Methods of producing vaccine compositions that elicit human leukocyte antigen class I restricted CD8 T cell responses against non-virion composition derived epitopes of viruses

Abstract

Methods for providing a vaccine composition capable of effectively inducing a systemic immune response and / or a topical immune response upon administration wherein the composition comprises a human leukocyte antigen class I (HLAI) restrictive epitope selected from the viral pathogen non-virion constituent protein (non-VIP) and thus elicits a CD8T cell response specific against viral infected cells.

Description

[0001] This application is a divisional application of Chinese patent application 202180031985.2. The filing date of the original application is April 27, 2021. The name of the invention is "Method for producing a vaccine composition that triggers human leukocyte antigen class I-restricted CD8 T cell responses against non-viral particle derived epitopes of viruses." Technical Field

[0002] The present invention relates to immunology and vaccines. More specifically, the present invention relates to methods for preparing vaccine compositions and vaccine compositions for use against viral pathogens. Specifically, the present invention relates to methods for producing administerable vaccine compositions that can be used as prophylactic or therapeutic agents for viral infections. Background Art

[0003] Viral vaccine composition

[0004] Currently, commercially available vaccine formulations primarily target the entire viral open reading frame (ORF) and typically seek to elicit a neutralizing immunoglobulin (Ig) response to confer immunity against the target viral pathogen. Vaccination strategies typically do not seek to specifically select HLA-restricted T-cell epitopes for inclusion in vaccine compositions. Typically, characterization of any HLA-restricted epitopes (HREs) is limited to those present in proteins comprising the virion or “virion-integral proteins” (VIPs), as T-cell responses are primarily observed against these specific viral ORF products in clinically manifested viral infections, and thus, they are hypothesized to confer productive T-cell-mediated cellular immunity. However, the inclusion of VIP-derived HREs in vaccine compositions is often intended to elicit a B-cell-driven Ig response and generally does not consider the life cycle of the target virus as it replicates and spreads within the infected host, nor how this life cycle elicits T-cell-mediated cellular immunity within the host. Therefore, when attempting to exploit or circumvent T-cell immunity in vaccine compositions, the potential for viral life cycle, viral immune escape, and immunopathology should be carefully considered.

[0005] Adaptive immune response to viral infection

[0006] The clinical manifestations of viral infection are often associated with viral immune escape and / or virus-induced immunopathology. These immunopathologies are often attributable to an over-response to "virally encoded proteins" (VEP). During natural viral infections with clinical manifestations, Ig production against VEP is often limited to VIPs available on the surface of viral particles 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 often 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, viral non-virion-integral proteins (non-VIPs) often drive subdominant T cell responses. Host immunity often engages in active immune escape of specific innate and adaptive responses driven by VEPs. In fact, viral non-VIPs often cause dysregulation of the virus-driven host innate immune response, thereby altering the course of downstream adaptive immune responses.

[0007] Challenges in specifying HLA-restricted epitopes for vaccine compositions

[0008] Due to the high diversity of HLA haplotypes in the human population, the standardization of viral HREs in vaccine compositions has been challenging. HLA allelic variants represent the most diverse gene family in higher organisms, with the genetic diversity of HLA genes conferring 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. The general lack of systematic and accurate tools to functionally analyze the highly complex processing and presentation of specific HREs has limited 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 adaptive immunity and immune tolerance in downstream T cell centers. The lack of systematic biotechnological tools to functionally analyze HRE-specific T cell responses has further hindered the targeted analysis of T cell responses against VEPs to determine whether they should be included in vaccine compositions.

[0009] HLA class I and II and related T cell responses

[0010] Two major classes of HLA, class I (HLA I) and class II (HLA II), typically trigger 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 primarily degraded by the proteasome and transported to the endoplasmic reticulum (ER) for processing. Within the endoplasmic reticulum, further proteolytic processing can occur during the process of enzyme-driven HLA I molecule loading HLAI-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, other processing and transport pathways are also present, enabling protein antigen degradation products derived from extracellular environment sampling to be delivered to the ER for HLA I cross-presentation. In contrast, HLA II molecules are only constitutively presented on the surface of professional APCs. HLA II molecules are largely restricted to presenting protein degradation products derived from sampling of the extracellular milieu in a process that involves endocytosis of the extracellular payload within endocytic vesicles, fusion with a distinct vesicular compartment containing the HLA II molecule, and a series of processing and chaperone enzymes that direct the loading of the HLA II molecule with the HRE and subsequent export to the cell surface for sampling by T cells.

[0011] HLA class I and CD8 T cell responses

[0012] The primary function of the CD8 T cell population is to function as cytotoxic lymphocytes (CTLs), mediating the execution of multiple cell death mechanisms against target cells upon detection of target cells presenting cognate HLA I-HRE epitopes, which activate the TCR carried by the relevant CD8 CTL clone. Therefore, detection of HLA I-HREs by CD8 T cells involves monitoring the intracellular proteome of nucleated cells, representing a central mechanism of adaptive cellular immunity in response to intracellularly expressed non-self proteins. This marks CD8 T cell responses that detect HLA I-HREs as a primary defense against abnormal proliferation and malignant cell transformation, as well as viral infections, and infections by bacteria, parasites, or other intracellular microorganisms. HLA I-HREs are relatively short peptides with more stringent physicochemical constraints than HLA II-HREs. The relatively restrictive nature of HLA I-HREs reflects the requirement for strict control of CD8 CTL adaptive responses to avoid tissue damage caused by spurious CD8 CTL responses to HLA I-HREs derived from self- and non-self antigenic components.

[0013] HLA class II and CD4 T cell responses

[0014] HLAII-HRE directed CD4 T cell responses are more complex and diverse than HLA1-HRE CD8 T cell responses. In general, CD4 T cells are considered "helper" cells that coordinate innate and adaptive immune responses by signaling to both leukocytes and non-leukocytes alike. With regard to adaptive immunity, HLAII-HRE CD4 T cell responses are necessary for driving B cell maturation to produce high-affinity Ig responses, and are also involved in differentiating CD8 T cells into, for example, memory cells. When detecting HLAII-HRE sampled from the extracellular space, CD4 T cells are crucial for coordinating adaptive immune responses to infections including extracellular microorganisms, primarily by driving the progression of humoral Ig responses through B cell maturation. For example, regulatory T cells (Tregs) responsible for immune tolerance to commensal microorganisms and food can also be primarily identified as CD4 T cells sampling HLAII-HRE. These HLAII-HRE CD4 Treg responses also play a critical role in quality control and inflammation resolution in both innate and adaptive immune responses to pathogenic microorganisms, for example by limiting the potential for virus-associated immunopathology.

[0015] Viral life cycle and host CD8 T cell responses

[0016] Viral infection of host cells results in the delivery of the viral genome and the expression of the encoded ORF, which can be divided into non-VIP and VIP. In general, non-VIP is responsible for viral replication and immune escape from host defense, while VIP is the protein that assembles into de novo synthesized virions within infected cells. Various types of immune escape mechanisms in certain viruses can also be attributed to VIP. In general, it can be said that non-VIP is mainly responsible for the processing and replication of the viral genome, coordinating viral genome packaging, controlling the occurrence of de novo synthesized virions and mediating immune escape.

[0017] Productive CD8 CTL priming against HLA-HREs expressed on infected cells is central to adaptive immunity to viral infection. Elimination of virus-infected cells by CD8 CTL action is considered a key aspect of viral clearance and the establishment of adaptive immunity. Infected host cells trigger a series of innate immune responses to attract CD8 T cells to sample HLA-HREs, whether by naive CD8 T cells initiating CD8 CTL adaptive immunity or by memory CD8 T cells evoking immunity to previous natural infection or vaccination. The primary intracellular innate immune response driving CD8 T cell recruitment and sampling is the interferon type I (IFN-I) pathway.

[0018] Viral non-VIPs are often ubiquitously expressed in cytoplasmic compartments and are therefore accessible to the HLA-I-HRE processing and presentation machinery, from which VIP is typically sequestered. This sequestration of VIP from HLA-I-HRE processing and presentation is particularly pronounced in enveloped viruses, where structural VIP is sequestered to membranes and vesicles during de novo virion synthesis. Therefore, CD8 CTL responses against viral non-VIPs can be considered a major driver of viral clearance in many forms of viral infection, by eliminating productively infected cells.

[0019] Professional APCs are potent inducers of CD8 CTL responses to HLAI-HREs. Indeed, DCs in particular possess mechanisms that promote cellular infection, thereby driving beneficial CD8 CTL responses. However, in the absence of productive professional APC infection, primary priming of CD8 CTL responses can be biased toward VIP through endocytosis of whole virions and trafficking of VIP to the ER for HLAI-HRE cross-presentation by DCs. This may represent a key driver of the immunodominant nature of CD8 T cell responses against VIP-derived HLAI-HREs in many clinically manifesting viral infections.

[0020] CD8 T cells are beneficial host responses to viral infection and immune evasion

[0021] The most productive CD8 T cell responses to viral infection are those that trigger CD8 CTL responses, which ultimately drive clearance of virally infected cells. This is primarily due to viral infection and the rapid triggering of innate immune responses by recognition of viral factors and genomes, with IFN-I playing a central 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 initiation of beneficial CD8 CTL responses against HLA-HREs presented by productively infected cells.

[0022] A key mechanism of viral immune evasion is the dysregulation and / or suppression of innate IFN-I responses. Many viral genomes encode numerous non-VIPs that disrupt IFN-I signaling. This facilitates viral immune evasion, particularly with respect to CD8 T cell detection of HLA-HREs in infected cells. This immune evasion mechanism allows virus-infected cells to evade CD8 CTL-driven clearance, while viral replication can proceed. This has significant implications for potentially unproductive or even deleterious HLA-HRE CD8 T cell responses during viral infection.

[0023] Non-productive CD8 T cell responses to viral infection

[0024] 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 CD8 CTL responses against viral non-VIP-derived HLAI-HRE, where the CD8 CTL responses against viral non-VIP-derived HLAI-HRE are most beneficial for viral clearance by eliminating infected cells. Crucially, this immune escape allows infected cells to produce and release new viral particles and amplify the infection. This inevitably 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 VIP, rather than the more productive non-VIP-derived HLAI-HER, unless the professional APCs themselves are subjected to productive viral infection.

[0025] The resulting immune escape of CD8 CTL responses against non-VIP-derived HLA-I-HREs presented by productively infected cells, and the emergence of an immunodominant response against VIP-derived HLA-I-HERs initiated by professional APCs, has significant implications for further downstream viral immune evasion. First, the dominance of CD8 T cell responses initiated by professional APCs shifts the CD8 CTL response away from productive non-VIP-derived HLA-I-HREs and toward VIP-derived HLA-I-HERs. Second, the immunodominance of VIP-derived HLA-I-HREs triggers a CD8 CTL response that promotes CD8 CTL elimination of professional APCs. Indeed, CD8 CTL-mediated elimination of professional APCs is thought to be a factor in resolving the inflammatory response to viral infection. However, during active viral infection, this can be viewed as a dysregulated immune response that drives exhaustion of professional APCs at the site of infection and in the draining lymph nodes (DLNs), resulting in a reduced capacity of professional APCs to coordinate the overall adaptive immune response, critically, the HLA-I-HRE / CD4 T cell response. This form of cumulative immune evasion of adaptive immunity, downstream of the initial immune evasion of CTL action against productively infected cells, is crucial in viral infections with a limited spectrum of host cells, particularly in the absence or inefficiency of professional APCs in productive infections.

[0026] CD4 T cells and Ig immune escape derived from dysregulated CD8 CTL responses

[0027] The accumulation of CD8 CTL responses against VIP-derived HLA I-HREs (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 HLA II-HRE-specific CD4 T cell responses, which are themselves crucial for the establishment of humoral Ig immunity. The immunodominant VIP-derived HLA I-HRE CD8 CTL responses that eliminate professional APCs from the site of infection (at the expense of responses against productive non-VIP-derived HLA I-HREs) also lead to a reduced availability of a professional APC platform in the DLN capable of orchestrating CD4 T cell responses. This inevitably leads to a reduced ability to effectively orchestrate 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 may also result in a reduced capacity of CD4 T cell responses to efficiently mediate the commitment of CD8 CTL responses to non-VIP-derived HLA I-HRE to memory differentiation and effectively establish sustained CD8-centric immunity.

[0028] Ig-driven immunopathology and vaccine-related diseases

[0029] The goal of most current vaccine strategies is to provide a composition of essentially complete VIP, typically within a whole inactivated or attenuated virus, with the central goal being to elicit the production of neutralizing antibodies. However, without the support of a coordinated HRE T cell response, these strategies typically result in poor neutralizing antibody titers and incomplete immunity to the target viral pathogen. In addition, some vaccine strategies have led to worsening immunopathology in animals and human subjects, partly due to dysregulation of Ig responses downstream of the misdirected HRE-specific T cell response to VIP. In some cases, both developed vaccination strategies and natural infections have found immunopathology associated with self-production of neutralizing antibodies, particularly in respiratory mucosal infections, where high-titer antibody secretion can lead to excessive mucus secretion and obstructive respiratory distress. Due to the high complexity of the HRE repertoire during viral infection, the high diversity of HLA haplotypes between individuals, and an incomplete understanding of the overall adaptive immune response to viral infection, these vaccine-related disease phenomena are not yet understood and are difficult to resolve.

[0030] Prospects for viral vaccine compositions that elicit selected CD8 T cell responses

[0031] There is a large body of evidence supporting the key role of CD8 CTL responses in preventing the establishment and clearance of viral infections. In fact, animal models have clearly demonstrated that antigen-specific CD8 T cells have a significant protective effect against bolus viral challenge. The above observations point to a vaccine strategy in which selected non-VIP-derived HLA I-HREs are provided in the vaccine composition to selectively elicit beneficial CD8 CTL responses. To this end, the vaccine composition is intended to provide a frontline defense against invading viruses while further avoiding the potential non-productive or harmful initiation of CD4 and Ig responses, which can lead to the exacerbation of immunopathology following subsequent natural viral infection.

[0032] To date, it has been challenging to analyze HRE-specific T cell responses with sufficient depth and precision to identify the precise HREs for effective vaccine compositions designed to elicit favorable T cell responses. Emerging biotechnology methods now allow, among other things, in-depth, rapid, and accurate functional analysis of HREs in addition to T cell responses to these antigens (see, for example, WO2018083316, WO2018083317, WO2018083339, and WO2018083318). Summary of the Invention

[0033] The present invention provides methods for producing administerable vaccine compositions useful as prophylactic or therapeutic agents for viral infections. In particular, the present invention provides vaccine compositions that are effective in inducing a systemic and / or local immune response upon administration, wherein the composition comprises a human leukocyte antigen class I (HLAI)-restricted epitope (HLAI-HRE) selected from non-virion-integral proteins (non-VIP) of viral pathogens, thereby eliciting a CD8 T cell response specific for virally infected cells.

[0034] The present invention provides methods for selecting non-VIP-derived HLA I-HREs, which are used to produce vaccine compositions that selectively elicit CD8 CTL responses that are beneficial for viral immunity and clearance while avoiding unproductive or deleterious CD8 CTL responses to VIP-derived HLA I-HREs.

[0035] This approach involves selecting HLA I-HREs for inclusion in vaccine compositions designed to elicit selective CD8 CTL responses against virus-infected cells while avoiding spurious CD8 CTL responses driven by viral immune escape mechanisms and downstream immunopathology.

[0036] Specifically, the present invention provides methods for producing vaccine compositions that can be administered as prophylactic or therapeutic agents for viral infections. The present invention particularly relates to vaccine compositions that are capable of effectively inducing a systemic and / or local immune response upon administration, wherein the composition comprises one or more non-VIP-derived HLA-I-HREs selected from target viral pathogens while avoiding HLA-I-HREs derived from VIP, thereby eliciting a restricted and highly specific CD8 T cell response specifically against virally infected cells.

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

[0038] a. Identifying a non-virion protein-derived human leukocyte antigen class I restricted epitope (non-VIP derived HLAI-HRE) from a viral pathogen, wherein it is desired to obtain a vaccine composition against the viral pathogen;

[0039] b. Classifying the immunogenicity of the identified non-VIP-derived HLA I-HRE in naive 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;

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

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

[0042] The immunogenicity of non-VIP derived HLA I-HREs in donors without prior infection or confirmed prior infection should be determined for HLA I-HREs that have been confirmed to be processed and presented in a cell model infected with a productive virus to confirm the availability of the epitope in the normal viral life cycle of the infected cell. That is, the use of a single expressed viral ORF, ORF fragment, or 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 targeted 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 a viral pathogen.

[0043] Systematic comparative analysis of non-VIP-derived HLA I-HRE immune responses can be performed in the following human subject cohorts:

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

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

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

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

[0048] The priority order of non-VIP-derived HLA I-HREs identified in each subject category for inclusion in the vaccine composition was 1>2>3>4.

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

[0050] These vaccine formulations can be used to treat and prevent known pathogens, as well as to rapidly develop responses to new pathogens. In addition, knowledge of non-VIP-derived HLA I-HREs enables the development of diagnostic programs for clinical and epidemiological surveillance. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 .Initial infection with SARS-CoV-2 leads to the escape of innate immunity mediated by virus-encoded proteins, which mediate the escape of early adaptive CD8 T cell responses.

[0052] Figure 2 .Infection established during the initial immune escape period leads to delayed recruitment of the immune response.

[0053] Figure 3 The late initiation of the adaptive immune response results in a bias towards sampling of accumulated virions by professional APCs, as well as a directed T cell response against HLA-restricted antigens derived from virion constituent proteins. This is a properly directed response for HLAII / CD4 helper and B cell responses. This may be counterproductive to the HLA1 / CD8 CTL response, which targets non-virion proteins that are highly expressed in productively infected cells, rather than those present on virions.

[0054] Figure 4 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, resulting in dysregulated T and B cell responses, which can enhance the cytokine storm in severe Covid-19 manifestations and result in an overall weak neutralizing antibody response to SARS-CoV-2 infection.

[0055] Figure 5 A) Selected core HLA-A, B, and C alleles, B) The proportion of each ethnic group predicted to carry at least n core alleles, and C) The cumulative probability of observing at least n core alleles in a given ethnic group. DETAILED DESCRIPTION

[0056] The present invention is described in the following clauses which list aspects and embodiments:

[0057] 1. A method for producing a vaccine composition for use against a viral pathogen, comprising:

[0058] a. Identifying a non-virion protein-derived human leukocyte antigen class I restricted epitope (non-VIP derived HLAI-HRE) from a viral pathogen, wherein it is desired to obtain a vaccine composition against the viral pathogen;

[0059] b. Classifying the immunogenicity of the identified non-VIP-derived HLA I-HRE in naive 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;

[0060] c. Select non-VIP-derived HLA I-HREs with confirmed immunogenicity in donors without prior target virus infection or with observed CD8 T cell responses in donors with confirmed active, latent, or resolved target virus infection;

[0061] d. Incorporating the selected non-VIP-derived HLA I-HRE into a vaccine composition.

[0062] 2. The method of claim 1 , wherein a plurality of non-VIP derived HLA I-HREs are selected for inclusion in the vaccine composition to represent one or more HLA I-HERs of the selected HLA I alleles, wherein the selected HLA I alleles represent those alleles carried by at least 60% of individuals in the target population for which the vaccine composition is designed.

[0063] 3. The method of claim 1 , wherein one or more non-VIP derived HLA I-HREs are selected for inclusion in the vaccine composition to represent one or more HLA I-HERs in one or more selected HLA I alleles, wherein the selected one or more HLA I alleles are carried by the individual for whom the vaccine composition is designed.

[0064] 4. The method of any one of items 1 to 3, wherein the vaccine composition comprises one or more vaccination vectors selected from the group consisting of:

[0065] a. Recombinant non-replicating or replicating viral vectors;

[0066] b. virus-like particles;

[0067] c. recombinant RNA constructs with or without modified nucleotides;

[0068] d. recombinant DNA constructs with or without modified nucleotides;

[0069] e. Recombinant proteins with or without modified amino acids;

[0070] f. Synthetic polypeptides with or without modified amino acids.

[0071] 5. The method of claim 4, wherein the one or more vaccination vectors are selected from a, b or c, and wherein the selected non-VIP derived HLAI-HRE is incorporated into an expression construct that does not allow expression of functional non-VIP proteins in host cells upon vaccine delivery, so as to avoid the immune escape activity of the viral non-VIP.

[0072] 6. The method of claim 5, wherein the selected non-VIP-derived HLAI-HRE is provided by one or more of the following means:

[0073] a. introducing point mutations and / or sequence insertions and / or sequence deletions that inactivate protein function within the full-length non-VIP ORF;

[0074] b. constructing a synthetic nucleic acid sequence comprising a non-VIP ORF fragment encoding the selected HLAI-HRE in a tandem construct;

[0075] c. Constructing a synthetic nucleic acid sequence containing a non-VIP ORF fragment encoding the selected HLA I-HRE within a carrier protein sequence.

[0076] 7. The method of claim 4, wherein the one or more vaccination 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 a carrier protein or polypeptide.

[0077] 8. The method of any one of items 1 to 7, wherein the viral pathogen is selected from adenovirus, alphavirus, arbovirus, Borna disease, bunyavirus, calicivirus, genital warts, coronavirus, coxsackievirus, cytomegalovirus, dengue virus, leukopenia, Epstein-Barr virus, erythema infectiosum, hantavirus, viral hemorrhagic fever, viral hepatitis, herpes simplex virus, herpes zoster virus, HIV, infectious mononucleosis, influenza, Lassa fever virus, measles, mumps, molluscum contagiosum, paramyxovirus, phlebitis, polyomavirus, poxvirus, retrovirus, Rift Valley fever, rubella, chronic disease virus, smallpox, subacute sclerosing panencephalitis, tumor virus infection, West Nile virus, yellow fever virus, rabies virus and respiratory syncytial virus.

[0078] 9. The method of claim 8, wherein the viral pathogen is a coronavirus, such as SARS-Cov2.

[0079] 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.

[0080] 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 commitment of non-VIP-derived HLA1 HRE-specific CD8 T cell responses to memory differentiation.

[0081] 12. The method of claim 10, wherein the one or more B cell / immunoglobulin epitopes are selected from the VIP protein of the target viral pathogen and expressed on the surface of the viral particles.

[0082] 13. The method of any one of items 10 and 12, wherein the one or more B cell / immunoglobulin epitopes are modified to remove the HLA I-HRE from VIP to avoid eliciting a CD8 T cell response against the VIP protein upon vaccine delivery.

[0083] 14. The method of claim 11, wherein the one or more HLAII-HREs may comprise sequences derived from a target viral pathogen, or may be synthetic or naturally occurring HLAII-HE epitopes that, when included in a vaccine composition, can promote beneficial CD4 T cell responses to support B cell maturation and / or CD8 T cell memory commitment.

[0084] 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.

[0085] 16. The method of any one of items 4 to 15, wherein the vaccine vector is further formulated into a vaccine formulation for administration to a human or veterinary subject, wherein the vaccine formulation further comprises a pharmacologically suitable excipient.

[0086] 17. A vaccine composition for use against viral pathogens, prepared by the method described in any one of items 1 to 16.

[0087] 18. A vaccine formulation comprising the vaccine composition according to item 17 and at least one pharmaceutically acceptable excipient.

[0088] 19. The vaccine formulation of claim 18, further comprising at least one vaccine adjuvant.

[0089] 20. A method for vaccinating a human or animal subject to provide immunity against a virus, comprising administering the vaccine composition of item 17, the vaccine formulation of any one of items 18-19, or the vaccine composition prepared by the method of any one of items 1-16.

[0090] 21. A method for treating a human or animal subject having an acute, chronic or latent viral infection by eliciting an immune response by immunizing the human or animal subject with the vaccine composition described in item 17, the vaccine formulation described in any one of items 18-19, or the vaccine composition prepared by the method described in any one of items 1-16.

[0091] 22. A method for eliciting a CD8 T cell response against a viral infection in a human or animal subject, comprising administering the vaccine composition of item 17, the vaccine formulation of any one of items 18-19, or the vaccine composition prepared by the method of any one of items 1-16.

[0092] 23. The method of 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.

[0093] 24. The method of any one of items 20-22, wherein the vaccine is administered intranasally.

[0094] 25. The method of any one of items 20-22, wherein the vaccine is administered intramuscularly.

[0095] 26. The method of any one of items 20-22, wherein the vaccine is administered intradermally.

[0096] 27. The method of 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.

[0097] 28. The method of any one of items 20 to 27, wherein the prime-boost vaccines are administered by the same route.

[0098] 29. The method of any one of items 20 to 27, wherein the prime-boost vaccine is delivered intramuscularly and the boost vaccine is delivered intranasally.

[0099] 30. The method of any one of items 27-29, wherein the priming and boosting vaccine compositions, vaccine vectors and / or vaccine formulations may be the same or different.

[0100] Example

[0101] Example 1: SARS-CoV-2 infection immunopathogenesis model illustrating the advantages of non-VIP-derived HLAI-HRE vaccine compositions

[0102] Several years ago, several cases of pneumonia of unknown etiology were identified. These pneumonia cases were subsequently linked to a novel virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). On March 11, 2020, the World Health Organization declared SARS-CoV-2 the cause of COVID-19, now considered a pandemic. The disease is characterized by respiratory tract infection that, depending on severity, can lead to acute respiratory distress syndrome. Elderly subjects with SARS-CoV-2 infection have a significantly worse prognosis compared with younger individuals, suggesting that a diminished 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 consensus model for SARS-CoV-2 immunopathogenesis that has direct implications for determining current and past infection status in non-elderly subjects, as well as for understanding the severity of COVID-19 in elderly subjects. This model has implications for the design of vaccine compositions that selectively contain non-VIP-derived HLA-HREs.

[0103] The development of viral vaccines has long been hampered by a lack of understanding of antigen-specific T cell responses to natural viral infection 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 by viral proteins. Furthermore, the lack of tools to rapidly and unambiguously detect human T cell responses to these multiple HLA-restricted antigens is a serious limitation.

[0104] Several respiratory viral infections are associated with acute inflammatory responses in the respiratory mucosa, often with dysregulated T-cell responses. Historically, RSV, SARS-CoV, and even invasive influenza strains have been implicated as drivers of severe inflammatory syndromes in the respiratory mucosa. Now, subjects infected with SARS-CoV-2, which represents the most severe clinical manifestations of Covid-19, experience a cytokine storm that ultimately leads to disease. These severe immunopathologies are apparently driven by an overactive T-cell response during viremia.

[0105] Historical experience with vaccine-associated exacerbations of illness during attempts to vaccinate against respiratory viral infections suggests that current efforts to deploy vaccines against novel coronaviruses carry significant risks. RSV has been recognized for decades as a high unmet medical need, and early attempts to vaccinate against RSV in the 1960s resulted in exacerbations of acute respiratory distress in vaccine recipients following subsequent natural infection. Studies of this phenomenon in subsequent decades have implicated antigen-specific CD8 and CD4 T-cell responses as equally involved in driving viral clearance and immunopathology. Similarly, experimental vaccines against SARS-CoV, the early novel coronavirus responsible for the 2003 SARS outbreak, resulted in severe inflammatory responses during developmental vaccine studies, which were attributed in part to anti-spike immunoglobulins.

[0106] In support of the significant role of T cell responses in coronavirus infections, studies of SARS-CoV infection have shown that in recovered subjects, B cell responses are weak, but antigen-specific T cell responses are durable—a trend mirrored in the SARS-CoV-2 pandemic. Furthermore, a prominent feature of SARS is lymphopenia, presumably driven by the recruitment of massive T cells to the respiratory mucosa, in both SARS-CoV and SARS-CoV-2 infections. Combined with the clear correlation between lymphopenia and poor prognosis in Covid-19 patients, and the emerging details of T cell immunopathology in severe Covid-19, there is a clear need for precise analysis of T cell responses in subjects infected with SARS-CoV-2. This is required to assess the safety and efficacy of current vaccine candidates and to directly inform the development of next-generation vaccines.

[0107] Summary of SARS-CoV-2 Immunopathogenesis Models

[0108] Initial immune evasion by CD8 CTLs during the early stages of infection leads to the establishment of infection, which is more likely in elderly subjects, as their naive CD8 T-cell pool is known to be reduced due to declining thymic output. At this stage, an effective early CD8-mediated response can clear the infection even asymptomatically, without the subsequent production of detectable high-affinity neutralizing antibodies, but can establish memory T cells. Notably, otherwise healthy, non-elderly subjects often present with asymptomatic SARS-CoV-2 infection or very mild symptoms that are resolved by low-level and transient Ig responses.

[0109] Initial immune escape results in the escape of detection of "non-virion component proteins" (non-VIP) HLA-I restricted epitopes, which is ideally required for the elimination of virus-infected cells.

[0110] Accumulation of danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) during infection establishment and epithelial cell death leads to the characteristic recruitment of immune cells infiltrating into the infected respiratory mucosa.

[0111] Initial immune evasion allows virions to accumulate and then be sampled by professional APCs, which themselves may not become productively infected.

[0112] Professional APCs process and present virion component proteins (VIPs) to trigger HLA I / CD8 and HLA II / CD4 responses.

[0113] Detection of VIP by the HLAII / CD4 response initiates a CD4 helper response to support B cell maturation against viral surface proteins sampled by B cells, leading to a productive adaptive immune response with the production of neutralizing antibodies. In many, but not all, cases, the infection can resolve as a result.

[0114] HLA-I / CD8 responses that detect VIP are not ideally suited to target epithelial or other cells with productive viral infection, but these responses can target professional APCs that cross-present HLA-I restricted antigens from virus-infected cells.

[0115] Misdirected CD8 CTL responses against VIP can lead to the depletion of professional APCs at sites of infection and in the DLN, resulting in a global breakdown in the coordination of T-cell responses and loss of support for B-cell maturation.

[0116] Poor B-cell responses may explain the discordant antibody responses in subjects with SARS-CoV-2 infection and those with clinical manifestations of Covid-19. Therefore, the use of serology alone in the clinical management and epidemiology of patients is unreliable for policymaking, as T-cell responses should also be considered.

[0117] Vaccination strategies targeting large numbers of VIPs, such as the spike protein in SARS-CoV-2, can elicit counterproductive CD8 CTL responses and exacerbate disease during natural infection, particularly in vaccinees carrying HLA alleles that confer susceptibility to responses to these specific HLA-restricted epitopes.

[0118] 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 that can drive viral clearance early during viral encounter and infection without the risk of ADE or misdirection of deleterious VIP-mediated CD8 CTL responses would be beneficial.

[0119] Detailed model of SARS-CoV-2 immunopathogenesis

[0120] This section outlines a four-step model of a dysregulated immune response initiated by initial immune escape of CD8 CTL responses early in infection through virus-mediated suppression of innate immune signaling in infected cells. Innate immune signaling by professional APCs can subsequently be suppressed even in the absence of productive infection of these cells. Overall, initial immune escape by infected cells redirects CD8 CTL responses toward virion component proteins (VIPs). Immune escape leads to poor clearance of virus-infected cells, in addition to early initiation of CD8 CTL-driven exhaustion of professional APCs, which are typically associated with resolution of the inflammatory response after significant viral clearance. This model of immunopathogenesis can explain many clinical observations associated with severe Covid-19 clinical manifestations, with the exception of 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 by detecting CD8 T cell effector and memory populations to more reliably detect current mild or past infection, compared to unreliable serological detection of apparently weak and transient Ig responses in young individuals infected with SARS-CoV-2. This model supports the exclusive and selective use of non-VIP-derived HLA I-HREs in vaccine compositions for the prevention or treatment of SARS-CoV-2 infection, where eliciting CD8 CTL responses against non-VIP-derived HLA I-HREs is considered necessary and sufficient for protective immunity against SARS-CoV-2 and a range of other viral infections.

[0121] Step 1: Initial SARS-CoV-2 infection and immune escape of CD8 T cells. Figure 1 .

[0122] Figure 1 This suggests that initial SARS-CoV-2 infection leads to the escape of innate immunity mediated by virus-encoded proteins, which in turn mediates the escape of early adaptive CD8 T cell responses.

[0123] A) Virus particle entry into target epithelial cells occurs through docking of the ACE2 complex, involving pre-cleavage by furin in addition to the action of host cell serine proteases, particularly TMPRSS2, for spike complex processing and host cell entry from endocytic compartments.

[0124] B) Entry into the host cell results in complex transcription of the viral genome, producing virus-encoded proteins (VEP).

[0125] C) It is known that the virion component protein (VIP) of coronavirus includes factors that are responsible for immune escape activity that are bound to structural proteins and non-structural proteins. It is believed that the delivery of VIP that confers immune escape activity confers immediate suppression of the innate immune response 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.

[0126] D) Non-virion component proteins (non-VIP) produced by viral genome transcription can further participate in the suppression of host cell innate immunity.

[0127] E) The primary means by which VEPs drive innate immune suppression is by blocking the IFN type I (IFN I) response.

[0128] F) Suppression of the IFN1 response in infected host cells leads to reduced recruitment and sampling of naive CD8 T cells, thereby reducing their ability to detect and respond to viral HLA-restricted epitopes during the early stages of infection. Therefore, the relative availability of naive and activated memory CD8 T cells is crucial for clearance of early infection at this stage.

[0129] G) By immune evasion of frontline CD8 T cell responses, infected cells are free to execute viral genome replication and de novo virion generation.

[0130] H) Infected cells release de novo synthesized virions, further amplifying host cell infection.

[0131] Step 2: Virus spread and immune activation. Figure 2 .

[0132] Figure 2 This suggests that infection established during the initial immune escape period leads to delayed recruitment of the immune response.

[0133] A) Accumulation of cellular dysfunction, VEP expression, viral genome replication, and loss of membrane integrity leads to the release of danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs).

[0134] 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.

[0135] C) De novo synthesized virions released from infected host cells infect adjacent epithelial cells and initiate viral spread to other host cells.

[0136] Step 3: Late initiation of adaptive immunity and misdirected CD8 CTL responses. Figure 3 .

[0137] Figure 3 This suggests that the late onset of the adaptive immune response leads to a biased sampling of accumulated virions by professional APCs and a directed T cell response against HLA-restricted antigens derived from virion components. This is a properly directed response for HLA II / CD4 helper and B cell responses. This can productively balance the HLA I / CD8 CTL response, which targets non-virion proteins that are highly expressed in productively infected cells, rather than those present on virions.

[0138] A) Cell death in chronically infected epithelial cells leads to mucosal permeabilization, cellular infiltration of the lung lumen, edema, and the onset of pneumonia. In particular, alveolar macrophages are crucial for this process and are responsible for phagocytosis of infected cells and viral particles.

[0139] B) Viral dissemination increases virion production within the host and elevates extracellular virion titers, allowing further spread of the infection and sampling of virions by professional APCs and B cells to initiate deeper adaptive immune responses typical of any form of infection.

[0140] C) DCs sample accumulated virions through an endocytic mechanism, providing VIPs for processing and presentation of HLA-restricted epitopes. It is currently unclear whether DCs are productively infected, with conflicting reports in the literature. However, it is clear that delivery of virions to DCs partially inhibits intrinsic signaling, with DCs exposed to virions exhibiting a marked lack of activation. This DC activation typically triggers increased processing and presentation of HLA-I and HLA-II restricted epitopes. In the absence of this activation, overall detection of HLA-restricted epitopes by CD4 and CD8 T cells is insensitive—biasing activation of T cell responses toward the most abundant proteins presented by virions (i.e., VIPs).

[0141] D) Trafficking of VIP sampled from the extracellular milieu for processing and presentation of HLA-I restricted epitopes.

[0142] E) In the absence of productive infection of DCs and in the presence of DC activation inhibition, naive CD8 T cells sampling HLA-restricted epitopes presented by DCs activated primarily against VIPs rather than non-VIPs. This could be considered incorrect immune priming, as CD8 CTL-mediated clearance of virus-infected cells would ideally target non-VIPs, which are abundantly expressed on productively infected cells.

[0143] F) Processing of VIP sampled from the extracellular milieu results in presentation of HLAII-restricted epitopes.

[0144] G) Naive CD4 T cells sampling HLAII-restricted epitopes presented by DCs are activated primarily against VIP rather than non-VIP. This can be considered as correct immune priming, as CD4 helper effector function against VIP is required for B cell maturation toward neutralizing antibody production.

[0145] H) B cell sampling of virions via the BCR initiates a humoral response with the support of intact CD4 helper effectors, providing first low-affinity antibodies to VIP, with the normal progression of clonal selection and haplotype switching.

[0146] I) The virus further spreads to cells expressing ACE2, notably including alveolar macrophages.

[0147] Step 4: Misdirected CD8 CTL responses lead to global immune dysregulation. Figure 4 .

[0148] Figure 4 We show 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, resulting in dysregulated T and B cell responses, which can enhance the cytokine storm in severe Covid-19 manifestations and contribute to an overall weak neutralizing antibody response to SARS-CoV-2 infection.

[0149] A) Apoptotic and necrotic cells are marked by an early antibody response and undergo phagocytosis by macrophages. Further destruction of epithelial cells leads to severe tissue damage in the respiratory mucosa and accumulated mucus production, accompanied by cellular infiltration, edema, and worsening pneumonia.

[0150] B) Materials phagocytosed by macrophages can be presented by these professional APCs to promote VIP-derived HLA-restricted epitopes. Alveolar macrophages are known to express ACE2 and are believed to be infected. Due to the strong expression of innate PAMP receptors in macrophages and the general enhancement of innate immune pathways, 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 viral particles that are also phagocytosed, these cells can also sensitize the CTL response that has been triggered against VIP by continuously phagocytosing the abundant VIP proteins in infected and dying cells.

[0151] C) Cumulative CTL responses against VIP-derived HLA1-restricted epitopes may contribute to macrophage depletion by antigen-specific CTL action during dysregulated immune responses.

[0152] D) Sustained DC sampling of virions during viremia supports that these cells cross-present VIP-derived HLA1-restricted epitopes.

[0153] E) Cumulative CTL responses against VIP-derived HLA-I restricted epitopes can contribute to the exhaustion of macrophages by antigen-specific CTLs during dysregulated immune responses, leading to a lack of professional APCs in MALT and DLNs, thereby contributing to dysregulated T and B cell responses, resulting in cumulative immune dysfunction and cytokine storm.

[0154] F) Depletion of DCs in MALT and DLN results in dysregulated and inefficient CD4 T cell responses and a lack of CD4 helper effector functions to drive B cell maturation toward high-quality and diverse neutralizing antibody responses.

[0155] Epidemiological significance of SARS-CoV-2 infection and other viral infections in humans

[0156] 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 mechanisms behind this are unclear, but dysregulated B cell maturation responses mediated by exhaustion of professional APCs due to misdirected CD8 CTL activity would result in the inability to reliably detect antibody responses even in convalescent Covid-19 patients.

[0157] Furthermore, just as phagocytic and endocytic sampling by macrophages and DCs, respectively, can contribute to partial immunosuppression of antigen-presenting activity, B cells can be susceptible to virus-mediated suppression during viremia. Indeed, BCR-driven B cell endocytosis during viral epitope sampling would result in the internalization of virions that include innate immunosuppressive factors expressed within their envelope. This alone suggests that immunosuppressive VIPs could leak through membrane fusion events between host cells and the viral envelope, imparting innate immunosuppression to B cells and thereby impairing overall B-cell maturation. Of course, in the absence of productive infection and destruction of macrophages, DCs, or B cells, virion sampling delivers high doses of VIP-derived HLA-I-restricted epitopes, which could render these professional APC populations susceptible to misdirected CTL action, which could be detrimental to the overall adaptive immune response by limiting support for antigen-specific CD4 T-cell responses and B-cell maturation.

[0158] Numerous studies have linked productive and nonproductive infection of professional APC populations to dysregulated IFN responses and reduced maturation of professional APCs, which can be linked to direct inhibition of innate signaling pathways by VEPs. This could contribute to poor overall immunopathology and exacerbate the impact of cumulative adaptive immune dysfunction in individuals with overt COVID-19.

[0159] Regardless of the precise mechanisms underlying adaptive immune dysregulation, it is clear from emerging reports that serology is unreliable in subjects with asymptomatic or mild Covid-19 presentations. Furthermore, it is unclear to what extent subjects with SARS-CoV-2 infection who are asymptomatic or have subclinical disease mount a detectable antibody response. Instead, it appears that most subjects who have cleared subclinical SARS-CoV-2 do so by generating sufficient CD8 CTL responses against VEPs and may never mount an antibody response against VIPs.

[0160] It is currently unknown whether subjects with mild infection generate CD8 CTL responses against other VIP- and / or non-VIP-derived HLA-restricted epitopes. Given the historical lack of depth in analyzing antigen-specific T cell responses, the role of CD8 CTL responses against specific VIP- and non-VIP-derived HLA-I-restricted epitopes in viral clearance during subclinical infection with any virus in human subjects is unknown.

[0161] Risks of vaccines encoding VIP

[0162] This article is not intended to explain the general risk of adverse events when deploying VIP-centric vaccines in the population. However, as discussed above with respect to the immunopathological mechanisms underlying severe Covid-19 manifestations, there is a specific risk associated with eliciting sub-neutralizing, and indeed transient, neutralizing Ig responses in vaccine recipients using VIP-centric compositions.

[0163] Primarily, incomplete or transient Ig-driven protection of vaccine recipients against subsequent natural SARS-CoV-2 infection could contribute to vaccine-associated disease exacerbation through two distinct mechanisms. First, subneutralizing antibody responses could lead to antibody-dependent enhancement of Covid-19 by promoting professional APC uptake of partially opsonized (i.e., non-neutralized) virions and promoting misdirected CD8 CTL responses against VIP-derived HLA-restricted epitopes. Second, full-length VIP (including the “spike” protein) could equally elicit CD8 T-cell responses, which could lead to accelerated immunopathology and reduced capacity for viral clearance during subsequent natural infection.

[0164] Even if productive Ig-driven protective immunity exists in vaccinees using VIP-encoding vaccines, such vaccines simultaneously elicit potentially harmful CD8 CTL responses against VIP-derived HLA I-HRE, and the decline in Ig titers over time can lead to exacerbation of vaccine-associated disease because memory CD8 CTL populations remain in circulation long after protective Ig titers have subsided.

[0165] Indeed, despite decades of research, there is still a lack of effective vaccines against similar viral infections of the lower respiratory tract pneumonia, such as respiratory syncytial virus (RSV), and only incompletely productive vaccines against, for example, influenza. This could be due to the combination of viral vaccine composition and delivery, but given the above observations, it could equally be due to the dual priming of beneficial neutralizing antibody responses and deleterious CD8 CTL responses in inactivated viral vaccines or recombinant vaccines that deliver VIP with the goal of eliciting a neutralizing antibody response. The historical precedent of vaccine-associated disease exacerbation in RSV vaccination highlights the inherent risks of providing VIP (e.g., via inactivated viral vaccines) as the primary antigen source during vaccination, while VIP naturally cannot provide non-VIP antigenic targets.

[0166] In terms of the risks associated with VIP-centric vaccination strategies for the immunopathogenesis model proposed above, there is a natural hierarchy of risk associated with inactivated virus vaccines, full-length VIP, and VIP fragment (i.e., gene fragments or protein domains that present neutralizing antibody epitopes) vaccine compositions, simply in terms of the sequence space represented by each of these compositions, which provides VIP-derived HLA-I restricted epitopes to initiate potentially harmful CD8 CTL responses that can exacerbate disease in subsequent natural viral infection. In fact, for the emerging SARS-CoV-2 virus that has not yet been widely exposed to the human immune system, in the absence of long-term selective pressure from the human immune system, it is likely that a large number of HLA-I restricted epitopes will be present within any VEP. This means that the risk of vaccine-associated disease exacerbation is enhanced in vaccine strategies against SARS-CoV-2 compared to more established human viral pathogens with similar properties.

[0167] In any setting, especially with rapid vaccine deployment, a major logistical challenge in detecting vaccine-associated exacerbations of disease is that adverse events are not expected to occur at the time of the initial vaccine challenge but rather during random subsequent viral infections. Not only is this a technical challenge due to the complexity of systematically assessing T-cell responses to HLA-restricted antigens, but because of time and probability factors, potential adverse events may only become apparent in later studies, when many individuals have already undergone vaccine challenge. Furthermore, because VIP-derived HLA1-HRE-specific CD8 CTL memory cells persist far longer than significant VIP-directed Ig titers, such adverse events can occur months and years after the initial vaccine administration in the absence of subsequent booster vaccinations.

[0168] Next generation vaccine compositions containing non-VIP derived HLA I-HREs

[0169] With respect to the above observations presented, vaccine compositions containing selected non-VIP-derived HLAI-HREs are 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-centric vaccine strategies for SARS-CoV-2 (and indeed other invasive viral infections of the lower respiratory tract), it is proposed that highly selective inclusion of non-VIP-derived HLAI-HREs may be the safest and most productive means of vaccine deployment. That is, non-VIP-derived HLAI-HREs are necessary and sufficient to confer protective immunity through selective CD8 CTL responses against SARS-CoV-2 and a range of other human viral pathogens.

[0170] Vaccines that do not seek to elicit B-cell responses against VIPs but rather non-VIP antigen-specific CD8 T-cell responses could potentially have an enhanced safety profile. Indeed, given the complex nature of HLA-restricted antigen presentation in humans, it is reasonable to hypothesize that both vaccine efficacy and vaccine-related adverse events could be closely predicted by the recipient's HLA I haplotype and, to a lesser extent, HLA II haplotype.

[0171] In terms of adverse event management, quantification of T-cell responses against immunodominant HLA-restricted epitopes (presumably derived from VIPs) in patients and vaccine recipients can certainly be performed using established laboratory techniques (i.e., ELISPOT assays using primary samples). However, the identification, classification, and specification of HLA-I-restricted epitopes that elicit subdominant T-cell responses in clinically manifested SARS-CoV-2 infection cannot be addressed using such techniques due to poor sensitivity and resolution.

[0172] The high-resolution and systematic techniques for generating putative HLAI-restricted epitopes (i.e., non-VIP-derived HLAI-HREs) and their precise analysis of immunogenicity lay the foundation for the next generation of vaccines against the emerging SARS-CoV-2 and viruses with similar infectious life cycles. Given the limited payload capacity of modern recombinant RNA, DNA, and protein vaccine vectors, the compact nature of HLAI-HREs allows for maximal epitope coverage to match the HLA haplotypes of the target population to be vaccinated. Even with the most restrictive recombinant RNA vaccine vectors, a limited set of non-VIP-derived HLAI-HREs could achieve at least 60% HLAI allele coverage in the world population, particularly for rapid deployment of vaccines against emerging viral pathogens to confer population-wide protection.

[0173] In existing studies of similar invasive viral infections of the respiratory mucosa, several themes have emerged suggesting the possible presence of T cell immune dysregulation, in addition to T cell and immunoglobulin-related immunopathology.

[0174] An important perspective is that acute respiratory distress syndrome (ARDS) is associated with robust viral immunoglobulin responses in both natural infection and developmental vaccines. This implies that during persistent infection, robust HLA II / 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 infection control by HLA I / CD8 responses in susceptible individuals early in infection, followed by high viral titers driving robust HLA II / CD4 helper responses and B cell maturation. This aligns 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 naive T cell population decreases with age; however, the naive CD8 population also experiences a significantly more rapid relative decline compared to the naive CD4 population. Older subjects may develop immune dysregulation, in part due to a naturally declining CD8 T cell response associated with a naive CD8 T cell pool, which allows for progression of infection and viremia, driving B cell maturation and immunoglobulin-related immunopathology.

[0175] In support of this view of Covid-19 disease progression, subjects who recovered from SARS-CoV, the virus responsible for the 2003 SARS pandemic, were observed to have weak and transient virus-specific immune responses, lasting only a few months. In contrast, a significant CD8 T cell memory population was observed to persist one year after infection in recovered subjects.

[0176] Many current vaccine strategies for SARS-CoV-2 are focused on the spike protein as the key antigenic payload. Of note, this could be counterproductive if immunoglobulin-related immunopathology contributes to the severe inflammatory manifestations of Covid-19. Indeed, this appears to be the case in similar attempts to vaccinate against other viral infections of the respiratory mucosa.

[0177] Furthermore, an important aspect of the viral life cycle is that the spike protein is a poor reservoir for HLA-I-restricted epitopes during natural infection. Indeed, the spike protein is a membrane component that is produced and inserted into the ER membrane before being transported to vesicles where virion assembly and subsequent budding occur. This means that the spike protein is largely sequestered from the cytoplasmic pathways that are the primary contributors to antigen processing and cross-presentation of HLA-I-restricted epitopes. This not only limits the effective priming potential of CD8-centric immune priming, but the spike protein itself is more likely to drive HLA-II / CD4 priming and the initiation of immunoglobulin responses, thereby contributing to worsening vaccine-induced disease during subsequent natural infection.

[0178] Considering the above observations, it can be hypothesized that immunization with HLA-restricted epitopes to promote the induction of CD8 T cell immune responses is a safe and productive way to provide significant protection against SARS-CoV-2 infection. Highly defined payloads encoding HLA-I-restricted epitopes will help establish CD8 T cell memory responses as a frontline protection against natural infection while avoiding the potential for harmful induction of HLAII / CD4 or immunoglobulin responses. This approach requires the ability to accurately identify and characterize HLA-restricted epitopes and the CD8 T cell responses against these epitopes in natural infection.

[0179] A key feature of coronaviruses and other invasive viruses that infect the respiratory mucosa is the presence of regulatory proteins encoded in their genomes that subvert innate immunity within cells and potentially lead to dysregulation of T-cell immunity. Indeed, the SARS-CoV virus that caused the 2003 SARS pandemic encodes three ORFs capable of subverting innate immune responses leading to interferon production, which are similarly contained in the SARS-CoV-2 genome. These cytoplasmically accessible proteins represent ideal targets and the natural repertoire of HLA-I-restricted epitopes 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 CD8 T-cell response specific for HLA-I-restricted epitopes.

[0180] Given the clear central role of T cell immunity in SARS-CoV-2 immunity and immunopathology, safe and effective vaccine deployment must incorporate targeted immunity against HLA-restricted epitopes. This presents a significant challenge given the vast diversity of haplotypes across ethnic categories and geographic regions. Preparing for vaccine design and deployment in 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.

[0181] An important strategic aspect to consider is a more in-depth analysis of HLA-restricted epitopes of selected viral ORFs within the existing HLA allele core set and / or expanding this analysis to cover different HLA I haplotypes that are strategically important in the global SARS-CoV-2 pandemic.

[0182] A key point in controlling emerging pandemics moving forward is the ability to systematically assess the emergence of immune-evading viral strains. It should be noted that the novel coronavirus, SARS-CoV-2, likely arose after jumping from another animal species to humans, thus lacking extensive exposure to and selective pressure from the human immune system. It is reasonable to assume that a large number of HLAI-restricted epitopes will be presented by emerging SARS-CoV-2 strains, and that with widespread infection, new strains containing mutations that drive escape from the presentation of specific HLAI-restricted epitopes may emerge.

[0183] Example 2: Method for Determining Immunogenic Non-VIP-Derived HLA I-HREs for Inclusion in Vaccine Compositions

[0184] This example outlines the method for selecting non-VIP-derived HLA I-HREs for use in generating vaccine compositions using the SARS-CoV-2 viral infection outlined in Example 1.

[0185] This section outlines a method 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 cohorts: patients with severe Covid-19 manifestations, subjects with known infection but mild symptoms, and healthy donors who were not infected with SARS-CoV-2.

[0186] The goal of this overall analysis is to identify HLA-restricted epitopes that most effectively mediate viral clearance and potentially drive dysregulated antigen-specific T cell responses. These analyses will enable the next generation of valuable vaccines by providing critical data on antigen-specific T cell responses in uninfected subjects, patients with severe Covid-19, and patients whose infection resolves.

[0187] These example methods can be divided into 5 key work packages:

[0188] Work Package 1 (WP1)

[0189] SARS-CoV-2 HLA-I restricted epitope discovery

[0190] Work Package 2a (WP2a)

[0191] Antigen-specific CD8 T cell activation and TCR screening

[0192] Work Package 2b (WP2b)

[0193] HLA1 ​​multimer and TCR reagent library generation

[0194] Work Package 3 (WP3)

[0195] Validation of the HLA I antigen-specific T cell assay

[0196] Work Package 4 (WP4)

[0197] Conduct of studies with Covid-19 and uninfected subjects

[0198] SARS-CoV-2 HLA-I restricted epitope discovery – WP1

[0199] Purpose:

[0200] The first complete HLA-I restricted epitope scan of all SARS-CoV-2 ORFs was performed to identify functionally processed and cross-presented HLA-I restricted epitopes within the 16 most prevalent alleles in major markets.

[0201] Provides the basic dataset for selecting non-VIP derived HLAi-HREs used to perform studies in WP2a, WP2b, WP3 and WP4.

[0202] Techniques and methods:

[0203] In its most basic form, the engineered antigen-presenting cell (eAPC) system is a system for rapidly generating eAPC cell lines that express target analytes. 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 of HLA and antigen ORF constructs that are paired with genomic receptor sites within functionally engineered immortalized cell lines that represent "programmable" eAPCs (WO2018083316).

[0204] This eAPC platform provides high-throughput or high-content production of analyte eAPCs to power mass spectrometry (MS)-based methods to identify HLA-restricted antigens from integrated analyte ORFs within the context of the inherent HLA-I restriction repertoire derived from the eAPC protein itself.

[0205] This enables systematic analysis of analyte ORFs in a single HLA context (i.e., "monoallelic") by directly observing functionally processed and presented epitopes from the analyte sequence. Optionally, a series of expression constructs incorporating tandem minigene analytes can be used, along with an optional proteasome targeting motif that enhances cytoplasmic processing of the analyte protein in the expression system. This mass spectrometric readout is achieved by sample preparation using HLA capture from eAPC cell lysates, liquid chromatography fractionation of the sample, and then MS identification of HLA-restricted epitopes.

[0206] The set of 16 most common alleles formed the core working set with which HLA-restricted antigen discovery was performed. Figure 5 As shown, these were chosen to capture the best HLA coverage for major markets.

[0207] Deliverables:

[0208] A database of SARS-CoV-2 HLA-restricted epitopes presented by the 16 selected core HLA alleles.

[0209] Metadata includes relative strength scores (z scores) for the intrinsic HLA-restricted epitope repertoire of eAPCs and scores for the HLA allele-epitope consensus model.

[0210] Antigen-specific CD8 T cell activation and TCR screening – WP2a

[0211] Purpose:

[0212] The validated immunoresponsive HLA-restricted epitopes identified in WP1 were established by unbiased screening of uninfected and infected SARS-Cov-2 subjects.

[0213] The sensitivity of CD8 T cell activation-based and tetramer-based assays for the identified HLA-restricted SARS-CoV-2 epitopes was assessed.

[0214] Provide material for TCR validation and analysis standards in WP2b.

[0215] Techniques and methods:

[0216] The eAPC platform described in WP1 enables reliable and highly defined HLA-restricted antigen presentation to human cells from uninfected donors ( Primary CD8 T cells isolated from donors or patients are a key component for measuring HLA-restricted epitope responses in cell-based assays, further enhancing TCR discovery capabilities. Indeed, "monoallelic" eAPCs provide a reliable and reproducible format for stimulating and testing HLA-restricted epitope responses in T cell subsets, eliminating 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 infection.

[0217] This work package effectively deployed a set of techniques and methodologies;

[0218] 1. eAPC-based HLA allele-restricted stimulation of naive and memory CD8 T cell populations isolated from uninfected and infected SARS-CoV-2 subjects (WO2018083316).

[0219] 2. Quantification of HLA multimer-based reagents of HLA-restricted epitope-specific T cell responses following eAPC-stimulated growth.

[0220] 3. Tetramer-positive cells were single-cell sedimented by flow cytometry for downstream TCR clonal sequencing and validation.

[0221] 4. eAPC-based restimulation of outgrown primary CD8 T cell isolates to read out HLA-restricted epitope T cell responses through T cell activation markers (primarily IFNγ production).

[0222] The central purpose of the comprehensive assessment of immunogenicity of HLA-restricted epitopes is to provide an unbiased picture of the potential immunogenicity of 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.

[0223] It is important to note that all four techniques can be applied to a single clinical sample, conserving the use of original sample and significantly increasing throughput. This is further aided by the routine multiplexing of HLA multimer reagents in this analysis.

[0224] Regarding the restimulation method, after stimulating primary cells using an HLA allele-restricted eAPC-based system, the restimulation protocol was performed using eAPCs derived from a completely different cell line. This was designed to eliminate background signals encountered when using non-self APCs as the stimulation platform.

[0225] Deliverables:

[0226] A metadata layer on top of the HLA-I restricted epitope database generated in WP1 details the immunoresponsiveness potential of the HLA-restricted epitopes observed at the time of detection.

[0227] Preliminary evaluation of the sensitivity of cell activation-based and HLA-based flow cytometry assays for analyzing HLA-restricted epitope T cell responses in SARS-CoV-2 infection.

[0228] Validated TCR sequences targeting immunogenic HLA-restricted epitopes directly support WP2b, WP3, and WP4.

[0229] HLA1 ​​multimer and TCR reagent library generation – WP2b

[0230] Purpose:

[0231] An internally validated HLA multimer reagent library was constructed using matched TCR-expressing engineered TCR presenting cell (eTPC) assay standards.

[0232] The production of all HLA multimer reagents in the target library was operationalized to meet the reagent requirements of WP3 and WP4.

[0233] Generation of eTPC assay standards for expressed TCRs that can be used for flow cytometric assays based on HLA multimer reagents deployed in WP3.

[0234] Techniques and methods:

[0235] HLA multimer production technologies are highly standardized and well known to those skilled in the art; however, these methods involve challenging protein biochemistry involving protein folding and purification. The operationalization of these production workflows is driven by the need to provide reliable functional quality control standards in specific eTPC cell lines expressing TCRs.

[0236] This work package demonstrates a range of TCR molecular genetics and cell biology techniques to construct eTPC standards using materials derived from WP2a. These techniques can be summarized into four key workstreams:

[0237] 1. Semi-automated TCR α / β chain sequencing and bioinformatic filtering using proprietary primer libraries and software.

[0238] 2. Rapid PCR-independent TCR α / β ORF reconstruction using a TCR molecular genetics platform – the TCR ORF Reconstruction and Engineering System (TORES), which enables rapid and low-cost reconstruction of full-length TCR ORFs within expression vectors (WO2019016175 and WO2018083318).

[0239] 3. Perform eTPC-based high-throughput screening by integrating paired TCRα / β ORFs to screen HLA multimer reagents for target binding and / or interaction with eAPCs presenting target HLA and HLA I-HRE (WO2018083317 and WO2018083339 and WO2018083318).

[0240] 4. High-throughput preparation of eTPC standards expressing TCRs, with stable chemically fixed analytical standard reagent products (WO2018083317).

[0241] These approaches 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.

[0242] Deliverables:

[0243] HLA multimer reagent library for subsequent work package deployment.

[0244] eTPC-based analytical standard library for quality control of HLA multimer reagent preparation and internal controls for flow cytometry assay operationalization in WP3.

[0245] Operationalization of the HLA I antigen-specific T cell assay - WP3

[0246] Purpose:

[0247] Operationalization of the HLA multimer reagent-based assay for direct staining of peripheral blood and potentially liquid biopsies, with flow cytometric readout of target HLA allele and restricted epitope coverage, supports the study of antigen-specific T cell responses in WP4.

[0248] Operationalization of an assay based on eAPC-driven cell activation was used to support an indirect assay readout for parallel deployment of WP4.

[0249] Techniques and methods:

[0250] The technical and methodological aspects of the scalable deployment of reagent-based assays involve the use of analytical standards based on TCR-expressing eTPCs. 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 eTPCs expressing the specific TCR are chemically immobilized and stored as a stable reagent alongside the HLA multimer reagent stock for assay execution.

[0251] In cell-based activation assays, eAPC-driven HLA-restricted epitope stimulation of primary T cell isolates can directly detect the presence of antigen-specific T cell clones within a CD8 T cell memory population against viral epitopes. This assay is also sensitive enough to detect antigen-specific T cell clones within a naive cell population. These assays rely on peptide loading of monoallelic eAPC preparations expressing the desired HLA allele, followed by contact with individual-derived CD8 T cell isolates to stimulate memory and / or naive T cell populations. This enables relatively high-throughput, parallel analysis of HLA-restricted epitope-specific T cell responses in both memory and naive T cell compartments, compared to the use of primary APC sources in standard mixed leukocyte responses.

[0252] The current work package around the operationalization of reagent-based and activation-based assays involves accurate HLA typing of all subjects to assign the correct HLA multimer reagents and / or peptide-loaded monoallelic eAPCs to analyze individual subjects.

[0253] Deliverables:

[0254] HLA multimer reagent-based assays supporting the operationalization of WP4.

[0255] Operationalized cell activation-based assays supporting WP4 across selected HLA alleles and HLA-restricted epitope coverage.

[0256] Conduct of studies in patients and uninfected subjects - WP4

[0257] This work package aims to systematically study non-VIP derived HLA I-HRE responses in human subjects to identify immunogenic epitopes that drive viral clearance. The construction of tools and assays in WP1, WP2a, WP2b and WP3 enables these studies.

[0258] Purpose:

[0259] Comparative studies were performed 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.

[0260] Techniques and methods:

[0261] The current work package deploys reagents and assays developed in previous work packages, focusing on comparative analysis of antigen-specific T cell responses in subjects with known resolution of SARS-CoV-2 infection and no severe clinical manifestations, compared with individuals with severe Covid-19 presentation. In determining the primary antigen-specific CD8 T cell responses across different SARS-CoV-2 outcomes, the central goal 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.

[0262] Deliverables:

[0263] A systematic comparative analysis of non-VIP-derived HLA I-HRE immune responses was performed in the following categories of human subjects:

[0264] 5. Subjects with asymptomatic SARS-CoV-2 infection

[0265] 6. Subjects whose SARS-CoV-2 infection symptoms have resolved

[0266] 7. Subjects with severe Covid-19 disease

[0267] 8. Subjects known not to be infected with SARS-CoV-2

[0268] Non-VIP-derived HLA I-HREs identified in each subject category were prioritized for inclusion in the vaccine composition in the order 1>2>3>4.

[0269] definition

[0270] vaccine

[0271] A preparation generally comprising an antigenic component of a microorganism, virus, or other self or non-self substance, which, when administered to a human or veterinary subject, is intended to elicit an adaptive immune response in the host to provide immunity or tolerance to the target microorganism, virus, allergen, or other non-self or self substance.

[0272] Vaccine composition

[0273] The antigen and adjuvant delivered by the vaccine vector are intended to elicit a desired immune response when the vaccine is formulated and administered to a human or veterinary subject.

[0274] vaccine vectors

[0275] Vaccine vectors in the present context are defined as vehicles by which vaccine compositions are administered to human or veterinary subjects and include: recombinant non-replicating or replicating viral vectors; virus-like particles; recombinant RNA constructs; recombinant DNA constructs; recombinant proteins and / or protein complexes; and synthetic polypeptides.

[0276] vaccine adjuvants

[0277] Molecules or compounds with inherent immunomodulatory properties that, when co-administered with an antigen, are effective in enhancing the host antigen-specific immune response compared to the response produced when the antigen is administered alone. Some viral vectors and virus-like particle vectors are believed to have inherent adjuvant molecules and compounds, and additional co-stimulatory molecules can be encoded in the 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.

[0278] Vaccine preparations

[0279] The preparation for administration to human or animal subjects is a preparation in which the vaccine vector is formulated together with a pharmacologically suitable vaccine adjuvant and excipients, wherein the excipients may include: anti-adhesives, binders, coatings, pigments, disintegrants, flavors, glidants, lubricants, preservatives, adsorbents, sweeteners and vehicles.

[0280] Prime-boost vaccination

[0281] 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 responses, and then these immune responses are boosted with subsequent vaccine administrations. Prime-boost vaccine strategies can be administered in a homologous manner, wherein the vaccine composition, vaccine vector, and / or vaccine formulation, and / or vaccine route of administration are the same for the prime and boost administrations. Prime-boost vaccine strategies can also be implemented in a heterologous manner, wherein the vaccine composition, vaccine vector, and / or vaccine formulation, and / or vaccine route of administration are different for the prime and boost administrations.

[0282] antigen

[0283] A molecule, especially a biomolecule, that represents a target of the adaptive immune system; in the context of the B-cell and T-cell systems, antigens are detected by immunoglobulins and T-cell receptors, respectively.

[0284] Epitope

[0285] An epitope, also known as an antigenic determinant, is a portion of an antigen that is recognized by the adaptive immune system, particularly an immunoglobulin (Ig) or T-cell receptor (TCR). In other words, an epitope is the specific portion of an antigen that an immunoglobulin or T-cell receptor binds to.

[0286] Human leukocyte antigen (HLA)

[0287] The human leukocyte antigen (HLA) system or complex is a gene complex that encodes the major histocompatibility complex (MHC) proteins of jawed vertebrates. For clarity, the HLA nomenclature is used herein to describe the genes (i.e., HLA) and protein complexes (i.e., MHC).

[0288] Human leukocyte antigen class I (HLAI)

[0289] HLAs correspond to MHC class I (A, B, C, and E), which comprise the HLA class 1 group.

[0290] Human leukocyte antigen class II (HLA II)

[0291] HLAs corresponding to MHC class II (DP, DM, DO, DQ, and DR), which comprise the HLA class 2 group.

[0292] HLA-restricted epitopes (HREs)

[0293] Any epitope that is loaded into HLA molecules and presented on the cell surface for T-cell sampling (T-cellsampling) by TCR. Typically, for HLA I and II molecules, these epitopes represent peptides. Non-peptide molecules can also be loaded into HLA to be presented on the cell surface.

[0294] HLAI restricted epitope (HLAI-HRE)

[0295] Any epitope that is specifically loaded into an HLA1 molecule for export to the cell surface for T cell sampling.

[0296] HLA II restricted epitope (HLII-HRE)

[0297] Any epitope that is specifically loaded into an HLA II molecule for export to the cell surface for T cell sampling.

[0298] Virus-encoded protein (VEP)

[0299] All proteins encoded in the viral genome, including post-translational modifications of that protein.

[0300] Virus particles

[0301] The entire virus particle.

[0302] Virion component protein (VIP)

[0303] Any protein encoded by the viral genome that is present within the virion when it is released from an infected host cell following de novo virion generation.

[0304] Non-virion component proteins (non-VIP)

[0305] Any protein encoded by the viral genome that is expressed in infected host cells during the viral life cycle but is not present within virions.

[0306] Antigen-presenting cells

[0307] Any cell that expresses HLA-I molecules and is capable of loading HLA-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.

[0308] Professional antigen-presenting cells

[0309] Specialized APCs of the immune system constitutively express HLA II molecules and incorporate pathways in which sampling of extracellular components by pinocytosis, endocytosis, and phagocytosis leads to the presentation of HLA II-restricted epitopes. The major types of professional APCs are dendritic cells, macrophages, and B cells.

[0310] T cells

[0311] T lymphocytes or T cells refer to thymic differentiated lymphocytes that detect antigen epitopes through T cell receptors (TCRs) and mediate diverse adaptive immune functions.

[0312] TCR

[0313] Defined T cell immunoreceptors that are able to detect antigenic epitopes in an adaptive manner. There are α / β and γ / δ TCR systems, of which α / β are generally responsible for detecting HLA I and HLA II restricted epitopes.

[0314] naive T cells

[0315] Naive T cells are T cells that have differentiated in the bone marrow and successfully undergone the positive and negative processes of central selection in the thymus.

[0316] Effector T cells

[0317] CD4 helper or cytotoxic CD8 T cells that have been activated by encountering the productive epitope and are thus committed to influence a selected function of the T cell.

[0318] memory T cells

[0319] Committed effect T-cells can subsequently commit to a memory state, which may represent a quiescent cell state until a productive epitope is encountered again, thus representing a central mode of acquired T-cell immunity or tolerance to cognate antigen-derived epitopes.

[0320] CD8 T cells

[0321] T cells that express α / α, α / β, or β / β CD8 receptor dimers on their cell surface and are generally responsible for sampling HLA-HRE presented by APCs. The effector functions of CD8 T cells are most commonly cytotoxic but can further include regulatory and helper functions.

[0322] CD4 T cells

[0323] T cells that express the CD4 receptor on their cell surface and are generally responsible for sampling HLA II-HRE presented by APCs. The effector functions of CD4 T cells are highly diverse and not fully understood, but they are considered to be "helper" cells that detect diverse antigenic epitopes and coordinate the entire innate and adaptive immune system through a complex intracellular signaling network.

[0324] Cytotoxic T lymphocytes (CTLs)

[0325] T cells with cytotoxic effector functions are usually represented by CD8 T cells.

[0326] Helper T cells

[0327] A generalized definition used for those T cells that lack cytotoxic effector functions, most commonly referring to CD4 T cells.

[0328] T regulatory cells (Treg)

[0329] CD4 T cells are the most common T cells that specifically confer immune tolerance as central effector functions through a series of immunosuppressive signaling.

[0330] B cells

[0331] B lymphocytes, or B cells, are lymphocytes that differentiate in the bone marrow and successfully undergo positive and negative selection before migrating from the bone marrow. B cells are defined by a surface B cell receptor (BCR), which stands for membrane-bound immunoglobulin (Ig) and has a gene and protein structure similar to that of the TCR. The BCR is crucial for detecting Ig epitopes encountered for the first time, which triggers a gated B cell maturation process that requires continued epitope availability and a series of helper T cell inputs to progress and produce multiple forms of soluble immunoglobulins. B cells can also function as professional antigen-presenting cells, particularly in their ability to dock with helper T cells to receive signaling inputs.

[0332] Immunoglobulin (Ig)

[0333] Commonly referred to as antibodies, these soluble adaptive recognition molecules are produced by mature B cells and can mediate a range of effector functions in the host immune response by binding to cognate epitopes.

[0334] Neutralizing immunoglobulin (neutralizing Ig)

[0335] Immunoglobulins directed against epitopes usually derived from non-self antigens and serving as generalized effector functions for the neutralization of pathogens or non-self molecules.

[0336] Dendritic cells (DCs)

[0337] Professional antigen-presenting cells are central mediators of T cell and B cell responses.

[0338] macrophages

[0339] Professional antigen-presenting cells are central mediators of T- and B-cell responses and are typically defined by their phagocytic activity.

[0340] Autoantigens

[0341] Antigens derived from the host body that are involved in adaptive immunity.

[0342] non-self antigen

[0343] Antigens related to adaptive immunity that are derived from outside the host body usually refer to antigens from pathogens and allergens, but can also include antigens from food and commensal microorganisms.

[0344] Interferon type I (IFN-I)

[0345] A family of cytokines found in mammals that bind to interferon receptors and help regulate the immune system.

[0346] Draining lymph nodes (DLN)

[0347] Lymph nodes that drain target tissues, typically those that drain infected tissues, tissues containing dysplasia, malignancies, or tissues directly exposed to allergens, commensal microorganisms, and / or foods.

[0348] Open reading frame (ORF)

[0349] An in-frame nucleic acid can be transcribed to produce an ORF product or portion of a gene transcript.

[0350] Immunogenicity

[0351] A general term that denotes 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.

[0352] Active infection

[0353] Viral infection in which the virus continuously replicates and releases de novo synthesized virions from the infected host cell and infects other host cells.

[0354] Resolved infection

[0355] A viral infection that has been cleared by the host's immune system.

[0356] Latent infection

[0357] Viral infections have resulted in the host immune system suppressing viral replication and maintaining viral genomic material within host cells, which can undergo reactivation and de novo virion generation in the future to establish active infection.

[0358] Clinical manifestations

[0359] Viral infections with significant clinically relevant manifestations.

[0360] Attached boxes:

[0361] Figure 1 , Box 1:

[0362] Elderly subjects

[0363] The spectrum of immunodeficiency; notably a reduction in the naive T-cell repertoire.

[0364] Elderly subjects had lower naive CD8 T cell surveillance during the early stages of infection.

[0365] Escape from CD8 CTL activation early in infection.

[0366] Resulting in reduced and / or delayed potential to clear virus-infected cells.

[0367] Figure 2 , Box 1:

[0368] Systemic immune activation

[0369] Cellular injury releases DAMPs and PAMPs that are recognized by neighboring cells and macrophages to trigger cytokine and chemokine production.

[0370] Immune cell recruitment drives a more stringent sampling of cells by T cell populations, with higher numbers of dendritic cells arriving in MALT and DLN, and responses promoting sampling of CD8 and CD4 HLA-restricted epitopes.

[0371] Figure 2 , Box 2:

[0372] Viral spread

[0373] Immune evasion by frontline CD8 CTL activation against viral HLA-I restricted epitopes allows infection to establish.

[0374] Figure 3 , Box 1:

[0375] Virus particle accumulation

[0376] Viral dissemination within epithelial cells allows accumulation of virions for further viral dissemination and sampling by professional APCs and B cells.

[0377] Figure 3 , Box 2:

[0378] Adaptive response

[0379] Extensive immune cell recruitment skews adaptive CD8, CD4, and B cell responses toward VIP.

[0380] Figure 4 , Box 1:

[0381] Viremia and tissue damage

[0382] Phagocytosed epithelial cells provide VEPs for cross-presentation to CD8 T cells.

[0383] DCs sample high viral titers in the respiratory mucosa and draining lymph nodes for cross-presentation to CD8 CTLs.

[0384] Figure 4 , Box 2:

[0385] Dysregulated adaptive immune response

[0386] Initial immune escape based on suppression of innate immune responses leads to evasion of productively infected cells from early CD8 T cell detection and delayed CD8 CTL responses.

[0387] Viral infection, cell death, immune recruitment, and establishment of viremia drive the polarization of delayed adaptive CD8 CTL responses toward the highly abundant VIP-derived HLA-I-HRE.

[0388] CD8 CTL cytotoxic function leads to exhaustion of professional APCs at sites of infection and in the DLN, allowing for the rapid accumulation of tissue damage and a reduction in DCs to direct CD4 helper cell responses.

[0389] Dysregulation of T cell adaptive immunity leads to severe inflammation and cytokine storm, and weak CD4 T cell help for B cell maturation results in impaired antibody responses.

Claims

1. A method for producing a vaccine composition for use against a viral pathogen, the method comprising: a. Identifying a non-virion protein-derived human leukocyte antigen class I restricted epitope (non-VIP derived HLAI-HRE) from a viral pathogen, wherein it is desired to obtain a vaccine composition against the viral pathogen; b. Classifying the immunogenicity of the identified non-VIP-derived HLA I-HRE from step a in a population of naive CD8 T cells isolated from donors without prior target virus infection and / or in a population of memory CD8 T cells isolated from donors with confirmed active, latent, or resolved target virus infection; c. Selecting a non-VIP derived HLAI-HRE from step b that has confirmed immunogenicity in donors without prior target virus infection, or a non-VIP derived HLAI-HRE with observed CD8 T cell responses in donors with confirmed active, latent, or resolved target virus infection; d. Incorporating the selected non-VIP-derived HLA I-HRE from step c into a vaccine composition.

2. The method of claim 1 , wherein a plurality of non-VIP derived HLA I-HREs are selected for inclusion in the vaccine composition to represent one or more HLA I-HREs of selected HLA I alleles, wherein the selected HLA I alleles represent those alleles carried by at least 60% of individuals in the target population for which the vaccine composition is designed.

3. The method of claim 1 or 2, wherein one or more non-VIP derived HLA I-HREs are selected for inclusion in the vaccine composition to represent one or more HLA I-HERs in one or more selected HLA I alleles carried by the individual for whom the vaccine composition is designed.

4. The method according to any one of claims 1 to 3, wherein the vaccine composition comprises one or more vaccination vectors selected from the group consisting of: 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. A method according to claim 4, wherein the one or more vaccination vectors are selected from a, b, c or d according to claim 4, and wherein the selected non-VIP derived HLAI-HRE is incorporated into an expression construct that does not allow expression of functional non-VIP proteins in host cells upon vaccine delivery to avoid the immune escape activity of the viral non-VIP.

6. The method of claim 5, wherein the selected non-VIP derived HLA I-HRE is provided by one or more of the following means: a. introducing point mutations and / or sequence insertions and / or sequence deletions that inactivate protein function within the full-length non-VIP ORF; b. constructing a synthetic nucleic acid sequence comprising a non-VIP ORF fragment encoding the selected HLAI-HRE in a tandem construct; c. Constructing a synthetic nucleic acid sequence containing a non-VIP ORF fragment encoding the selected HLA I-HRE within a carrier protein sequence.

7. The method of claim 4, wherein the one or more vaccination vectors are selected from e or f according to claim 4, and wherein the recombinant protein or synthetic polypeptide comprises one or more non-VIP derived HLA1-HREs, and the protein or polypeptide molecule comprises tandem HLA1-HREs or encodes the HLA1-HER within a carrier protein or polypeptide.

8. The method of any one of claims 4-6, wherein the one or more vaccination vectors further encode one or more B cell / immunoglobulin epitopes to elicit a neutralizing Ig response.

9. The method according to any one of claims 4-6, wherein the one or more vaccination vectors further encode one or more selected HLAII-HRE epitopes to elicit CD4 T cell responses to support B cell maturation and neutralizing antibody production, and / or promote the commitment of non-VIP-derived HLA1 HRE-specific CD8 T cell responses to memory differentiation.

10. The method of claim 8, wherein the one or more B cell / immunoglobulin epitopes are modified to remove the HLA I-HRE from VIP to avoid eliciting a CD8 T cell response against the VIP protein upon vaccine delivery.

11. The method of any of the preceding claims, wherein step a of claim 1 comprises the use of standardized donor vectors of HLA and antigen ORF constructs paired with genomic acceptor sites within a functionally engineered immortalized cell line representing a programmable engineered antigen presenting cell (eAPC), followed by screening by a mass spectrometry (MS)-based approach to identify HLA-restricted antigens from integrated ORFs within the context of an inherent HLA I restriction repertoire derived from the eAPC protein itself, and enabling systematic analysis of ORFs in a single HLA context (monoallelic).

12. The method according to any one of the preceding claims, wherein step a of claim 1 further comprises using engineered TCR presenting cells (eTPCs) as a substep.

13. The method according to any of the preceding claims, wherein the selected non-VIP derived HLA I-HRE included in step d of claim 1 is a non-VIP derived HLA I-HRE from a donor without prior target virus infection, or a non-VIP derived HLA I-HRE to which a CD8 T cell response is observed in a donor with confirmed active and / or resolved target virus infection.

14. A vaccine composition for use against viral pathogens, prepared by the method of any one of claims 1 to 13.

15. A vaccine formulation comprising the vaccine composition of claim 14 and at least one pharmaceutically acceptable excipient.

16. A method of vaccinating a human or veterinary subject to provide immunity against a virus, comprising administering the vaccine composition of claim 14, the vaccine formulation of claim 15, or a vaccine composition prepared by the method of any one of claims 1 to 13.

17. A method for treating a human or veterinary subject suffering from an acute, chronic or latent viral infection by immunizing the human or veterinary subject with the vaccine composition of claim 14, the vaccine formulation of claim 15, or a vaccine composition prepared by the method of any one of claims 1 to 13 to elicit an immune response.

18. A method for eliciting a CD8 T cell response against a viral infection in a human or veterinary subject, comprising administering the vaccine composition of claim 14, the vaccine formulation of claim 15, or a vaccine composition prepared by the method of any one of claims 1 to 13.

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