cell populations isolated from uninfected and SARS-CoV-2-infected subjects (WO2018083316).

[0220] 2. HLA multimer reagent-based quantification of HLA-restricted epitope-specific T cell responses after eAPC stimulation and growth.

[0221] 3. Single-cell deposition of tetramer-positive cells by flow cytometry for downstream TCR clonotype sequencing and validation.

[0222] 4. eAPC-based restimulation of outgrown primary CD8 T cell isolates to read out HLA-restricted epitope T cell responses via T cell activation markers (primarily INFγ production).

[0223] The central aim of the comprehensive assessment of HLA-restricted epitope immunogenicity is to provide an unbiased landscape of potential immunogenicity for HLA-restricted epitopes encoded in the SARS-CoV-2 genome. This work package will further serve as a preliminary assessment of the sensitivity of tetramer-based and cell activation-based flow cytometry assays in uninfected and infected subjects.

[0224] It should be noted that all four of these technologies can be applied to a single clinical sample, which saves the use of the original sample and significantly increases throughput. This is further aided by the routine multiplexing of HLA multimer reagents in this analysis.

[0225] Regarding the restimulation method, after stimulating primary cells using an eAPC-based HLA allele-restricted system, the restimulation process is carried out using eAPCs constructed from a completely different cell line. This design is to eliminate background signals when using non-self APCs as the previously encountered stimulation platform.

[0226] Deliverables:

[0227] The metadata layer on the HLA-I restricted epitope database generated in WP1 details the immunogenic potential of HLA-restricted epitopes observed during detection.

[0228] Preliminary assessment of the sensitivity of HLA-restricted epitope T cell responses in SARS-CoV-2 infection analysis based on cell activation assays and HLA-based flow cytometry assays.

[0229] Validated TCR sequences against immunogenic HLA-restricted epitopes directly supporting WP2b, WP3, WP4.

[0230] Generation of HLA-I multimers and TCR reagent libraries – WP2b

[0231] Objective:

[0232] Use the matching engineered TCR-presenting cells (eTPCs) expressing TCRs analysis criteria to construct an internally validated HLA multimer reagent library.

[0233] Operationalize the generation of all HLA multimer reagents in the target library to meet the reagent requirements of WP3 and WP4.

[0234] Generate eTPC analysis criteria expressing TCRs for flow cytometry assays based on HLA multimer reagents to be deployed in WP3.

[0235] Techniques and methods:

[0236] HLA multimer generation techniques have been highly standardized and are well-known to those skilled in the art. However, these methods involve challenging protein biochemistry, including protein folding and purification. The operationalization of these generation workflows is driven by providing reliable functional quality control criteria in specific eTPC cell lines expressing TCRs.

[0237] This work package demonstrates a series of TCR molecular genetics and cell biology techniques to construct eTPC criteria using materials derived from WP2a. These techniques can be summarized into four key workflows:

[0238] 1. Semi-automatic TCRα / β chain sequencing and bioinformatics filtering using proprietary primer libraries and software.

[0239] 2. PCR-independent rapid TCRα / β ORF reconstruction using the TCR molecular genetics platform – TCR ORF Reconstruction and Engineering System (TORES) – which enables rapid and low-cost reconstruction of full-length TCR ORFs in expression vectors (WO2019016175 and WO2018083318).

[0240] 3. High-throughput screening based on eTPC is performed by integrating paired TCRα / β ORFs to screen for target binding of HLA multimer reagents and / or interaction with eAPCs presenting target HLA and HLAI-HRE (WO2018083317 and WO2018083339 and WO2018083318).

[0241] 4. High-throughput preparation of eTPC standards expressing TCR, with stable chemically fixed assay standard reagent products (WO2018083317).

[0242] These methods enhance the ability to generate large high-quality HLA and TCR reagent libraries that can be deployed for assay development and execution with unprecedented depth and precision.

[0243] Deliverables:

[0244] A library of HLA multimer reagents for deployment in subsequent work packages.

[0245] An eTPC-based assay standard library for quality control of HLA multimer reagent preparation and as an internal control for flow cytometry assay operationalization in WP3.

[0246] Operationalization of HLAI antigen-specific T cell assays - WP3

[0247] Objective:

[0248] To operationalize HLA multimer reagent-based assays for direct staining of peripheral blood and potentially liquid biopsies, with flow cytometry readouts of target HLA alleles and restricted epitope coverage to support the study of antigen-specific T cell responses in WP4.

[0249] To operationalize assays based on eAPC-driven cell activation for indirect assay readouts to support parallel deployment in WP4.

[0250] Techniques and methods:

[0251] The technical and methodological aspects of the scalable deployment method for reagent-based assays are to use eTPC-based assay standards expressing TCR. This not only allows for reliable production quality control of the reagents specified in WP2b but also serves as an internal positive control standard for these flow cytometry assays. The eTPC expressing a specific TCR is chemically fixed and stored as a stable reagent together with the HLA multimer reagent mother liquor for assay execution.

[0252] In cell activation-based assays, eAPC-driven HLA-restricted epitope stimulation of primary T cell isolates can directly detect the presence of antigen-specific T cell clones in the CD8 T cell memory population against viral epitopes. This detection method is also sensitive enough to detect antigen-specific T cell clones in naive cell populations. These assays rely on peptide loading of a monoallelic eAPC preparation expressing the desired HLA allele, followed by contact with CD8 T cell isolates from an individual to stimulate the memory and / or naive T cell population. This enables relatively high-throughput parallel analysis of HLA-restricted epitope-specific T cell responses in the memory and naive T cell compartments compared to using primary APC sources in standard mixed leukocyte responses.

[0253] Work around the operationalization of reagent-based and activation-based assays in the current work package involves accurate HLA typing of all subjects to assign the correct HLA multimer reagents and / or peptide-loaded monoallelic eAPCs for the analysis of individual subjects.

[0254] Deliverables:

[0255] HLA multimer reagent-based assays in support of the operationalization of WP4.

[0256] Cell activation-based assays in support of the operationalization of WP4 in terms of the selected HLA alleles and HLA-restricted epitope coverage.

[0257] Execution of patient and uninfected subject studies - WP4

[0258] This work package aims to conduct a systematic study of non-VIP-derived HLA-I-HRE responses in human subjects to identify immunogenic epitopes driving viral clearance. The tools and assays in WP1, WP2a, WP2b, and WP3 enable these studies.

[0259] Objective:

[0260] Conduct comparative studies in primary samples from uninfected and SARS-CoV-2-infected subjects to select immunogenic non-VIP-derived HLA-I-HREs suitable for inclusion in vaccine compositions.

[0261] Techniques and methods:

[0262] The current work package deploys the reagents and assays established in previous work packages, with a focus on a comparative analysis of antigen-specific T cell responses in subjects with known SARS-CoV-2 infection resolution and no severe clinical manifestations versus individuals with severe Covid-19 manifestations. In identifying the major antigen-specific CD8 T cell responses for different SARS-CoV-2 outcomes, the central aim is to identify non-VIP-derived HLA I-HREs that are confirmed to be immunogenic in human subjects to define vaccine compositions. The primary sample type in these studies is peripheral blood from HLA-typed subjects, as this type of analysis is relatively easy to scale up.

[0263] Deliverables:

[0264] Conduct a systematic comparative analysis of immune responses to non-VIP-derived HLA I-HREs in the following categories of human subjects:

[0265] 5. Subjects with asymptomatic SARS-CoV-2 infection

[0266] 6. Subjects with resolved symptoms of SARS-CoV-2 infection

[0267] 7. Subjects with severe Covid-19 disease

[0268] 8. Subjects known to be uninfected with SARS-CoV-2

[0269] Non-VIP-derived HLA I-HREs identified in each subject category are preferentially incorporated into the vaccine composition in the order 1>2>3>4.

[0270] Definitions

[0271] Vaccine

[0272] A preparation that generally contains antigenic components of microorganisms, viruses, or other self or non-self substances, which, when administered to human or veterinary subjects, is intended to elicit an adaptive immune response in the host to provide the host with immunity or tolerance to the target microorganism, virus, allergen, or other non-self or self substance.

[0273] Vaccine composition

[0274] Antigens and adjuvants delivered by a vaccine vector, which, when formulated and administered to human or veterinary subjects, are intended to elicit the desired immune response.

[0275] Vaccine vector

[0276] A vaccine vector in the context of this text is defined as a vector for administering a vaccine composition to a human or veterinary subject and includes: recombinant non-replicating or replicating viral vectors; virus-like particles; recombinant RNA constructs; recombinant DNA constructs; recombinant proteins and / or protein complexes; synthetic polypeptides.

[0277] Vaccine adjuvant

[0278] A molecule or compound having inherent immunomodulatory properties that, when co-administered with an antigen, can effectively enhance the host's antigen-specific immune response compared to the response generated when the antigen is administered alone. Some viral vectors and virus-like particle vectors are considered to have inherent adjuvant molecules and compounds, while additional co-stimulatory molecules can be encoded in nucleic acid, protein, and polypeptide sequences or conjugated to these antigenic biomolecules, which comprise the vaccine composition. Adjuvant molecules and compounds can further be included in the vaccine formulation.

[0279] Vaccine formulation

[0280] A preparation for administering to a human or veterinary subject, which is a formulation in which a vaccine vector is formulated together with a pharmacologically suitable vaccine adjuvant and excipients, where the excipients can include: anti-adhesives, binders, coatings, pigments, disintegrants, flavors, glidants, lubricants, preservatives, adsorbents, sweeteners, and vehicles.

[0281] Prime-boost vaccination

[0282] A sequential vaccination strategy in which a vaccine is delivered to a human or veterinary subject on multiple occasions to "prime" the desired antigen-specific immune response and then these immune responses are boosted with subsequent vaccine administrations. The prime-boost vaccine strategy can be administered in a homologous manner, where the vaccine composition, vaccine vector, and / or vaccine formulation and / or vaccine route can be the same for both the priming and boosting administrations. The prime-boost vaccine strategy can also be implemented in a heterologous manner, where the vaccine composition, vaccine vector, and / or vaccine formulation and / or vaccine route can be different for the priming and boosting administrations.

[0283] Antigen

[0284] A molecule, especially a biomolecule, that represents the target of the adaptive immune system; where in the context of the B-cell and T-cell systems, antigens are detected by immunoglobulins and T-cell receptors respectively.

[0285] Epitope

[0286] An epitope, also known as an antigenic determinant, is a part of an antigen that is recognized by the adaptive immune system, especially an immunoglobulin (Ig) or a T-cell receptor (TCR). That is, an epitope is the specific part of an antigen to which an immunoglobulin or a T-cell receptor binds.

[0287] Human leukocyte antigen (HLA)

[0288] The human leukocyte antigen (HLA) system or complex is a gene complex encoding the major histocompatibility complex (MHC) proteins in jawed vertebrates. For clarity, the HLA nomenclature is used herein to describe the genes (i.e., HLA) and protein complexes (i.e., MHC).

[0289] Human leukocyte antigen class I (HLA I)

[0290] The HLA corresponding to MHC class I (A, B, C, and E), which includes the HLA class I group.

[0291] Human leukocyte antigen class II (HLA II)

[0292] The HLA corresponding to MHC class II (DP, DM, DO, DQ, and DR), which includes the HLA class II group.

[0293] HLA-restricted epitope (HRE)

[0294] Any epitope that is loaded into an HLA molecule and presented on the cell surface for T-cell sampling by the TCR. Generally, for HLA I and II molecules, these epitopes represent peptides. Non-peptide molecules can also be loaded into HLA for presentation on the cell surface.

[0295] HLA I-restricted epitope (HLA I-HRE)

[0296] Any epitope that is specifically loaded into an HLA I molecule for export to the cell surface for T-cell sampling.

[0297] HLA II-restricted epitope (HL II-HRE)

[0298] Any epitope that is specifically loaded into an HLA II molecule for export to the cell surface for T-cell sampling.

[0299] Viral encoded protein (VEP)

[0300] All proteins encoded in the viral genome, including post-translational modifications of the protein.

[0301] Virus particle

[0302] The entire virus particle.

[0303] Virus particle constituent protein (VIP)

[0304] Any protein encoded by the viral genome that appears within the virus particle when the virus particle is released from an infected host cell following de novo virus particle production.

[0305] Non-viral particle constituent protein (non-VIP)

[0306] Any protein encoded by the viral genome that is expressed in an infected host cell during the viral life cycle but does not appear in the viral particle.

[0307] Antigen-presenting cell

[0308] Any cell that expresses HLA class I molecules and is capable of loading HLA class I-restricted epitopes and presenting these complexes on the cell surface for T cell sampling. Generally considered to be all nucleated cells of jawed vertebrates.

[0309] Professional antigen-presenting cell

[0310] Specialized APCs of the immune system that constitutively express HLA class II molecules and incorporate pathways in which extracellular component sampling by pinocytosis, endocytosis, and phagocytosis results in the presentation of HLA class II-restricted epitopes. The main types of professional APCs are dendritic cells, macrophages, and B cells.

[0311] T cell

[0312] T lymphocytes or T cells denote thymus-differentiated lymphocytes that detect epitopes via the T cell receptor (TCR) and mediate diverse adaptive immune functions.

[0313] TCR

[0314] Defined T cell immune receptor that is capable of detecting epitopes in an adaptive manner. There are α / β and γ / δ TCR systems, where α / β is generally responsible for detecting HLA class I and HLA class II-restricted epitopes.

[0315] Naive T cell

[0316] A naive T cell is a T cell that has differentiated in the bone marrow and has successfully undergone the positive and negative processes of central selection in the thymus.

[0317] Effector T cell

[0318] CD4 helper or cytotoxic CD8 T cells that have been activated by encountering productive epitopes and are thus committed to influencing the selected functions of the T cell.

[0319] Memory T cell

[0320] Committed effect T-cells can subsequently be committed to a memory state, which can represent a quiescent cell state until re-exposed to productive epitopes, and thus represents a central paradigm for acquired T cell immunity or tolerance to epitopes derived from associated antigens.

[0321] CD8 T cells

[0322] T cells that express α / α, α / β, or β / β CD8 receptor dimers on the cell surface and are generally responsible for sampling HLA I-HREs presented by APCs. The effector functions of CD8 T cells are most commonly cytotoxic, but can further include regulatory and helper functions.

[0323] CD4 T cells

[0324] T cells that express the CD4 receptor on the cell surface and are generally responsible for sampling HLA II-HREs presented by APCs. The effector functions of CD4 T cells are highly diverse and not fully understood, but are thought to be "helper" cells that detect diverse antigenic epitopes and coordinate the entire innate and adaptive immune systems through complex intracellular signaling networks.

[0325] Cytotoxic T lymphocytes (CTL)

[0326] T cells with cytotoxic effector functions, typically represented by CD8 T cells.

[0327] Helper T cells

[0328] A general definition for those T cells that do not have cytotoxic effector functions in effector T cell function, most commonly referring to CD4 T cells.

[0329] T regulatory cells (Tregs)

[0330] An effector T cell that specifically confers immune tolerance as a central effector function through a series of immunosuppressive signaling, can be represented by CD4 and CD8 T cells, but is most commonly CD4 T cells.

[0331] B cells

[0332] B lymphocytes, or B cells, are lymphocytes that differentiate in the bone marrow and have successfully undergone both positive and negative selection before leaving the bone marrow. B cells are defined by the surface B cell receptor (BCR), which represents membrane-bound immunoglobulin (Ig) and has a similar genetic and protein structure to the TCR. The BCR is crucial for detecting initially encountered Ig epitopes, and this initial encounter triggers the gating process of B cell maturation, which requires continuous epitope availability and a series of helper T cell inputs to progress and results in the production of various forms of soluble immunoglobulins. B cells can also act as professional antigen-presenting cells, especially in terms of their ability to dock with helper T cells to receive signaling inputs.

[0333] Immunoglobulin (Ig)

[0334] Often called antibodies, these soluble adaptive recognition molecules are produced by mature B cells and can mediate a series of effector functions in the host immune response by binding to associated epitopes.

[0335] Neutralizing immunoglobulin (neutralizing Ig)

[0336] Immunoglobulins that target epitopes usually derived from non-self antigens and serve as a generalized effector function for pathogen or non-self molecule neutralization.

[0337] Dendritic cells (DC)

[0338] Professional antigen-presenting cells that are central mediators of T cell and B cell responses.

[0339] Macrophages

[0340] Professional antigen-presenting cells that are central mediators of T cell and B cell responses and are usually defined by their phagocytic activity.

[0341] Self-antigen

[0342] Antigens derived from the host organism that are related to adaptive immunity.

[0343] Non-self antigen

[0344] Antigens derived from outside the host organism that are related to adaptive immunity, usually referring to antigens from pathogens and allergens, but can also include antigens from food and commensal microorganisms.

[0345] Type I interferons (IFN-I)

[0346] A family of cytokines found in mammals that bind to interferon receptors and help regulate the immune system.

[0347] Draining lymph nodes (DLN)

[0348] Lymph nodes draining the tissue of interest, usually refer to lymph nodes draining infected tissue, tissue containing dysplasia, malignancy or directly exposed to allergens, commensal microorganisms and / or food.

[0349] Open reading frame (ORF)

[0350] The part of a nucleic acid reading frame that can be transcribed to produce an ORF product or a gene transcript.

[0351] Immunogenicity

[0352] A general term indicating the potential of a given antigen or B-cell epitope or T-cell epitope to elicit a measurable response in the B-cell and T-cell systems, respectively.

[0353] Active infection

[0354] A viral infection in which the virus continuously replicates and releases de novo synthesized virions from infected host cells and infects other host cells.

[0355] Resolved infection

[0356] A viral infection that has been cleared by the host immune system.

[0357] Latent infection

[0358] A viral infection that has caused the host immune system to suppress viral replication and has led to the retention of viral genomic material within host cells, which can undergo reactivation and de novo virion production in the future to establish an active infection.

[0359] Clinical manifestation

[0360] A viral infection with significant clinically relevant manifestations.

[0361] Figure frame:

[0362] Figure 1 , Box 1:

[0363] Elderly subjects

[0364] The extent of immunodeficiency; significantly a reduction in the naive T cell repertoire.

[0365] Elderly subjects have lower naive CD8 T cell surveillance in the early stage of infection.

[0366] Escape CD8 CTL activation in the early stage of infection.

[0367] Result in a reduced and / or delayed potential to clear virus-infected cells.

[0368] Figure 2 , Box 1:

[0369] Systemic immune activation

[0370] Cell damage releases DAMPs and PAMPs that are recognized by neighboring cells and macrophages to trigger cytokine and chemokine production.

[0371] Immune cell recruitment drives more stringent sampling of cells by T cell populations. The arrival of higher numbers of dendritic cells in MALT and DLN and responses that facilitate sampling of CD8 and CD4 HLA-restricted epitopes.

[0372] Figure 2 , Box 2:

[0373] Viral transmission

[0374] Immune escape allowing establishment of infection by activation of frontline CD8 CTLs against viral HLA-I-restricted epitopes.

[0375] Figure 3 , Box 1:

[0376] Viral particle accumulation

[0377] Viral spread in epithelial cells allows accumulation of viral particles for further viral spread and sampling by professional APCs and B cells.

[0378] Figure 3 , Box 2:

[0379] Adaptive response

[0380] Widespread immune cell recruitment biases adaptive CD8, CD4, and B cell responses towards VIP.

[0381] Figure 4 , Box 1:

[0382] Viremia and tissue damage

[0383] Phagocytosed epithelial cells provide VEP for cross-presentation to CD8 T cells.

[0384] DCs sample high viral titers in the respiratory mucosa and draining lymph nodes for cross-presentation to CD8 CTLs.

[0385] Figure 4 , Box 2:

[0386] Dysregulated adaptive immune response

[0387] Initial immune escape based on inhibition of innate immune responses results in escape of productively infected cells from early CD8 T cell detection and delayed CD8 CTL responses.

[0388] Viral infection, cell death, immune recruitment, and the establishment of viremia drive the polarization of delayed adaptive CD8 CTL responses towards highly abundant VIP-derived HLAI-HREs.

[0389] CD8 CTL cytotoxic function leads to the depletion of professional APCs at the site of infection and in the DLN, allowing for the rapid accumulation of tissue damage and the reduction of DCs to direct CD4 helper cell responses.

[0390] Dysregulation of the coordination of T cell adaptive immunity leads to severe inflammation and cytokine storms. In addition, the weak help of CD4 T cells for B cell maturation results in impaired antibody responses.

Claims

1. A method for generating a vaccine composition against a viral pathogen, comprising: a. Identifying human leukocyte antigen class I-restricted epitopes derived from non-viral particle constituent proteins (non-VIP-derived HLAI-HREs) from a viral pathogen, wherein a vaccine composition against the viral pathogen is desired, said identification being performed using a standardized donor vector of HLA and antigen ORF constructs that are paired with genomic acceptor sites within a functionally engineered immortalized cell line representative of a programmable engineered antigen-presenting cell (eAPC); and then screening by a mass spectrometry (MS)-based method to identify HLA-restricted antigens from the integrated ORFs within the context of the endogenous HLAI-restricted repertoire derived from the eAPC protein itself; and enabling systematic analysis of the ORFs in a single HLA background (monoallelic); b. Classifying the immunogenicity of the identified non-VIP-derived HLAI-HREs from step a in naïve CD8 T cell populations isolated from donors without prior target virus infection and / or in memory CD8 T cell populations isolated from donors with confirmed active, latent, or resolved target virus infection; c. Selecting non-VIP-derived HLAI-HREs from step b that have confirmed immunogenicity in donors without prior target virus infection or non-VIP-derived HLAI-HREs that have observed CD8 T cell responses in donors with confirmed active, latent, or resolved target virus infection; d. Incorporating the selected non-VIP-derived HLAI-HREs from step c into the vaccine composition; and wherein the selected non-VIP-derived HLAI-HREs are provided by one or more of the following: i. Introducing point mutations and / or sequence insertions and / or sequence deletions that inactivate protein function within the full-length non-VIP ORF; ii. Constructing a synthetic nucleic acid sequence containing non-VIP ORF fragments encoding the selected HLAI-HREs in a tandem construct; iii. Constructing a synthetic nucleic acid sequence containing non-VIP ORF fragments encoding the selected HLAI-HREs within a vector protein sequence; wherein the non-viral particle constituent protein refers to any protein encoded by the viral genome that is expressed in an infected host cell during the viral life cycle but does not appear in the viral particle, wherein the viral pathogen is SARS-CoV-2.

2. The method according to claim 1, wherein multiple non-VIP-derived HLAI-HREs are selected and incorporated into the vaccine composition to represent one or more HLAI-HERs in the selected HLAI alleles, said selected HLAI alleles representing those alleles carried by at least 60% of the individuals within the target population for which the vaccine composition is designed.

3. The method according to claim 1, wherein one or more non-VIP-derived HLAI-HREs are selected and incorporated into the vaccine composition to represent one or more HLAI-HERs among the one or more selected HLAI alleles, and the one or more selected HLAI alleles are carried by the individual for whom the vaccine composition is designed.

4. The method according to claim 1, wherein the vaccine composition comprises one or more vaccination carriers selected from the following: a. Recombinant non-replicating or replicating viral vectors; b. Virus-like particles; c. Recombinant RNA constructs with or without modified nucleotides; d. Recombinant DNA constructs with or without modified nucleotides; e. Recombinant proteins with or without modified amino acids; f. Synthetic polypeptides with or without modified amino acids.

5. The method according to claim 4, wherein the one or more vaccination carriers are selected from a, b, c, or d according to claim 4, and the selected non-VIP-derived HLAI-HRE is incorporated into an expression construct that does not allow the expression of functional non-VIP protein in host cells during vaccine delivery to avoid the immune escape activity of the viral non-VIP.

6. The method according to claim 4, wherein the one or more vaccination carriers are selected from e or f according to claim 4, and the recombinant protein or synthetic polypeptide comprises one or more non-VIP-derived HLAI-HREs, and the protein or polypeptide molecule comprises tandem HLAI-HREs or encodes the HLAI-HER within a carrier protein or polypeptide.

7. The method according to claim 4, wherein the one or more vaccination carriers further encode one or more B cell / immunoglobulin epitopes to elicit a neutralizing Ig response.

8. The method according to claim 4, wherein the one or more vaccination carriers further encode one or more selected HLAII-HRE epitopes to elicit a CD4 T cell response to support B cell maturation and neutralizing antibody production, and / or to promote the differentiation of non-VIP-derived HLAI HRE-specific CD8 T cell responses into memory.

9. The method according to claim 7, wherein the one or more B cell / immunoglobulin epitopes are modified to remove HLAI-HRE from the VIP to avoid eliciting a CD8 T cell response against the VIP protein during vaccine delivery.

10. The method according to claim 1, wherein step a of claim 1 further comprises the sub-step of using engineered TCR-presenting cells (eTPC).

11. The method according to any one of claims 1-10, wherein the selected non-VIP-derived HLAI-HRE incorporated in step d of claim 1 is from a donor without prior target virus infection or has an observed CD8 T cell response in a donor with a confirmed active and / or resolved target virus infection.

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