Over the last two decades, there have been three deadly human outbreaks of coronaviruses (CoVs) caused by SARS-CoV, MERS-CoV, and SARS-CoV-2, which has caused the current COVID-19 global pandemic. All three deadly CoVs originated from bats and transmitted to humans via various intermediate animal reservoirs. It remains highly possible that other global COVID pandemics will emerge in the coming years caused by yet another spillover of a bat-derived SARS-like coronavirus (SL-CoV) into humans. Determining the Ag and the human B cells, CD4+ and CD8+ T cell epitope landscapes that are conserved among human and animal coronaviruses should inform in the development of future pan-coronavirus vaccines. In the current study, using several immunoinformatics and sequence alignment approaches, we identified several human B cell and CD4+ and CD8+ T cell epitopes that are highly conserved in 1) greater than 81,000 SARS-CoV-2 genome sequences identified in 190 countries on six continents; 2) six circulating CoVs that caused previous human outbreaks of the common cold; 3) nine SL-CoVs isolated from bats; 4) nine SL-CoV isolated from pangolins; 5) three SL-CoVs isolated from civet cats; and 6) four MERS strains isolated from camels. Furthermore, the identified epitopes: 1) recalled B cells and CD4+ and CD8+ T cells from both COVID-19 patients and healthy individuals who were never exposed to SARS-CoV-2, and 2) induced strong B cell and T cell responses in humanized HLA-DR1/HLA-A*02:01 double-transgenic mice. The findings pave the way to develop a preemptive multiepitope pan-coronavirus vaccine to protect against past, current, and future outbreaks.

The next generation of needle-free mucosal vaccines is being rationally designed according to rules that govern the way in which the epitopes are recognized by and stimulate the genital mucosal immune system. We hypothesized that synthetic peptide epitopes extended with an agonist of Toll-like receptor 2 (TLR-2), that are abundantly expressed by dendritic and epithelial cells of the vaginal mucosa, would lead to induction of protective immunity against genital herpes. To test this hypothesis, we intravaginally (IVAG) immunized wild-type B6, TLR-2 (TLR2^−/−) or myeloid differentiation factor 88 deficient (MyD88^−/−) mice with a herpes simplex virus type 2 (HSV-2) CD8⁺ T-cell peptide epitope extended by a palmitic acid moiety (a TLR-2 agonist). IVAG delivery of the lipopeptide generated HSV-2-specific memory CD8⁺ cytotoxic T cells both locally in the genital tract draining lymph nodes and systemically in the spleen. Moreover, lipopeptide-immunized TLR2^−/− and MyD88^−/− mice developed significantly less HSV-specific CD8⁺ T-cell response, earlier death, faster disease progression, and higher vaginal HSV-2 titers compared to lipopeptide-immunized wild-type B6 mice. IVAG immunization with self-adjuvanting lipid-tailed peptides appears to be a novel mucosal vaccine approach, which has attractive practical and immunological features.

Lipopeptides, a form of peptide immunogens, are currently under intense investigation as human vaccines for many infectious pathogens and cancers. However, the cellular and molecular mechanisms of lipopeptide immunogenicity are only partially understood. We have investigated the influence of the lipid content on the immunogenicity of lipopeptides using the herpes simplex virus type 1 (HSV‐1) gD_1–23 peptide as a model antigen. Totally synthetic lipopeptides were constructed by covalent attachment to the peptide backbone of either N^ϵ‐palmitoyl‐lysine (palmitoyl‐lipidated peptide, palmitoyl‐LP) or cholesterol‐lysine (cholesterol‐lipidated peptide, cholesterol‐LP). Immunization of mice with the palmitoyl‐LP, but not with its cholesterol‐LP analog, induced a strong T cell‐dependent protective immunity against lethal HSV‐1 infection. Analysis of cytokine profiles and IgG2a/IgG1 ratios revealed that a dominant Th1‐type immune response was stimulated by the palmitoyl‐LP, as opposed to a Th2 response generated by its cholesterol‐LP analog. The palmitoyl‐LP was efficiently taken up in vitro by immature dendritic cells (DC) in a time‐ and dose‐dependent manner, and induced phenotypic maturation and production of pro‐inflammatory cytokines by DC. Finally, DC stimulated with the palmitoyl‐LP induced antigen‐specific T cell responses through the Toll‐like receptor‐2 pathway. These findings have important implications for the development of effective lipopeptide immunization strategies against infectious pathogens.

The molecular characterization of the epitope repertoire on herpes simplex virus (HSV) antigens would greatly expand our knowledge of HSV immunity and improve immune interventions against herpesvirus infections. HSV glycoprotein D (gD) is an immunodominant viral coat protein and is considered an excellent vaccine candidate antigen. By using the TEPITOPE prediction algorithm, we have identified and characterized a total of 12 regions within the HSV type 1 (HSV-1) gD bearing potential CD4⁺T-cell epitopes, each 27 to 34 amino acids in length. Immunogenicity studies of the corresponding medium-sized peptides confirmed all previously known gD epitopes and additionally revealed four new immunodominant regions (gD_49-82, gD_146-179, gD_228-257, and gD_332-358), each containing naturally processed epitopes. These epitopes elicited potent T-cell responses in mice of diverse major histocompatibility complex backgrounds. Each of the four new immunodominant peptide epitopes generated strong CD4⁺Th1 T cells that were biologically active against HSV-1-infected bone marrow-derived dendritic cells. Importantly, immunization of H-2^dmice with the four newly identified CD4⁺Th1 peptide epitopes but not with four CD4⁺Th2 peptide epitopes induced a robust protective immunity against lethal ocular HSV-1 challenge. These peptide epitopes may prove to be important components of an effective immunoprophylactic strategy against herpes.

Following ocular herpes simplex virus 1 (HSV-1) infection of C57BL/6 mice, HSV-specific (HSV-gB _498–505 tetramer ⁺ ) CD8 ⁺ T cells are induced, selectively retained in latently infected trigeminal ganglia (TG), and appear to decrease HSV-1 reactivation. The HSV-1 latency-associated transcript (LAT) gene, the only viral gene that is abundantly transcribed during latency, increases reactivation. Previously we found that during latency with HSV-1 strain McKrae-derived viruses, more of the total TG resident CD8 T cells expressed markers of exhaustion with LAT ⁺ virus compared to LAT ⁻ virus. Here we extend these findings to HSV-1 strain 17syn+-derived LAT ⁺ and LAT ⁻ viruses and to a virus expressing just the first 20% of LAT. Thus, the previous findings were not an artifact of HSV-1 strain McKrae, and the LAT function involved mapped to the first 1.5 kb of LAT. Importantly, to our knowledge, we show here for the first time that during LAT ⁺ virus latency, most of the HSV-1-specific TG resident CD8 T cells were functionally exhausted, as judged by low cytotoxic function and decreased gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) production. This resulted in LAT ⁻ TG having more functional HSV-gB _498–505 tetramer ⁺ CD8 ⁺ T cells compared to LAT ⁺ TG. In addition, LAT expression, in the absence of other HSV-1 gene products, appeared to be able to directly or indirectly upregulate both PD-L1 and major histocompatibility complex class I (MHC-I) on mouse neuroblastoma cells (Neuro2A). These findings may constitute a novel immune evasion mechanism whereby the HSV-1 LAT directly or indirectly promotes functional exhaustion (i.e., dysfunction) of HSV-specific CD8 ⁺ T cells in latently infected TG, resulting in increased virus reactivation.

We introduced a novel humanized HLA-A*0201 transgenic (HLA Tg) rabbit model to assess the protective efficacy of a human CD8⁺ T cell epitope-based vaccine against primary ocular herpes infection and disease. Each of the three immunodominant human CD8⁺ T cell peptide epitopes from HSV-1 glycoprotein D (gD_53–61, gD_70–78, and gD_278–286) were joined with a promiscuous human CD4⁺ T cell peptide epitope (gD_49–82) to construct three separate pairs of CD4–CD8 peptides. Each CD4–CD8 peptide pair was then covalently linked to an N^ε-palmitoyl–lysine residue via a functional base lysine amino group to construct CD4–CD8 lipopeptides. HLA Tg rabbits were immunized s.c. with a mixture of the three CD4–CD8 HSV-1 gD lipopeptides. The HSV-gD–specific T cell responses induced by the mixture of CD4–CD8 lipopeptide vaccine and the protective efficacy against acute virus replication and ocular disease were determined. Immunization induced HSV-gD_49–82–specific CD4⁺ T cells in draining lymph node (DLN); induced HLA-restricted HSV-gD_53–61, gD_70–78, and gD_278–286–specific CD8⁺ T cells in DLN, conjunctiva, and trigeminal ganglia and reduced HSV-1 replication in tears and corneal eye disease after ocular HSV-1 challenge. In addition, the HSV-1 epitope-specific CD8⁺ T cells induced in DLNs, conjunctiva, and the trigeminal ganglia were inversely proportional with corneal disease. The humanized HLA Tg rabbits appeared to be a useful preclinical animal model for investigating the immunogenicity and protective efficacy of human CD8⁺ T cell epitope-based prophylactic vaccines against ocular herpes. The relevance of HLA Tg rabbits for future investigation of human CD4–CD8 epitope-based therapeutic vaccines against recurrent HSV-1 is discussed.