Pressured cytotoxic T cell epitope strength among SARS-CoV-2 variants correlates with COVID-19 severity.

Heterogeneity in susceptibility among individuals to COVID-19 has been evident through the pandemic worldwide. Cytotoxic T lymphocyte (CTL) responses generated against pathogens in certain individuals are known to impose selection pressure on the pathogen, thus driving emergence of new variants. In this study, we probe the role played by host genetic heterogeneity in terms of HLA-genotypes in determining differential COVID-19 severity in patients. We use bioinformatic tools for CTL epitope prediction to identify epitopes under immune pressure. Using HLA-genotype data of COVID-19 patients from a local cohort, we observe that the recognition of pressured epitopes from the parent strain Wuhan-Hu-1 correlates with COVID-19 severity. We also identify and rank list HLA-alleles and epitopes that offer protectivity against severe disease in infected individuals. Finally, we shortlist a set of 6 pressured and protective epitopes that represent regions in the viral proteome that are under high immune pressure across SARS-CoV-2 variants. Identification of such epitopes, defined by the distribution of HLA-genotypes among members of a population, could potentially aid in prediction of indigenous variants of SARS-CoV-2 and other pathogens.

Synthesis of Killer T Cell Epitopes for Peptide-Based Vaccine of SARS-CoV-2

COVID-19 is a current global pandemic, which has prompted many countries to develop ways to deal with it. Peptides have many medicinal and diagnostic benefits, so recently, many researchers have been developing peptide-based vaccines against COVID-19. In peptide-based vaccines, peptides act as specific antigens that will provide a faster immune response because they do not go through the process of cutting proteins in the Major Histocompatibility complex (MHC) antigen-presenting cells (APC) and can be directly presented outside the cells so that they can be recognized by the host killer T cells (CTL). Vaccine development can be accelerated with the help of immunoinformatics to predict specific epitopes to induce CTL. We have predicted the CTL epitope through the immunoinformatic method. This study aims to synthesize candidate CTL epitopes as a candidate for the SARS-CoV-2 vaccine using the SPPS method with the Fmoc/t-Bu strategy. In this study, two CTL epitopes were synthesized through a conventional solid-phase peptide synthesis (SPPS) method, and another CTL epitope was synthesized using a semi-automated peptide synthesizer. The SPPS method is faster because the purification is only carried out at the final stage, while the Fmoc/t-Bu strategy was applied because it provides a mild reaction condition. Both synthetic approaches were compared. The semi-automated peptide synthesizer made the synthesis faster and more efficient due to using an inert gas (N2) during the synthesis. The synthetic peptides were characterized by TOF-ESI-MS. The three peptides showed ion peaks at m/z 1137.5509 (M+H)+, 1064.3468 (M+H)+, and 916.5859 (M+H)+, indicating correct molecular ion peaks for EILDITPCSF, IPIGAGICASY, and FIAGLIAIV, respectively. © 2022 National Information and Documentation Center.

Identification of HLA-A*24:02-Restricted CTL Candidate Epitopes Derived from the Nonstructural Polyprotein 1a of SARS-CoV-2 and Analysis of Their Conservation Using the Mutation Database of SARS-CoV-2 Variants.

COVID-19 vaccines are currently being administered worldwide and playing a critical role in controlling the pandemic. They have been designed to elicit neutralizing antibodies against Spike protein of the original SARS-CoV-2, and hence they are less effective against SARS-CoV-2 variants with mutated Spike than the original virus. It is possible that novel variants with abilities of enhanced transmissibility and/or immunoevasion will appear in the near future and perfectly escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Several lines of evidence suggest the contribution of CTLs on the viral control in COVID-19, and CTLs target a wide range of proteins involving comparatively conserved nonstructural proteins. Here, we identified 22 HLA-A*24:02-restricted CTL candidate epitopes derived from the nonstructural polyprotein 1a (pp1a) of SARS-CoV-2 using computational algorithms, HLA-A*24:02 transgenic mice and the peptide-encapsulated liposomes. We focused on pp1a and HLA-A*24:02 because pp1a is relatively conserved and HLA-A*24:02 is predominant in East Asians such as Japanese. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by a number of mutations in the Sequence Read Archive database of SARS-CoV-2 variants. The information of such conserved epitopes might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any SARS-CoV-2 variants by the induction of both anti-Spike neutralizing antibodies and CTLs specific for conserved epitopes. IMPORTANCE COVID-19 vaccines have been designed to elicit neutralizing antibodies against the Spike protein of the original SARS-CoV-2, and hence they are less effective against variants. It is possible that novel variants will appear and escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Here, we identified 22 HLA-A*24:02-restricted CTL candidate epitopes derived from the nonstructural polyprotein 1a (pp1a) of SARS-CoV-2. We focused on pp1a and HLA-A*24:02 because pp1a is conserved and HLA-A*24:02 is predominant in East Asians. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by mutations in the database of SARS-CoV-2 variants. The information might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any variants.

SARS-CoV-2-Seronegative Subjects Target CTL Epitopes in the SARS-CoV-2 Nucleoprotein Cross-Reactive to Common Cold Coronaviruses.

The beta-coronavirus SARS-CoV-2 induces severe disease (COVID-19) mainly in elderly persons with risk factors, whereas the majority of patients experience a mild course of infection. As the circulating common cold coronaviruses OC43 and HKU1 share some homologous sequences with SARS-CoV-2, beta-coronavirus cross-reactive T-cell responses could influence the susceptibility to SARS-CoV-2 infection and the course of COVID-19. To investigate the role of beta-coronavirus cross-reactive T-cells, we analyzed the T-cell response against a 15 amino acid long peptide (SCoV-DP15: DLSPRWYFYYLGTGP) from the SARS-CoV-2 nucleoprotein sequence with a high homology to the corresponding sequence (QLLPRWYFYYLGTGP) in OC43 and HKU1. SCoV-DP15-specific T-cells were detected in 4 out of 23 (17.4%) SARS-CoV-2-seronegative healthy donors. As HIV-1 infection is a potential risk factor for COVID-19, we also studied a cohort of HIV-1-infected patients on antiretroviral therapy. 44 out of these 116 HIV-1-infected patients (37.9%) showed a specific recognition of the SCoV-DP15 peptide or of shorter peptides within SCoV-DP15 by CD4+ T-cells and/or by CD8+ T-cells. We could define several new cross-reactive HLA-I-restricted epitopes in the SARS-CoV-2 nucleoprotein such as SPRWYFYYL (HLA-B*07, HLA-B*35), DLSPRWYFYY (HLA-A*02), LSPRWYFYY (HLA-A*29), WYFYYLGTGP and WYFYYLGT. Epitope specific CD8+ T-cell lines recognized corresponding epitopes within OC43 and HKU1 to a similar degree or even at lower peptide concentrations suggesting that they were induced by infection with OC43 or HKU1. Our results confirm that SARS-CoV-2-seronegative subjects can target SARS-CoV-2 not only by beta-coronavirus cross-reactive CD4+ T-cells but also by cross-reactive CD8+ cytotoxic T-cells (CTL). The delineation of cross-reactive T-cell epitopes contributes to an efficient epitope-specific immunomonitoring of SARS-CoV-2-specific T-cells. Further prospective studies are needed to prove a protective role of cross-reactive T-cells and their restricting HLA alleles for control of SARS-CoV-2 infection. The frequent observation of SARS-CoV-2-reactive T-cells in HIV-1-infected subjects could be a reason that treated HIV-1 infection does not seem to be a strong risk factor for the development of severe COVID-19.

Identification of a dominant CD8+ CTL epitope in the SARS-associated coronavirus 2 spike protein.

Coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2, has been spreading throughout the world. To date, there are still no approved human vaccines for this disease. To develop an effective vaccine, the establishment of animal models for evaluating post-vaccination immune responses is necessary. In this study, we have identified a CTL epitope in the SARS-CoV-2 spike (S) protein that could be used to measure the cellular immune response against this protein. Potential predicted CTL epitopes of the SARS-CoV-2 S protein were investigated by immunizing BALB/c mice with a recombinant of the receptor-binding domain (RBD) of the S protein. Then, CD8+ T cells specific for S-RBD were detected by stimulating with potential epitope peptides and then measuring the interferon-gamma production. Truncation of this peptide revealed that S-RBD-specific CD8+ T cells recognized a H2-Dd-restricted S526-533 peptide. In conclusion, this animal model is suitable for evaluating the immunogenicity of SARS-CoV-2 vaccines.

Glycosylation, ligand binding sites and antigenic variations between membrane glycoprotein of COVID-19 and related coronaviruses.

A new coronavirus strain has wreaked havoc on human lives so the WHO was declared as a pandemic since 20th March 2020. The Membrane glycoprotein MP spans the viral envelope and it has a highly conserved glycosylation sequence. AIM: Our study goal was to find out the N-glycosylation, ligand binding sites, and antigenic variations between COVID-19 and other associated viruses. METHODS: We performed In silico methodologies for serial analysis at both an operational and result/output level is assessed and compared study factors. RESULTS: We detected high similarity in sequence alignment for >89% between COVID-19 MP and other MP of CoVs. Prediction of N-glycosylation and cytotoxic T-cell epitopes, we identified precisely sites between SARS-CoV-2 MP and Pangolin CoV MP 100%. We also didn't obtain any similarity in ligand binding site residues between MP sequences. Our study didn't reveal any similarity in CTL epitope predication between coronaviruses under study using the CTLPred server. CONCLUSIONS: Our results exhibit that the membrane glycoprotein of SARS-CoV-2 is closely associated with predecessor SARS-CoVs specifically Pngolin CoV. Prediction of novel CTL epitopes may substantial scopes for the expansion of a peptide-based vaccine for the inhibition virion assembly of SARS-CoV-2.

Una nueva cepa de coronavirus está causando estragos en la humanidad, por lo que la OMS declaró la situación de pandemia el 20 de marzo de 2020. La glicoproteína de membrana MP atraviesa la envoltura viral, y tiene una secuencia de glicosilación altamente conservada. OBJETIVO: El objetivo de nuestro estudio fue averiguar la N-glicosilación, los sitios de unión y las variaciones antigénicas entre COVID-19 y el resto de virus asociados. MÉTODOS: Realizamos metodologías in silico para análisis de series, tanto a nivel operativo como de resultados, y valoramos y comparamos los factores de estudio. RESULTADOS: Detectamos una gran similitud en cuanto a la alineación de secuencia para > 89% entre la MP de COVID-19 y otras MP de CoV. Prediciendo la N-glicosilación y los epítopos de las células T citotóxicas identificamos con precisión del 100% los sitios entre MP de SARS-CoV-2 y MP de CoV de pangolín. No obtuvimos ninguna similitud en cuanto a los residuos del sitio de unión del ligando entre las secuencias de MP. Nuestro estudio no reveló ninguna similitud en la predicción del epítope CTL entre los coronavirus estudiados, utilizando el servidor CTLPred. CONCLUSIONES: Nuestros resultados muestran que la glicoproteína de membrana de SARS-CoV-2 está estrechamente asociada a los SARS-CoV anteriores, específicamente CoV de pangolín. La predicción de los nuevos epítopos CTL puede definir sustancialmente la expansión de una vacuna basada en péptidos para la inhibición del ensamblaje del virión de SARS-CoV-2.