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Understanding the evolutionary patterns and geographic spread of SARS-CoV-2 variants, particularly Omicron, is essential for effective public health responses. This study focused on the genomic analysis of the Omicron variant in Cordoba, Argentina from 2021 to 2022. Phylogenetic analysis revealed the dominant presence of BA.1 and BA.2 lineages, with BA.5 emerging earlier than BA.4, aligning with observations from other regions. Haplotype network analysis showed significant genetic divergence within Omicron samples, forming distinct clusters. In comparison to global datasets, we identified mutations in the Omicron genomes (A27S, Y145D, and L212I) situated within the NTD region of the Spike protein. These mutations, while not widespread globally, showed higher prevalence in our region. Of particular interest were the Y145D and L212I substitutions, previously unreported in Argentina. In silico analysis revealed that both mutations impact the binding affinity of T-cell epitopes to HLA type I and II alleles. Notably, these alleles are among the most common in the Argentinian population, with some associated with protection against and others with susceptibility to SARS-CoV-2 infection. These findings strongly suggest that these prevalent mutations likely influence the immunogenicity of the Spike protein and contribute to immune evasion mechanisms. This study provides valuable insights into the genomic dynamics of the Omicron variant in Cordoba, Argentina and highlights unique mutations with potential implications for COVID-19 vaccines.

Visceral leishmaniasis (VL) is a tropical disease that can be fatal if acute and untreated. Diagnosis is difficult, the treatment is toxic and prophylactic vaccines do not exist. Leishmania parasites express hundreds of proteins and several of them are relevant for the host's immune system. In this context, in the present study, 10 specific T-cell epitopes from 5 parasite proteins, which were identified by antibodies in VL patients’ sera, were selected and used to construct a gene codifying the new chimeric protein called rCHI. The rCHI vaccine was developed and thoroughly evaluated for its potential effectiveness against Leishmania infantum infection. We used monophosphoryl lipid A (MPLA) and polymeric micelles (Mic) as adjuvant and/or delivery system. The results demonstrated that both rCHI/MPLA and rCHI/Mic significantly stimulate an antileishmanial Th1-type cellular response, with higher production of IFN- γ , TNF- α , IL-12 and nitrite in vaccinated animals, and this response was sustained after challenge. In addition, these mice significantly reduced the parasitism in internal organs and increased the production of IgG2a isotype antibodies. In vivo and in vitro toxicity showed that rCHI is safe for the mammalians, and the recombinant protein also induced in vitro lymphoproliferative response and production of Th1-type cytokines by human cells, which were collected from healthy subjects and treated VL patients. These data suggest rCHI plus MPLA or micelles could be considered as a vaccine candidate against VL.

With the development of novel vaccine strategies, T-cell epitope-based vaccines have become promising prophylactic and therapeutic tools against infectious diseases that cannot be controlled via traditional vaccines. T-cell epitope-based vaccines leverage specific immunogenic peptides to elicit protective T-cell responses against infectious pathogens. Compared to traditional vaccines, they provide superior efficacy and safety, minimizing the risk of adverse side effects. In this review, we summarized and compared the prediction and identification methods of T-cell epitopes. By integrating bioinformatic prediction and experimental validation, efficient and precise screening of T-cell epitopes can be achieved. Importantly, we delved into the development approaches to diverse T-cell epitope-based vaccines, comparing their merits and demerits, as well as discussing the prevalent challenges and perspectives in their applications. This review offers fresh perspectives for the formulation of safe and efficacious epitope-based vaccines for the devastating diseases against which no vaccines are currently available.

Recurrent pregnancy loss (RPL), defined as two or more pregnancy losses before the 24th week of gestation, affects 1%-3% of women worldwide. Approximately, 40% of RPL cases are secondary RPL (sRPL), where women have given birth before facing pregnancy losses. The underlying causes of RPL remain unclear, but immune-related factors may play a role. Previously, a randomised controlled trial using immunoglobulin (IVIG) in sRPL women with a history of four pregnancy losses performed in our RPL unit did not show significant effects of IVIG treatment overall. Yet, some evidence suggests potential benefits for a subset of sRPL patients. In the cohort used for the randomised controlled trial, we examined the role of maternal HLA class II-presented fetal HLA-derived epitopes in sRPL using the predicted indirectly recognisable HLA epitopes (PIRCHE-II) algorithm. In the placebo group, sRPL mothers with an anti-HLA antibody response had higher PIRCHE-II scores when having a live birth compared with sRPL women who experienced another pregnancy loss. This difference was not observed in the IVIG-treated group. Furthermore, as a proxy for T-cell memory, the number of overlapping peptides between the two paternal haplotypes in couples having live births without treatment displayed a larger number of overlapping peptides. This effect was primarily driven by class II-derived peptides. These results suggest that specific combinations of sRPL mothers and fathers, particularly those with an anti-HLA antibody response, may generate higher PIRCHE-II scores, which could contribute to successful live births. Understanding these immune interactions may provide insights for personalised diagnostic and therapeutic strategies in sRPL., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: To date, no studies have investigated the potential reactivation of human endogenous retroviruses (HERVs) in the pathogenesis of antiphospholipid syndrome (APS). HERV-derived syncytin-1 and syncytin-2 are localized in the plasma membrane of cells and physiologically expressed during pregnancy. The current study aimed to determine whether the epitopes of syncytins can trigger an immune response leading to APS in genetically predisposed individuals., Methods: The TepiTool, ABCpred, and DiscoTope servers were utilized to predict T-cell and B-cell epitopes by inputting the FASTA sequences and 3D structures of syncytin-1, syncytin-2, and β2-glycoprotein I (β2GPI), which served as a reference antigen for APS. T-cell epitopes were selected based on their binding to a panel of human leukocyte antigen (HLA) class II alleles associated with APS according to the literature. Epitope predictions for the different proteins were statistically compared using GraphPad Prism., Results: For syncytin-1, we identified a total of 721 T-cell epitopes, 51 linear B-cell epitopes, and up to 40 conformational epitopes. For syncytin-2, we predicted 705 T-cell epitopes and 28 linear B-cell epitopes, but a lower number of conformational epitopes, which also exhibited lower B-cell receptor (BCR)-binding scores. The predicted T-cell and B-cell conformational epitopes of both syncytin-1 and syncytin-2 demonstrated significantly higher binding affinity to selected HLA alleles and BCR compared with β2GPI. Furthermore, syncytin-1 exhibited significantly higher immunogenicity than syncytin-2., Conclusions: Both syncytin-1 and syncytin-2 are computationally endowed with potential epitopes that may activate either T cells or B cells in individuals genetically predisposed to APS. While these findings may illuminate the possible role of HERVs in the development of APS, they warrant validation in further laboratory studies.

Dengue virus poses a significant global health challenge, particularly in tropical and subtropical regions. Despite the urgent demand for vaccines in the control of the disease, the two approved vaccines, Dengvaxia and TV003/TV005, there are current questions regarding their effectiveness due to an increased risk of antibody-dependent enhancement (ADE) and reduced protection. These challenges have underscored the need for further development of improved vaccines for Dengue Virus. This study presents a new design using an in silico approach to generate a more effective dengue vaccine. Initially, our design process began with the collection of Dengue polyprotein sequences from 10 representative countries worldwide. And then conserved fragments of viral proteins were retrieved as the bases for epitope screening. The selection of epitopes was then carried out with criteria such as antigenicity, immunogenicity, and binding affinity with MHC molecules, while the exclusion criteria were according to their allergenicity, toxicity, and potential for antibody-dependent enhancement. We then constructed a core antigen with the selected epitopes and linked the outcomes with distinct adjuvant proteins, resulting in three candidate vaccines: PSDV-1, PSDV-2, and PSDV-3. Among these, PSDV-2 was selected for further validation due to its superior physicochemical and structural properties. Extensive simulations demonstrated that PSDV-2 exhibited strong binding to pattern recognition receptors, high stability, and robust immune induction, confirming its potential as a high-quality vaccine candidate. For its recombinant expression, a plasmid was subsequently designed. Our new vaccine design offers a promising additional option for Dengue virus protection. Further experimental validations will be conducted to confirm its protective efficacy and safety.

Introduction: This immunoinformatic study identified potential epitopes from the envelopment polyprotein (Gn/Gc) of Rift Valley fever virus (RVFV), a pathogenic virus causing severe fever in humans and livestock. Effective vaccination is crucial for controlling RVFV outbreaks. The identification of suitable epitopes is crucial for the development of safe and effective vaccines., Methodology: Protein sequences were obtained from the UniProt database, and evaluated through VaxiJen v2.0 to predict the B and T-cell epitopes within the RVFV glycoprotein. Gn/Gc protein sequences were analyzed with bioinformatics tools and algorithms. The predicted T-cell and B-cell epitopes were evaluated for antigenicity, allergenicity, and toxicity by the VaxiJen v2.0 system, AllerTop v2.0, and ToxinPred server, respectively., Results: We employed computational methods to screen the RVFV envelopment polyprotein encompassing N-terminal and C-terminal glycoprotein segments, to discover antigenic T- and B-cell epitopes. Our analysis unveiled multiple potential epitopes within the RVFV glycoprotein, specifically within the Gn/Gc protein sequences. Subsequently, we selected eleven cytotoxic T-lymphocytes (CTL) and four helper T-lymphocytes (HTL) for population coverage analysis, which collectively extended to cover 97.04% of the world's population, representing diverse ethnicities and regions. Notably, the CTL epitope VQADLTLMF exhibited binding affinity to numerous human leukocyte antigen (HLA) alleles. The identification of glycoprotein (Gn/Gc) epitopes through this immunoinformatic study bears significant implications for advancing the development of an effective RVFV vaccine., Conclusions: These findings provide valuable insights into the immunological aspects of the disease and may contribute towards the development of broad-spectrum antiviral therapies targeting other RNA viruses with similar polymerase enzymes., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2024 Tanjir Hosen, Saaimatul Huq, Mohammad Abdullah-Al-Shoeb, Shahidul Islam, Muhammad Abul Kalam Azad.)

The E6 and E7 proteins of the high risk human papillomaviruses (HR HPVs) play a key role in the oncogenesis associated with papillomavirus infection. Data on the variability of these proteins are limited, and the factors affecting their variability are still poorly understood. We analyzed the variability of the currently known sequences of the HPV type 16 (HPV16) E6 and E7 proteins, taking into account their geographic origin and year of sample collection, as well as the direction of their evolution in the major geographic regions of the world. All sequences belonging to HPV16 genome fragments encoding the E6 and E7 oncoproteins were downloaded from the NCBI GenBank database on October 6, 2022. Samples were filtered according to the following parameters: the sequence has to include at least one of the two whole open reading frames, and given date of collection, and the country of origin. A total of 3651 full-genome nucleotide sequences encoding the E6 protein and 4578 full-genome nucleotide sequences encoding the E7 protein were sampled. The nucleotide sequences obtained after sampling and alignment were converted to amino acid sequences and analyzed using the MEGA11, R, RStudio, Jmodeltest 2.1.20, BEAST v1.10.4, Fastcov, and Biostrings software. The highest variability in the E6 protein was recorded for amino acid (AA) residues in the positions 17, 21, 32, 85, and 90. The most variable in E7 were aa positions 28, 29, 51, and 77. The samples were divided geographically into five heterogeneous groups as derived from Africa, Europe, America, South-West and South Asia, and South-East Asia. Unique amino acid substitutions (AA-substitutions) in the E6/E7 proteins of HPV16, presumably characteristic to certain ethnic groups, were identified for a number of countries. They weare mainly localized in the sites of known B- and T-cell epitopes and relatively rarely the domains critical for in structure and protein function. The revealed differences in AA-substitutions in different ethnic groups and their colocalization with the clusters of B- and T-cell epitopes suggested their possible relation to the geographical distribution of alleles and haplotypes of the major histocompatibility complex (HLA). This may lead to the recognition of a different set of B- and T-cell epitopes of the virus in different geographic areas, resulting in the regional differences in the direction of epitopic drift. Phylogenetic analysis of the nucleotide sequences encoding the E6 protein of HPV16 revealed a common ancestor, confirmed regional clustering of the E6 protein sequences sharing common AA-substitutions, and identified cases of reversion of individual AA-substitutions when the change of geographical localization. For the E7 protein, such analysis was not possible due to the high sequence homology. Covariance analysis of the pooled of E6 and E7 sequences revealed that there was no associations between amino acid residues in any aa position within E6 or E7 as well as aa positions of E6 and E7 proteins. The data presented here are important for the development of universal therapeutic vaccines against HPV of high carcinogenic risk.

Background: T-cell responses can be protective or detrimental during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; however, the underlying mechanism is poorly understood., Methods: In this study, we screened 144 15-mer peptides spanning the SARS-CoV-2 spike, nucleocapsid (NP), M, ORF8, ORF10, and ORF3a proteins and 39 reported SARS-CoV-1 peptides in peripheral blood mononuclear cells (PBMCs) from nine laboratory-confirmed coronavirus disease 2019 (COVID-19) patients (five moderate and four severe cases) and nine healthy donors (HDs) collected before the COVID-19 pandemic. T-cell responses were monitored by IFN-γ and IL-17A production using ELISA, and the positive samples were sequenced for the T cell receptor (TCR) β chain. The positive T-cell responses to individual SARS-CoV-2 peptides were validated by flow cytometry., Results: COVID-19 patients with moderate disease produced more IFN-γ than HDs and patients with severe disease (moderate vs. HDs, p < 0.0001; moderate vs. severe, p < 0.0001) but less IL-17A than those with severe disease ( p < 0.0001). A positive correlation was observed between IFN-γ production and T-cell clonal expansion in patients with moderate COVID-19 (r = 0.3370, p = 0.0214) but not in those with severe COVID-19 (r = -0.1700, p = 0.2480). Using flow cytometry, we identified that a conserved peptide of the M protein (Peptide-120, P120) was a dominant epitope recognized by CD8+ T cells in patients with moderate disease., Conclusion: Coordinated IFN-γ production and clonal expansion of SARS-CoV-2-specific T cells are associated with disease resolution in COVID-19. Our findings contribute to a better understanding of T-cell-mediated immunity in COVID-19 and may inform future strategies for managing and preventing severe outcomes of SARS-CoV-2 infection.

The HLA-B*35 alleles have been associated with a slow or rapid progression of HIV-1 infection. However, the mechanisms related to HIV-1 progression have yet to be entirely understood. Several reports indicate that the binding affinity between the HLA-I molecule and peptides could be associated with an increased CD8 + T-cell response. Novel HLA-B*35-restricted mutated variants have been described from HSNQVSQNY (HY9) and HPVHAGPIA (HA9) epitopes. Bioinformatic analysis has indicated that these mutated epitopes show low and high binding affinity towards HLA-B*35, respectively. However, the polyfunctionality of CD8 + T-cells stimulated with these mutated and wild-type epitopes has yet to be reported. The results suggest that the low-binding affinity H124 N/S125 N/N126S mutated peptide in the HY9 epitope induced a lower percentage of CD107a + CD8 + T-cells than the wild-type epitope. Instead, the high-binding affinity peptides I223V and I223A in the HA9 epitope induced a significantly higher frequency of polyfunctional CD8 + T-cells. Also, a higher proportion of CD8 + T-cells with two functions, with Granzyme B + Perforin + being the predominant profile, was observed after stimulation with mutated peptides associated with high binding affinity in the HA9 epitope. These results suggest that the high-affinity mutated peptides induced a more polyfunctional CD8 + T-cell response, which could be related to the control of viral replication., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Paula Velilla-Hernandez reports financial support was provided by Colombia Ministry of Science Technology and Innovation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Complications due to HCMV infection or reactivation remain a challenging clinical problem in immunocompromised patients, mainly due to insufficient or absent T-cell functionality. Knowledge of viral targets is crucial to improve monitoring of high-risk patients and optimise antiviral T-cell therapy. To expand the epitope spectrum, genetically-engineered dendritic cells (DCs) and fibroblasts were designed to secrete soluble (s)HLA-A*11:01 and infected with an HCMV mutant lacking immune evasion molecules (US2-6 + 11). More than 700 HLA-A*11:01-restricted epitopes, including more than 50 epitopes derived from a broad range of HCMV open-reading-frames (ORFs) were identified by mass spectrometry and screened for HLA-A*11:01-binding using established prediction tools. The immunogenicity of the 24 highest scoring new candidates was evaluated in vitro in healthy HLA-A*11:01 + /HCMV + donors. Thus, four subdominant epitopes and one immunodominant epitope, derived from the anti-apoptotic protein UL36 and ORFL101C (A11 SAL ), were identified. Their HLA-A*11:01 complex stability was verified in vitro. In depth analyses revealed highly proliferative and cytotoxic memory T-cell responses against A11 SAL , with T-cell responses comparable to the immunodominant HLA-A*02:01-restricted HCMVpp65 NLV epitope. A11 SAL -specific T cells were also detectable in vivo in immunosuppressed transplant patients and shown to be effective in an in vitro HCMV-infection model, suggesting their crucial role in inhibiting viral replication and improvement of patient's outcome. The developed in vitro pipeline is the first to utilise genetically-engineered DCs to identify naturally presented immunodominant HCMV-derived epitopes. It therefore offers advantages over in silico predictions, is transferable to other HLA alleles, and will significantly expand the repertoire of viral targets to improve therapeutic options., (© 2024 The Author(s). HLA: Immune Response Genetics published by John Wiley & Sons Ltd.)

Food allergies mediated by specific IgE (sIgE) have a significant socioeconomic impact on society. Evaluating the IgE cross-reactivity between allergens from different allergen sources can enable the better management of these potentially life-threatening adverse reactions to food proteins and enhance food safety. A novel banana fruit allergen, S-adenosyl-L-homocysteine hydrolase (SAHH), has been recently identified and its recombinant homolog was heterologously overproduced in E. coli . In this study, we performed a search in the NCBI (National Center for Biotechnology Information) for SAHH homologs in ryegrass, latex, and kiwifruit, all of which are commonly associated with pollen-latex-fruit syndrome. In addition, Western immunoblot analysis was utilized to identify the cross-reactive IgE to banana SAHH in the sera of patients with a latex allergy, kiwifruit allergy, and ryegrass allergy. ClustalOmega analysis showed more than 92% amino acid sequence identity among the banana SAHH homologs in ryegrass, latex, and kiwifruit. In addition to five B-cell epitopes, in silico analysis predicted eleven T-cell epitopes in banana SAHH, seventeen in kiwifruit SAHH, twelve in ryegrass SAHH, and eight in latex SAHH, which were related to the seven-allele HLA reference set (HLA-DRB1*03:01, HLA-DRB1*07:01, HLA-DRB1*15:01, HLA-DRB3*01:01, HLA-DRB3*02:02, HLA-DRB4*01:01, HLA-DRB5*01:01). Four T-cell epitopes were identical in banana and kiwifruit SAHH (positions 328, 278, 142, 341), as well as banana and ryegrass SAHH (positions 278, 142, 96, and 341). All four SAHHs shared two T-cell epitopes (positions 278 and 341). In line with the high amino acid sequence identity and B-cell epitope homology among the analyzed proteins, the cross-reactive IgE to banana SAHH was detected in three of three latex-allergic patients, five of six ryegrass-allergic patients, and two of three kiwifruit-allergic patients. Although banana SAHH has only been studied in a small group of allergic individuals, it is a novel cross-reactive food allergen that should be considered when testing for pollen-latex-fruit syndrome.

Introduction: Human leukocyte antigen (HLA)-DPB1 mismatches during hematopoietic stem cell transplantation (HSCT) with an unrelated donor result in an increased risk for the development of graft-versus-host disease (GvHD). The number of CD8 + T-cell epitopes available for indirect allorecognition as predicted by the PIRCHE algorithm has been shown to be associated with GvHD development. As a proof of principle, PIRCHE-I predictions for HLA-DPB1 mismatches were validated in vitro and in vivo., Methods: PIRCHE-I analysis was performed to identify HLA-DPB1-derived peptides that could theoretically bind to HLA-A*02:01. PIRCHE-I predictions for HLA-DPB1 mismatches were validated in vitro by investigating binding affinities of HLA-DPB1-derived peptides to the HLA-A*02:01 in a competition-based binding assay. To investigate the capacity of HLA-DPB1-derived peptides to elicit a T-cell response in vivo, mice were immunized with these peptides. T-cell alloreactivity was subsequently evaluated using an interferon-gamma ELISpot assay., Results: The PIRCHE-I algorithm identified five HLA-DPB1-derived peptides (RMCRHNYEL, YIYNREEFV, YIYNREELV, YIYNREEYA, and YIYNRQEYA) to be presented by HLA-A*02:01. Binding of these peptides to HLA-A*02:01 was confirmed in a competition-based peptide binding assay, all showing an IC 50 value of 21 μm or lower. The peptides elicited an interferon-gamma response in vivo., Conclusion: Our results indicate that the PIRCHE-I algorithm can identify potential immunogenic HLA-DPB1-derived peptides present in recipients of an HLA-DPB1-mismatched donor. These combined in vitro and in vivo observations strengthen the validity of the PIRCHE-I algorithm to identify HLA-DPB1 mismatch-related GvHD development upon HSCT., Competing Interests: The authors of this manuscript have conflicts of interest to disclose. The UMC Utrecht has filed a patent application on the prediction of an alloimmune response against mismatched HLA. E.S. is listed as an inventor on these patents. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Background: The clinical routine test of HBV-specific T cell reactivity is still limited due to the high polymorphisms of human leukocyte antigens (HLA) in patient cohort and the lack of universal detection kit, thus the clinical implication remains disputed., Methods: A broad-spectrum peptide library, which consists of 103 functionally validated CD8 + T-cell epitopes spanning overall HBsAg, HBeAg, HBx and HBpol proteins and fits to the HLA polymorphisms of Chinese and Northeast Asian populations, was grouped into eight peptide pools and was used to establish an ELISpot assay for enumerating the reactive HBV-specific T cells in PBMCs. Totally 294 HBV-infected patients including 203 ones with chronic hepatitis B (CHB), 13 ones in acute resolved stage (R), 52 ones with liver cirrhosis (LC) and 26 ones with hepatocellular carcinoma (HCC) were detected, and 33 CHB patients were longitudinally monitored for 3 times with an interval of 3-5 months., Results: The numbers of reactive HBV-specific T cells were significantly correlated with ALT level, HBsAg level, and disease stage (R, CHB, LC and HCC), and R patients displayed the strongest HBV-specific T cell reactivity while CHB patients showed the weakest one. For 203 CHB patients, the numbers of reactive HBV-specific T cells presented a significantly declined trend when the serum viral DNA load, HBsAg, HBeAg or ALT level gradually increased, but only a very low negative correlation coefficient was defined (r = - 0.21, - 0.21, - 0.27, - 0.079, respectively). Different Nucleotide Analogs (NUCs) did not bring difference on HBV-specific T cell reactivity in the same duration of treatment. NUCs/pegIFN-α combination led to much more reactive HBV-specific T cells than NUCs monotherapy. The dynamic numbers of reactive HBV-specific T cells were obviously increasing in most CHB patients undergoing routine treatment, and the longitudinal trend possess a high predictive power for the hepatitis progression 6 or 12 months later., Conclusion: The presented method could be developed into an efficient reference method for the clinical evaluation of cellular immunity. The CHB patients presenting low reactivity of HBV-specific T cells have a worse prognosis for hepatitis progression and should be treated using pegIFN-α to improve host T-cell immunity., (© 2024. The Author(s).)

Zika virus is an infectious virus, that belongs to Flaviviridae family, which is transferred to humans through mosquito vectors and severely threatens human health; but, apart from available resources, no effective and secure vaccine is present against Zika virus, to prevent such infections. In current study, we employed structural vaccinology approach to design an epitope-based vaccine against Zika virus, which is biocompatible, and secure and might trigger an adaptive and innate immune response by using computational approaches. We first retrieved the protein sequence from National Center for Biotechnology Information (NCBI) database and carried out for BLAST P. After BLAST P, predicted protein sequences were shortlisted and checked for allergic features and antigenic properties. Final sequence of Zika virus, with accession number (APO40588.1) was selected based on high antigenic score and non-allergenicity. Final protein sequence used various computational approaches including antigenicity testing, toxicity evaluation, allergenicity, and conservancy assessment to identify superior B-cell and T-cell epitopes. Two B-cell epitopes, five MHC-six MHC-II epitopes and I were used to construct an immunogenic multi-epitope-based vaccine by using suitable linkers. A 50S ribosomal protein was added at N terminal to improve the immunogenicity of vaccine. In molecular docking, strong interactions were presented between constructed vaccine and Toll-like receptor 9 (- 1100.6 kcal/mol), suggesting their possible relevance in the immunological response to vaccine. The molecular dynamics simulations ensure the dynamic and structural stability of constructed vaccine. The results of C-immune simulation revealed that constructed vaccine activate B and T lymphocytes which induce high level of antibodies and cytokines to combat Zika infection. The constructed vaccine is an effective biomarker with non-sensitization, nontoxicity; nonallergic, good immunogenicity, and antigenicity, however, experimental assays are required to verify the results of present study., (© 2024. The Author(s).)

Objective: The objective of the present study was to design a multi-epitope protein from A. lumbricoides and APD allergens and to evaluate its IgE reactivity preliminarily., Methods: Using computational tools, a molecule containing multiple "T" epitopes of allergens derived from A. lumbricoides and APD was designed " in silico " This multi-epitope protein (MP1) was expressed using an E. coli system and purified by affinity chromatography using Ni-NTA agarose. Anti-MP1 and anti-HDM extract IgE reactivity was evaluated by Dot-Blot and indirect ELISA from sera of HDM-allergic patients and non-allergic individuals from Barranquilla-Colombia. Allergic individuals had a positive skin test to a standardized battery of inhaled allergens (EUROLINE - Ref: DP 3704-1601-1 E) and mite- specific IgE., Results: Multi-epitope (MP1) protein was expressed and purified with high purity. Dot-Blot result showed that all sera from allergic patients showed lower IgE reactivity to MP1 compared to HDM extract. By ELISA, significantly lower concentrations of anti-MP1 IgE (Median: 270.86 ng/ml; IQR: 90.3) were observed in contrast to anti-HDM IgE levels (Median: 988.5 ng/ml; IQR: 1117.6) in sera of patients allergic to HDM., Conclusions: A protein composed of multiple epitopes of A. lumbricoides and HDM allergens was designed, expressed, and purified. Preliminary Dot-Blot results suggest that this molecule shows hypoallergenic properties with very low IgE reactivity compared to mite extract. Further functional studies are needed to understand better the immune response induced by this molecule.

Objective: To evaluate the IgE reactivity of sera in patients suffering from type 1 diabetes (T1D), lupus nephritis (LN) and juvenile idiopathic arthritis (JIA) against a molecule constructed from T epitopes of A. lumbricoides allergens., Methods: We designed and expressed a synthetic multi-epítope protein named MP1 from A. lumbricoides and house dust mites allergens. By indirect ELISA, we evaluated IgE-reactivity to MP1 and to the whole-body extract of Ascaris lumbricoides in 45 sera from Colombian Caribbean patients with lupus nephritis (LN; n=25), type 1 diabetes (T1D; n=10) and Juvenil idiopathic arthritis (JIA; n=10). Individuals with poly autoimmunity were excluded. All patients were referred to the study by their specialist doctor., Results: IgE to whole-body extract of A. lumbricoides showed the following median concentrations., 484.2 ng/ml (IQR: 203.4) in JIA patients, 325.6 ng/ml (IQR: 179.3) in individuals with LN, and 424.7 ng/ml (IQR: 80.1) in the T1D group. On the other hand, IgE-reactivity to MP1 was 126.4 ng/ml (IQR: 90.9) in JIA patients, 130.7 ng/ml (IQR: 94.8) in an individual with LN, and 148.8 ng/ml (IQR: 102.1) in the T1D group. Although no statistical differences were observed between patient groups, the IgE to MP1 in all patients (n: 45) (IgE median: 134.2 ng/ml; IQR: 100) were significantly less compared to Ascaris extract (IgE median: 380.7 ng/ml; IQR: 175.8); ( W: 0.732; p-value: 1.034x10 -7 )., Conclusions: These preliminary results suggest that MP1 showed antigenic properties with low IgE- reactivity, compared to Ascaris lumbricoides extracted in individuals with autoimmune diseases. Further studies are needed to understand better the immune response induced by this molecule.

The class I HLA genotype has been widely recognized as a factor influencing HIV disease progression in treatment-naïve subjects. However, little is known regarding its role in HIV disease course and how it influences the size of the viral reservoir once anti-retroviral therapy (ART) is started. Here, leveraging on cutting-edge bioinformatic tools, we explored the relationship between HLA class I and the HIV reservoir in a cohort of 90 people living with HIV (PLWH) undergoing ART and who achieved viral suppression. Analysis of HLA allele distribution among patients with high and low HIV reservoir allowed us to document a predominant role of HLA-B and -C genes in regulating the size of HIV reservoir. We then focused on the analysis of HIV antigen (Ag) repertoire, by investigating immunogenetic parameters such as the degree of homozygosity, HLA evolutionary distance and Ag load. In particular, we used two different bioinformatic algorithms, NetMHCpan and MixMHCpred, to predict HLA presentation of immunogenic HIV-derived peptides and identified HLA-B*57:01 and HLA-B*58:01 among the highest ranking HLAs in terms of total load, suggesting that their previously reported protective role against HIV disease progression might be linked to a more effective viral recognition and presentation to Cytotoxic T lymphocytes (CTLs). Further, we speculated that some peptide-HLA complexes, including those produced by the interaction between HLA-B*27 and the HIV Gag protein, might be particularly relevant for the efficient regulation of HIV replication and containment of the HIV reservoir. Last, we provide evidence of a possible synergistic effect between the CCR5 ∆32 mutation and Ag load in controlling HIV reservoir., (© 2023 The Authors. HLA: Immune Response Genetics published by John Wiley & Sons Ltd.)

Identification and characterization of CD8 + T-cells is important to determine their role in protecting and clearing viral infections. Here we provide details of the peptide-MHC (pMHC) tetramers-based approach to identify antigen-specific T-cells in human and murine samples. This method provides ex vivo quantification and functional characterization of T-cells reactive to specific viral antigens derived from CMV and rotavirus in human blood and in murine intestinal lamina propria samples, respectively., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Numerous tools exist for biological sequence comparisons and search. One case of particular interest for immunologists is finding matches for linear peptide T cell epitopes, typically between 8 and 15 residues in length, in a large set of protein sequences. Both to find exact matches or matches that account for residue substitutions. The utility of such tools is critical in applications ranging from identifying conservation across viral epitopes, identifying putative epitope targets for allergens, and finding matches for cancer-associated neoepitopes to examine the role of tolerance in tumor recognition., Results: We defined a set of benchmarks that reflect the different practical applications of short peptide sequence matching. We evaluated a suite of existing methods for speed and recall and developed a new tool, PEPMatch. The tool uses a deterministic k-mer mapping algorithm that preprocesses proteomes before searching, achieving a 50-fold increase in speed over methods such as the Basic Local Alignment Search Tool (BLAST) without compromising recall. PEPMatch's code and benchmark datasets are publicly available., Conclusions: PEPMatch offers significant speed and recall advantages for peptide sequence matching. While it is of immediate utility for immunologists, the developed benchmarking framework also provides a standard against which future tools can be evaluated for improvements. The tool is available at https://nextgen-tools.iedb.org , and the source code can be found at https://github.com/IEDB/PEPMatch ., (© 2023. The Author(s).)

Background: Cow milk (CM) allergy is the most prevalent food allergy in young children in the United States and Great Britain. Current diagnostic tests are either unreliable (IgE test and skin prick test) or resource-intensive with risks (food challenges)., Objective: We sought to determine whether allergen-specific T cells in CM-allergic (CMA) patients have a distinct quality and/or quantity that could potentially be used as a diagnostic marker., Methods: Using PBMCs from 147 food-allergic pediatric subjects, we mapped T-cell responses to a set of reactive epitopes in CM that we compiled in a peptide pool. This pool induced cytokine responses in in vitro cultured cells distinguishing subjects with CMA from subjects without CMA. We further used the pool to isolate and characterize antigen-specific CD4 memory T cells using flow cytometry and single-cell RNA/TCR sequencing assays., Results: We detected significant changes in the transcriptional program and clonality of CM antigen-specific (CM+) T cells elicited by the pool in subjects with CMA versus subjects without CMA ex vivo. CM+ T cells from subjects with CMA had increased percentages of FOXP3 + cells over FOXP3 - cells. FOXP3 + cells are often equated with regulatory T cells that have suppressive activity, but CM+ FOXP3 + cells from subjects with CMA showed significant expression of interferon-responsive genes and dysregulated chemokine receptor expression compared with subjects without CMA, suggesting that these are not conventional regulatory T cells. The CM+ FOXP3 + cells were also more clonally expanded than the FOXP3 - population. We were further able to use surface markers (CD25, CD127, and CCR7) in combination with our peptide pool stimulation to quantify these CM+ FOXP3 + cells by a simple flow-cytometry assay. We show increased percentages of CM+ CD127 - CD25 + cells from subjects with CMA in an independent cohort, which could be used for diagnostic purposes. Looking specifically for T H 2 cells normally associated with allergic diseases, we found a small population of clonally expanded CM+ cells that were significantly increased in subjects with CMA and that had high expression of T H 2 cytokines and pathogenic T H 2/T follicular helper markers., Conclusions: Overall, these findings suggest that there are several differences in the phenotypes of CM+ T cells with CM allergy and that the increase in CM+ FOXP3 + cells is a potential diagnostic marker of an allergic state. Such markers have promising applications in monitoring natural disease outgrowth and/or the efficacy of immunotherapy that will need to be validated in future studies., (Copyright © 2023 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The transport of peptides from the cytoplasm to the endoplasmic reticulum (ER) by transporters associated with antigen processing (TAP) is a critical step in the intracellular presentation of cytotoxic T lymphocyte (CTL) epitopes. The development and application of computational methods, especially deep learning methods and new neural network strategies that can automatically learn feature representations with limited knowledge, provide an opportunity to develop fast and efficient methods to identify TAP-binding peptides. Herein, this study presents a comprehensive analysis of TAP-binding peptide sequences to derive TAP-binding motifs and preferences for N-terminal and C-terminal amino acids. A novel recurrent neural network (RNN)-based method called DeepTAP, using bidirectional gated recurrent unit (BiGRU), was developed for the accurate prediction of TAP-binding peptides. Our results demonstrated that DeepTAP achieves an optimal balance between prediction precision and false positives, outperforming other baseline models. Furthermore, DeepTAP significantly improves the prediction accuracy of high-confidence neoantigens, especially the top-ranked ones, making it a valuable tool for researchers studying antigen presentation processes and T-cell epitope screening. DeepTAP is freely available at https://github.com/zjupgx/deeptap and https://pgx.zju.edu.cn/deeptap., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Fig. 2 was created with BioRender.com., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

T-cell immunity, mediated by CD4 + and CD8 + T cells, represents a cornerstone in the control of viral infections. Virus-derived T-cell epitopes are represented by human leukocyte antigen (HLA)-presented viral peptides on the surface of virus-infected cells. They are the prerequisite for the recognition of infected cells by T cells. Knowledge of viral T-cell epitopes provides on the one hand a diagnostic tool to decipher protective T-cell immune responses in the human population and on the other hand various prophylactic and therapeutic options including vaccination approaches and the transfer of virus-specific T cells. Such approaches have already been proven to be effective against various viral infections, particularly in immunocompromised patients lacking sufficient humoral, antibody-based immune response. This review provides an overview on the state of the art as well as current studies regarding the identification and characterization of viral T-cell epitopes and approaches of clinical application. In the first chapter in silico prediction tools and direct, mass spectrometry-based identification of viral T-cell epitopes is compared. The second chapter provides an overview of commonly used assays for further characterization of T-cell responses and phenotypes. The final chapter presents an overview of clinical application of viral T-cell epitopes with a focus on human immunodeficiency virus (HIV), human cytomegalovirus (HCMV) and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), being representatives of relevant viruses., Competing Interests: Declaration of Competing Interest A.N. and J.S.W. are listed as inventors on patent applications of the COVID-19T cell activator CoVac-1, application number: PCT/EP 20 190 070.1 and of SARS-CoV-2 CD8+ und CD4+ T cell epitopes, Application number: PCT/EP 20 169 047.6. A.N. is listed as inventor on a patent application of HCMV-derived peptides, application number: EP2020/074114. The other author declares no competing interests., (Copyright © 2023. Published by Elsevier Ltd.)

Background: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an ultra-rare autoimmune disorder caused by autoantibodies against ADAMTS13. A strong association of DRB1∗11 with iTTP and DRB1∗11-restricted T-cell epitopes in ADAMTS13 have been reported in Europeans, whereas we previously found DRB1∗08:03 as a susceptible allele in Japanese., Objectives: The limited information is available regarding a susceptible allele and its T-cell epitopes in Japanese patients with iTTP., Materials and Methods: We conducted a reanalysis on iTTP-predisposing alleles using 3 distinct Japanese control groups. Subsequently, a novel human leukocyte antigen (HLA)-peptide expression assay (MHC-density assay) was used to identify the presentation of 24 ADAMTS13-derived peptides, including the regions that were identified previously by MHC-peptidome analysis and/or T-cell assays or predicted by NetMHCIIpan-4.0, to DRB1∗08:03 and DRB1∗11:01., Results: We reconfirmed the strong association of DRB1∗08:03 with iTTP, as well as the absence of the secondary risk alleles and protective alleles in Japanese iTTP, which altogether reveal that the HLA association pattern is completely different between the European and Japanese iTTP. MHC-density assay found the 3 ADAMTS13-derived peptides in the spacer domain as a potential strong binder to DRB1∗08:03. Moreover, 6 peptides in the metalloprotease, spacer, sixth thrombospondin-1 repeat, and CUB domains in ADAMTS13 showed increased presentation by both DRB1∗08:03 and DRB1∗11:01., Conclusion: Altogether, the findings of distinct HLA-DR association with iTTP across populations and the presentation of common peptides by DRB1∗08:03 and DRB1∗11:01 suggest that the same ADAMTS13-derived peptides might be presented and trigger the activation of autoreactive CD4 + T cells, leading to production of anti-ADAMTS13 autoantibodies by autoreactive B cells., (Copyright © 2022 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Citrullinated proteins and anti-citrullinated protein antibodies (ACPAs) play an important role in the pathogenesis of rheumatoid arthritis (RA). It has been suggested that during inflammation or dysbiosis, bacteria could initiate production of ACPAs. Most patients with RA are seropositive for ACPAs, but these antibodies have overlapping reactivity to different posttranslational modifications (PTMs). For initiation and development of RA, T lymphocytes and T cell epitopes are still required. In this study, we evaluated the ability of bacterial L-asparaginase to modify RA-related T cell epitopes within type II collagen (CII259-273 and CII311-325), as well as whether these modified epitopes are recognized by ACPAs from RA patients. We included 12 patients with early RA and 11 healthy subjects selected according to predefined specific criteria. LC-MS/MS analyses revealed that the bacterial L-asparaginase can modify investigated T cell epitopes. ELISA tests showed cross-reactivity of ACPA positive sera from early RA patients towards the enzymatically modified immunodominant T cell epitopes within type II collagen (CII), but not to the modified irrelevant peptides. These data suggest that the cross-reactive ACPAs recognize the "carbonyl-Gly-Pro" motif in CII. Moreover, the T cell recognition of the modified major immunodominant T cell epitope Gal264-CII259-273 was not affected. This epitope was still able to activate autoreactive T cells from early RA patients. It is likely that such modifications are the missing link between the T cell priming and the development of anti-modified protein antibodies (AMPAs). Our results provide additional information on the etiology and pathogenesis of RA., Competing Interests: The authors declare no conflict of interest., (Copyright © 2023 Termedia.)

In the worrisome scenarios of various waves of SARS-CoV-2 pandemic, a comprehensive bioinformatics pipeline is essential to analyse the virus genomes in order to understand its evolution, thereby identifying mutations as signature SNPs, conserved regions and subsequently to design epitope based synthetic vaccine. We have thus performed multiple sequence alignment of 4996 Indian SARS-CoV-2 genomes as a case study using MAFFT followed by phylogenetic analysis using Nextstrain to identify virus clades. Furthermore, based on the entropy of each genomic coordinate of the aligned sequences, conserved regions are identified. After refinement of the conserved regions, based on its length, one conserved region is identified for which the primers and probes are reported for virus detection. The refined conserved regions are also used to identify T-cell and B-cell epitopes along with their immunogenic and antigenic scores. Such scores are used for selecting the most immunogenic and antigenic epitopes. By executing this pipeline, 40 unique signature SNPs are identified resulting in 23 non-synonymous signature SNPs which provide 28 amino acid changes in protein. On the other hand, 12 conserved regions are selected based on refinement criteria out of which one is selected as the potential target for virus detection. Additionally, 22 MHC-I and 21 MHC-II restricted T-cell epitopes with 10 unique HLA alleles each and 17 B-cell epitopes are obtained for 12 conserved regions. All the results are validated both quantitatively and qualitatively which show that from genetic variability to synthetic vaccine design, the proposed pipeline can be used effectively to combat SARS-CoV-2., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Reports on T-cell cross-reactivity against SARS-CoV-2 epitopes in unexposed individuals have been linked with prior exposure to the human common cold coronaviruses (HCCCs). Several studies suggested that cross-reactive T-cells response to live attenuated vaccines (LAVs) such as BCG (Bacillus Calmette-Guérin), OPV (Oral Polio Vaccine), and MMR (measles, mumps, and rubella) can limit the development and severity of COVID-19. This study aims to identify potential cross-reactivity between SARS-CoV-2, HCCCs, and LAVs in the context of T-cell epitopes peptides presented by HLA (Human Leukocyte Antigen) alleles of the Indonesian population. SARS-CoV-2 derived T-cell epitopes were predicted using immunoinformatics tools and assessed for their conservancy, variability, and population coverage. Two fully conserved epitopes with 100% similarity and nine heterologous epitopes with identical T-cell receptor (TCR) contact residues were identified from the ORF1ab fragment of SARS-CoV-2 and all HCCCs. Cross-reactive epitopes from various proteins of SARS-CoV-2 and LAVs were also identified (15 epitopes from BCG, 7 epitopes from MMR, but none from OPV). A majority of the identified epitopes were observed to belong to ORF1ab, further suggesting the vital role of ORF1ab in the coronaviruses family and suggesting it as a candidate for a potential universal coronavirus vaccine that protects against severe disease by inducing cell mediated immunity.

Background: Bovine leukemia virus (BLV) is an oncogenic delta-retrovirus causing bovine leucosis. Studies on BLV have shown the association with human breast cancer. However, the exact molecular mechanism is neither known nor their appropriate preventative measure to halt the disease initiation and progression. In this study, we designed a multi-epitope vaccine against BLV using a computational analyses., Methods: Following a rigorous assessment, the vaccine was constructed using the T-cell epitopes from each BLV-derived protein with suitable adjuvant and linkers. Both physicochemistry and immunogenic potency as well as the safeness of the vaccine candidate were assessed. Population coverage was done to evaluate the vaccine probable efficiency in eliciting the immune response worldwide. After homology modeling, the three-dimensional structure was refined and validated to determine the quality of the designed vaccine. The vaccine protein was then subjected to molecular docking with Toll-like receptor 3 (TLR3) to evaluate the binding efficiency followed by dynamic simulation for stable interaction., Results: Our vaccine construct has the potential immune response and good physicochemical properties. The vaccine is antigenic and immunogenic, and has no allergenic or toxic effect on the human body. This novel vaccine contains a significant interactions and binding affinity with the TLR3 receptor., Conclusions: The proposed vaccine candidate would be structurally stable and capable of generating an effective immune response to combat BLV infections. However, experimental evaluations are essential to validate the exact safety and immunogenic profiling of this vaccine., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Vaccination is the most effective means of protecting people from influenza virus infection. The effectiveness of existing vaccines is very limited due to antigenic drift of the influenza virus. Therefore, there is a requirement to develop a universal vaccine that provides broad and long-lasting protection against influenza. CD8+ T-cell response played a vital role in controlling influenza virus infection, reducing viral load, and less clinical syndrome. In this study, we optimized the HA sequences of human seasonal influenza viruses (H1N1, H3N2, Victoria, and Yamagata) by designing multivalent vaccine antigen sets using a mosaic vaccine design strategy and genetic algorithms, and designed an HA mosaic cocktail containing the most potential CTL epitopes of seasonal influenza viruses. We then tested the recombinant mosaic antigen, which has a significant number of potential T-cell epitopes. Results from genetic evolutionary analyses and 3D structural simulations demonstrated its potential to be an effective immunogen. In addition, we have modified an existing neutralizing antibody-based seasonal influenza virus vaccine to include a component that activates cross-protective T cells, which would provide an attractive strategy for improving human protection against seasonal influenza virus drift and mutation and provide an idea for the development of a rationally designed influenza vaccine targeting T lymphocyte immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Zhao, Wang, Li, Sun and Shu.)

While antibodies garner the lion's share of attention in SARS-CoV-2 immunity, cellular immunity (T cells) may be equally, if not more important, in controlling infection. Both CD8 + and CD4 + T cells are elicited earlier and are associated with milder disease, than antibodies, and T-cell activation appears to be necessary for control of infection. Variants of concern (VOCs) such as Omicron have escaped the neutralizing antibody responses after two mRNA vaccine doses, but T-cell immunity is largely intact. The breadth and patient-specific nature of the latter offers a formidable line of defense that can limit the severity of illness, and are likely to be responsible for most of the protection from natural infection or vaccination against VOCs which have evaded the antibody response. Comprehensive searches for T-cell epitopes, T-cell activation from infection and vaccination of specific patient groups, and elicitation of cellular immunity by various alternative vaccine modalities are here reviewed. Development of vaccines that specifically target T cells is called for, to meet the needs of patient groups for whom cellular immunity is weaker, such as the elderly and the immunosuppressed. While VOCs have not yet fully escaped T-cell immunity elicited by natural infection and vaccines, some early reports of partial escape suggest that future VOCs may achieve the dreaded result, dislodging a substantial proportion of cellular immunity, enough to cause a grave public health burden. A proactive, rather than reactive, solution which identifies and targets immutable sequences in SARS-CoV-2, not just those which are conserved, may be the only recourse humankind has to disarm these future VOCs before they disarm us., Competing Interests: Competing interests: The author declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: AY is President of InvVax., (© The Author(s), 2022.)

COVID-19 brought scenes from sci-fi movies into real life. Infected individuals include asymptomatic cases to severe disease leading to death, suggesting the involvement of the genetic constitution of populations and pathogens contributing to differential individuals' outcomes. To investigate shared immunogenic features between SARS-CoV-2 targets and other coronaviruses, we modeled their peptides in 3D structures of HLA-A*02:01 (pMHC), comparing their molecular surfaces These structures were also compared with a panel of epitopes from unrelated viruses, looking for potential triggers conferring cross-protection in uninfected individuals. As expected, SARS-CoV 1 and 2 peptides share molecular and physicochemical features, providing an explanation for the verified experimental immunogenicity among them. Surprisingly, even discordant sequences from human coronaviruses 229E, OC43 and epitopes from unrelated viruses involved in endemic human infections exhibit similar fingerprints of immunogenicity with SARS-CoV-2 peptides. The same approach indicates a conserved CD8+ T cell recognition between Wuhan SARS-CoV-2 sequences and altered peptides from Variants of Concern. Examination of structural data over epitope sequence analysis here could explain how previous infections may produce a heterologous immunity response in a global scale against emergent diseases such as Covid-19, mitigating its full lethal potential, and paves the way for the development of wide spectrum vaccine development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Antonio, Meireles, Bragatte and Vieira.)

Computational prediction to screen potential vaccine candidates has been proven to be a reliable way to provide guarantees for vaccine discovery in infectious diseases. As an important class of organisms causing infectious diseases, pathogenic eukaryotes (such as parasitic protozoans) have evolved the ability to colonize a wide range of hosts, including humans and animals; meanwhile, protective vaccines are urgently needed. Inspired by the immunological idea that pathogen-derived epitopes are able to mediate the CD8 + T-cell-related host adaptive immune response and with the available positive and negative CD8 + T-cell epitopes (TCEs), we proposed a novel predictor called CD8TCEI-EukPath to detect CD8 + TCEs of eukaryotic pathogens. Our method integrated multiple amino acid sequence-based hybrid features, employed a well-established feature selection technique, and eventually built an efficient machine learning classifier to differentiate CD8 + TCEs from non-CD8 + TCEs. Based on the feature selection results, 520 optimal hybrid features were used for modeling by utilizing the LightGBM algorithm. CD8TCEI-EukPath achieved impressive performance, with an accuracy of 79.255% in ten-fold cross-validation and an accuracy of 78.169% in the independent test. Collectively, CD8TCEI-EukPath will contribute to rapidly screening epitope-based vaccine candidates, particularly from large peptide-coding datasets. To conduct the prediction of CD8 + TCEs conveniently, an online web server is freely accessible (http://lab.malab.cn/∼hrs/CD8TCEI-EukPath/)., Competing Interests: XZ was employed by the company Beidahuang Industry Group General Hospital. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor QZ declared a past co-authorship with the author RSH., (Copyright © 2022 Hu, Wu, Zhang, Zhou and Zhang.)

Currently, there is no licensed vaccine to protect against human visceral leishmaniasis (VL), a potentially fatal disease caused by infection with Leishmania parasites. In the current study, a recombinant chimeric protein ChimT was developed based on T-cell epitopes identified from the immunogenic Leishmania amastigote proteins LiHyp1, LiHyV, LiHyC and LiHyG. ChimT was associated with the adjuvants saponin (Sap) or monophosphoryl lipid A (MPLA) and used to immunize mice, and their immunogenicity and protective efficacy were evaluated. Both ChimT/Sap and ChimT/MPLA induced the development of a specific Th1-type immune response, with significantly high levels of IFN-γ, IL-2, IL-12, TNF-α and GM-CSF cytokines produced by CD4+ and CD8+ T cell subtypes (p < 0.05), with correspondingly low production of anti-leishmanial IL-4 and IL-10 cytokines. Significantly increased (p < 0.05) levels of nitrite, a proxy for nitric oxide, and IFN-γ expression (p < 0.05) were detected in stimulated spleen cell cultures from immunized and infected mice, as was significant production of parasite-specific IgG2a isotype antibodies. Significant reductions in the parasite load in the internal organs of the immunized and infected mice (p < 0.05) were quantified with a limiting dilution technique and quantitative PCR and correlated with the immunological findings. ChimT/MPLA showed marginally superior immunogenicity than ChimT/Sap, and although this was not statistically significant (p > 0.05), ChimT/MPLA was preferred since ChimT/Sap induced transient edema in the inoculation site. ChimT also induced high IFN-γ and low IL-10 levels from human PBMCs isolated from healthy individuals and from VL-treated patients. In conclusion, the experimental T-cell multi-epitope amastigote stage Leishmania vaccine administered with adjuvants appears to be a promising vaccine candidate to protect against VL.

The COVID-19 pandemic emerged in 2020 and has caused an unprecedented burden to all countries in the world. SARS-CoV-2 continues to circulate and antigenically evolve, enabling multiple reinfections. To address the issue of the virus antigenic variability, T cell-based vaccines are being developed, which are directed to more conserved viral epitopes. We used live attenuated influenza vaccine (LAIV) virus vector to generate recombinant influenza viruses expressing various T-cell epitopes of SARS-CoV-2 from either neuraminidase (NA) or non-structural (NS1) genes, via the P2A self-cleavage site. Intranasal immunization of human leukocyte antigen-A*0201 (HLA-A2.1) transgenic mice with these recombinant viruses did not result in significant SARS-CoV-2-specific T-cell responses, due to the immunodominance of NP 366 influenza T-cell epitope. However, side-by-side stimulation of peripheral blood mononuclear cells (PBMCs) of COVID-19 convalescents with recombinant viruses and LAIV vector demonstrated activation of memory T cells in samples stimulated with LAIV/SARS-CoV-2, but not LAIV alone. Hamsters immunized with a selected LAIV/SARS-CoV-2 prototype were protected against challenge with influenza virus and a high dose of SARS-CoV-2 of Wuhan and Delta lineages, which was confirmed by reduced weight loss, milder clinical symptoms and less pronounced histopathological signs of SARS-CoV-2 infection in the lungs, compared to LAIV- and mock-immunized animals. Overall, LAIV is a promising platform for the development of a bivalent vaccine against influenza and SARS-CoV-2.

Peptides are widely used for the diagnostics, prevention, and therapy of certain human diseases. How useful can they be for the disease caused by the SARS-CoV-2 coronavirus? In this review, we discuss the possibility of using synthetic and recombinant peptides and polypeptides for prevention of COVID-19 via blocking the interaction between the virus and its main receptor ACE2, as well as components of antiviral vaccines, in particular, against new emerging virus variants.

Peptide immunotherapy (PIT) represents a safe and efficacious therapeutic regimen with in-consequential side-effects. The present study aims to identify T-cell epitopes of Per a 5 allergen, a delta class GST from Periplaneta americana and investigate effect of peptide treatment in murine model of cockroach allergen-mediated hyper-reactivity. The epitopes (TC-P1, TC-P2, and TC-P3) were identified as promiscuous MHC-II binders by MHC-Pred, ProPred, and IEDB analysis tool. Murine model of cockroach allergic hyper-reactivity was generated in Balb/c mice. A marked reduction in cellular infiltration in lungs (3-fold compared with Non-IT) was observed in T3-IT group as evidenced by total leucocyte count in BALF and histology. Specific IgE levels were reduced 3-fold in T2-IT and T3-IT compared with Non-IT with increase in IgG2a levels. IL-4 and IL-13 were reduced upto 2.5-fold in treatment groups compared with Non-IT group. Splenocytes revealed significant increase in levels of CD4+FoxP3+ T cells in TC-P1 and TC-P2 mice demonstrating a systemic shift towards Tregs. Peptide treatment downregulated NF-kB signalling in lung and enhanced the levels of immune-regulatory molecules α1-antitrypsin and elafin. Our results indicate that TC-P1 and TC-P3 alter Th2 cytokine milieu and antibody isotype ratio to suppress allergic inflammation. PIT modulates local and systemic mechanisms to resolve inflammation and possess potential for treatment in cockroach allergy., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The recent success of monoclonal antibody checkpoint inhibitor therapies that enhance the ability of CD8 + T cells to detect cancer-related antigenic peptides has refocused the need to fully understand the repertoire of peptides being presented to the immune system. Whilst the peptide ligandome presented by cell surface human leucocyte antigen class I (HLA-I) molecules on cancer cells has been studied extensively, the ligandome of extracellular vesicles (EVs) remains poorly defined. Here, we report the HLA-I ligandome of both the cell surface and EVs from eight breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-361, MDA-MB-415, MDA-MB-453, HCC 1806, HCC 1395, and HCC 1954), and additionally the melanoma cell line ESTDAB-056 and the multiple myeloma line RPMI 8226. Utilizing HLA-I immunoisolation and mass spectrometry, we detected a total of 6574 peptides from the cell surface and 2461 peptides from the EVs of the cell lines studied. Within the EV HLA-I ligandome, we identified 150 peptides derived from tumour associated antigenic proteins, of which 19 peptides have been shown to elicit T-cell responses in previous studies. Our data thus show the prevalence of clinically relevant tumour-associated antigenic peptides in the HLA-I ligandome presented on EV., (© 2022 The Authors. Immunology published by John Wiley & Sons Ltd.)

Celiac disease (CeD) is an autoimmune enteropathy induced by prolamin and glutelin proteins in wheat, barley, rye, and triticale recognized by genetically restricted major histocompatibility (MHC) receptors. Patients with CeD must avoid consuming these proteins. Regulators in Europe and the United States expect an evaluation of CeD risks from proteins in genetically modified (GM) crops or novel foods for wheat-related proteins. Our database includes evidence-based causative peptides and proteins and two amino acid sequence comparison tools for CeD risk assessment. Sequence entries are based on the review of published studies of specific gluten-reactive T cell activation or intestinal epithelial toxicity. The initial database in 2012 was updated in 2018 and 2022. The current database holds 1,041 causative peptides and 76 representative proteins. The FASTA sequence comparison of 76 representative CeD proteins provides an insurance for possible unreported epitopes. Validation was conducted using protein homologs from Pooideae and non-Pooideae monocots, dicots, and non-plant proteins. Criteria for minimum percent identity and maximum E -scores are guidelines. Exact matches to any of the 1,041 peptides suggest risks, while FASTA alignment to the 76 CeD proteins suggests possible risks. Matched proteins should be tested further by CeD-specific CD4/8+ T cell assays or in vivo challenges before their use in foods., Competing Interests: RG declares limited funding from six biotechnology companies from 2009 to 2012 for support of the database management. Unilever SEAC and NuSeed Americas provided limited funding to the AllergenOnline.org database from 2018 to 2021. These companies did not contribute to or see the article prior to submission. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Amnuaycheewa, Abdelmoteleb, Wise, Bohle, Ferreira, Tetteh, Taylor and Goodman.)

Coronavirus disease 2019 (COVID-19) is the most devastating pandemic of the century, which is still far from over. The remarkable success of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines is the working hope, but the evolving variants are the huge concern that can turn the tide. Potential immune escape mutations (PIEMs) in the past and circulating variants were not studied at large scale (all available data). Hence, the conservation of antigenic determinants (epitopes) was analyzed in all available sequences of SARS-CoV-2 according to time (months), proteins, hosts, and variants. Numerous highly conserved B- and T-cell epitopes were identified in 24 proteins of SARS-CoV-2. A decrease in the conservation of epitopes with time was observed in almost all proteins, which was more rapid in neutralizing epitopes. Delta variant still has the highest PIEM in the circulating strains, which pose threat to the effectiveness of current vaccines. The inclusion of identified, highly conserved, and important epitopes in subunit vaccines can increase vaccine effectiveness against evolving variants. Trends in the conservation of epitopes in different proteins, hosts, and variants with time may also help to inspire the counter measure against the current pandemic., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jaiswal and Lee.)

Since the first outbreak of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, its high infectivity led to its prevalence around the world in an exceptionally short time. Efforts have been made to control the ongoing outbreak, and among them, vaccine developments are going on high priority. New clinical trials add to growing evidence that vaccines from many countries were highly effective at preventing SARS-CoV-2 virus infection. One of them is B cell-based vaccines, which were common during a pandemic. However, neutralizing antibody therapy becomes less effective when viruses mutate. In order to tackle the problem, we focused on T-cell immune mechanism. In this study, the mutated strains of the virus were selected globally from India (B.1.617.1 and B.1.617.2), United Kingdom (B.1.1.7), South Africa (B.1.351), and Brazil (P.1), and the overlapping peptides were collected based on mutation sites of S-protein. After that, residue scanning was used to predict the affinity between overlapping peptide and HLA-A*11:01, the most frequent human leukocyte antigen (HLA) allele among the Chinese population. Then, the binding free energy was evaluated with molecular docking to further verify the affinity changes after the mutations happen in the virus genomes. The affinity test results of three epitopes on spike protein from experimental validation were consistent with our predicted results, thereby supporting the inclusion of the epitope 374 FSTFKCYGL 382 in future vaccine design and providing a useful reference route to improve vaccine development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mei, Gu, Shen, Lin and Li.)

CD8 + T-cells play a crucial role in the control of HIV replication. HIV-specific CD8 + T-cell responses rapidly expand since the acute phase of the infection, and it has been observed that HIV controllers harbor CD8 + T-cells with potent anti-HIV capacity. The development of CD8 + T-cell-based vaccine against HIV-1 has focused on searching for immunodominant epitopes. However, the strong immune pressure of CD8 + T-cells causes the selection of viral variants with mutations in immunodominant epitopes. Since HIV-1 mutations are selected under the context of a specific HLA-I, the circulation of viral variants with these mutations is highly predictable based on the most prevalent HLA-I within a population. We previously demonstrated the adaptation of circulating strains of HIV-1 to the HLA-A*02 molecule by identifying mutations under positive selection located in GC9 and SL9 epitopes derived from the Gag protein. Also, we used an in silico prediction approach and evaluated whether the mutations found had a higher or lower affinity to the HLA-A*02. Although this strategy allowed predicting the interaction between mutated peptides and HLA-I, the functional response of CD8 + T-cells that these peptides induce is unknown. In the present work, peripheral blood mononuclear cells from 12 HIV-1 + HLA-A*02:01 + individuals were stimulated with the mutated and wild-type peptides derived from the GC9 and SL9 epitopes. The functional profile of CD8 + T-cells was evaluated using flow cytometry, and the frequency of subpopulations was determined according to their number of functions and the polyfunctionality index. The results suggest that the quality of the response (polyfunctionality) could be associated with the binding affinity of the peptide to the HLA molecule, and the functional profile of specific CD8 + T-cells to mutated epitopes in individuals under cART is maintained., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sánchez-Martínez, Acevedo-Sáenz, Alzate-Ángel, Álvarez, Guzmán, Roman, Urcuqui-Inchima, Cardona-Maya and Velilla.)

Vibrio harveyi belongs to the family Vibrionaceae of class Gammaproteobacteria. Around 12 Vibrio species can cause gastroenteritis (gastrointestinal illness) in humans. A large number of bacterial particles can be found in the infected cells, which may cause death. Despite these devastating complications, there is still no cure or vaccine for the bacteria. As a result, we used an immunoinformatics approach to develop a multi-epitope vaccine against the most pathogenic hemolysin gene of V. harveyi. The immunodominant T- and B-cell epitopes were identified using the hemolysin protein. We developed a vaccine employing three possible epitopes: cytotoxic T-lymphocytes, helper T-lymphocytes, and linear B-lymphocyte epitopes, after thorough testing. The vaccine was developed to be antigenic, immunogenic, and non-allergenic, as well as have a better solubility. Molecular dynamics simulation revealed significant structural stiffness and binding stability. In addition, the immunological simulation generated by computers revealed that the vaccination might elicit immune reactions Escherichia coli K12 as a model, codon optimization yielded ideal GC content and a higher codon adaptation index value, which was then included in the cloning vector pET2+ (a). Altogether, our experiment implies that the proposed peptide vaccine might be a good option for vibriosis prophylaxis.

Porphyromonas gingivalis, a prominent pathogen responsible for acute periodontal diseases, is widely studied by the scientific community for its successful evasion of the host immune system. P. gingivalis is associated with rheumatoid arthritis, dementia, and Alzheimer's. The pathogen successfully survives itself against the heavy load of conventional antibiotics because of its ability to evade the host immune system. Subtractive proteomics and reverse vaccinology approaches were employed in order to prioritize the best proteins for vaccine designing. Three vaccine candidates with Uniprot ID: Q7MWZ2 (histidine Kinase), Q7MVL1 (Fe (2+) transporter), and Q7MWZ2 (Capsular polysaccharide transport protein) were identified for vaccine designing. These proteins are antigenic and essential for pathogen survival. A wide range of immunoinformatics tools was applied for the prediction of epitopes, B, and T cells, for the vaccine candidate proteins. Molecular docking of the predicted epitopes against the MHC molecules were carried out. In-silico vaccine was constructed using carefully evaluated epitopes and consequently modeled for docking with human Toll-like receptor 2. Chain C of Pam3CSK4 (PDB ID; 2Z7X) was linked to the vaccine as an adjuvant to boost immune response towards the vaccine. For stability evaluation of the vaccine-TLR-2 docked complex, Molecular Dynamics simulations were performed. The reverse-translated nucleotide sequence cloned in Eschericia coli to attain the maximal expression of the vaccine protein. The maximal expression was ensured by CAI score of 0.96. The current vaccine requires future experimental validation to confirm its effectiveness. The vaccine developed will be helpful to protect against P. gingivalis associated infections.Communicated by Ramaswamy H. Sarma.

Dengue outbreaks are a serious public health concern that occurs on a regular basis in various locations of India. According to the Government of India's National Center for Vector-Borne Disease Control, a total of 1,23,106 dengue cases were identified in India as of October 2021. The currently available dengue vaccine was found to be ineffective against all serotypes of the virus. Dengue virus serotype 2 was reported to be the sole predominant serotype in Eastern Uttar Pradesh, India. An epitope-based peptide vaccine is believed to be safe and effective against all serotypes of the dengue virus. In this work, an epitope-based peptide vaccine based on envelope protein against the dengue virus was developed using the reverse vaccinology method. T-cell epitopes present in the envelope protein were screened using different immunoinformatic tools. Epitopes predicted by all servers were chosen and additionally picked out on the grounds of their antigenic reactivity, immunogenicity, toxicity, and allergenicity assessment. Three potent T cell epitopes as IVQPENLEY, ILIGVVITW, and DTAWDFGSL were screened, which binds with HLA-B*35:01, HLA-B*58:01, HLA-A*26:01 alleles, respectively. To build a 3D structure model of epitopes and alleles, the PepstrMod and Swiss-Model servers were used. Predicted epitopes and HLA alleles were docked using the HPEPDOCK server to confirm binding ability. These anticipated epitopes were found to cover the greatest number of populations in India and around the world. These identified epitopes have a high potential for eliciting an immune response in the development of a vaccine against the dengue virus, while further experimental validation is required for final confirmation., Supplementary Information: The online version contains supplementary material available at 10.1007/s10989-022-10402-4., Competing Interests: Conflict of interestI confirm that the authors hereby declare they that have no conflict of interest., (© The Author(s), under exclusive licence to Springer Nature B.V. 2022.)

SARS-CoV-2 and its variants caused the COVID-19 pandemic. Vaccines that target conserved regions of SARS-CoV-2 and stimulate protective T-cell responses are important for reducing symptoms and limiting the infection. Seven cytotoxic (CTL) and five helper T-cells (HTL) epitopes from ORF1ab were identified using NetCTLpan and NetMHCIIpan algorithms, respectively. These epitopes were generated from ORF1ab regions that are evolutionary stable as reflected by zero Shannon's entropy and are presented by 56 human leukocyte antigen (HLA) Class I and 22 HLA Class II, ensuring good coverage for the Indonesian and world population. Having fulfilled other criteria such as immunogenicity, IFNγ inducing ability, and non-homology to human and microbiome peptides, the epitopes were assembled into a vaccine construct (VC) together with β-defensin as adjuvant and appropriate linkers. The VC was shown to have good physicochemical characteristics and capability of inducing CTL as well as HTL responses, which stem from the engagement of the vaccine with toll-like receptor 4 (TLR4) as revealed by docking simulations. The most promiscuous peptide 899 WSMATYYLF 907 was shown via docking simulation to interact well with HLA-A*24:07, the most predominant allele in Indonesia. The data presented here will contribute to the in vitro study of T-cell epitope mapping and vaccine design in Indonesia.

Identification of relevant epitopes is crucial for the development of subunit peptide vaccines inducing neutralizing and cellular immunity against SARS-CoV-2. Our aim was the characterization of epitopes in the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein to generate a peptide vaccine. Epitope mapping using a panel of 10 amino acid overlapped 15-mer peptides covering region 401-515 from RBD did not identify linear epitopes when tested with sera from infected individuals or from RBD-immunized mice. However, immunization of mice with these 15-mer peptides identified four peptides located at region 446-480 that induced antibodies recognizing the peptides and RBD/S1 proteins. Immunization with peptide 446-480 from S protein formulated with Freund's adjuvant or with CpG oligodeoxinucleotide/Alum induced polyepitopic antibody responses in BALB/c and C56BL/6J mice, recognizing RBD (titres of 3 × 10 4 -3 × 10 5 , depending on the adjuvant) and displaying neutralizing capacity (80-95% inhibition capacity; p  <   0.05) against SARS-CoV-2. Murine CD4 and CD8T-cell epitopes were identified in region 446-480 and vaccination experiments using HLA transgenic mice suggested the presence of multiple human T-cell epitopes. Antibodies induced by peptide 446-480 showed broad recognition of S proteins and S-derived peptides belonging to SARS-CoV-2 variants of concern. Importantly, vaccination with peptide 446-480 or with a cyclic version of peptide 446-488 containing a disulphide bridge between cysteines 480 and 488, protected humanized K18-hACE2 mice from a lethal dose of SARS-CoV-2 (62.5 and 75% of protection; p  <   0.01 and p  <   0.001, respectively). This region could be the basis for a peptide vaccine or other vaccine platforms against Covid-19.

Background: Pulmonary tuberculosis (PTB) is still a major health problem worldwide. The espD has a potential to be a new biomarker because it is important for the espA, espC, and ESX-1 protein secretion system that are actively expressed in active multiplication of Mycobacterium tuberculosis complex., Methods: A total of 55 sputum samples and 41 culture isolates from newly diagnosed PTB patients at Dr. Soetomo Academic Hospital were collected from September 2016 to April 2019. The tested samples using polymerase chain reaction targeted 555 bp of espD gene and sequencing. Clone Manager Version 6 and NCBI BLAST were used to align the gene sequence against wild-type M. tuberculosis. The prediction of T-cell epitope in espD gene was detected by GENETYX. The three-dimensional (3D) structure of espD was modeled by SWISS-MODEL and I-TASSER and was visualized with PyMOL., Results: From 55 sputum samples, 43 (78.18%) showed positive results, and all culture isolates showed positive results. In addition, all sequenced samples showed 100% homolog with M. tuberculosis H37Rv gene without detected variant or mutation. There were four T-cell epitopes that could be obtained. The 3D model had a I-TASSER confidence score of 3.91 with estimated RMSD of approximately 14.5 Å. The structure consists of a main fold of a three-stranded antiparallel β-sheet and a long α-helix surrounded by several minor secondary structures., Conclusions: This study provides a brief information about the sequence, epitope prediction, and 3D structure of EspD protein from M. tuberculosis strains in Indonesia., Competing Interests: None

Background: Coronavirus disease 2019 (COVID-19) continues to be a major public health challenge globally. The identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived T-cell epitopes is of critical importance for peptide vaccines or diagnostic tools of COVID-19., Methods: In this study, several SARS-CoV-2-derived human leukocyte antigen (HLA)-I binding peptides were predicted by NetMHCpan-4.1 and selected by Popcover to achieve pancoverage of the Chinese population. The top 5 ranked peptides derived from each protein of SARS-CoV-2 were then evaluated using peripheral blood mononuclear cells from unexposed individuals (negative for SARS-CoV-2 immunoglobulin G)., Results: Seven epitopes derived from 4 SARS-CoV-2 proteins were identified. It is interesting to note that most (5 of 7) of the SARS-CoV-2-derived peptides with predicted affinities for HLA-I molecules were identified as HLA-II-restricted epitopes and induced CD4+ T cell-dependent responses. These results complete missing pieces of pre-existing SARS-CoV-2-specific T cells and suggest that pre-existing T cells targeting all SARS-CoV-2-encoded proteins can be discovered in unexposed populations., Conclusions: In summary, in the current study, we present an alternative and effective strategy for the identification of T-cell epitopes of SARS-CoV-2 in healthy subjects, which may indicate an important role in the development of peptide vaccines for COVID-19., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) kills thousands of people worldwide every day, thus necessitating rapid development of countermeasures. Immunoinformatics analyses carried out here in search of immunodominant regions in recently identified SARS-CoV-2 unannotated open reading frames (uORFs) have identified eight linear B-cell, one conformational B-cell, 10 CD4+ T-cell, and 12 CD8+ T-cell promising epitopes. Among them, ORF9b B-cell and T-cell epitopes are the most promising followed by M.ext and ORF3c epitopes. ORF9b 40-48 (CD8+ T-cell epitope) is found to be highly immunogenic and antigenic with the highest allele coverage. Furthermore, it has overlap with four potent CD4+ T-cell epitopes. Structure-based B-cell epitope prediction has identified ORF9b 61-68 to be immunodominant, which partially overlaps with one of the linear B-cell epitopes (ORF9b 65-69 ). ORF3c CD4+ T-cell epitopes (ORF3c 2-16 , ORF3c 3-17 , and ORF3c 4-18 ) and linear B-cell epitope (ORF3c 14-22 ) have also been identified as the candidate epitopes. Similarly, M.ext and 7a.iORF1 (overlap with M and ORF7a) proteins have promising immunogenic regions. By considering the level of antigen expression, four ORF9b and five M.ext epitopes are finally shortlisted as potent epitopes. Mutation analysis has further revealed that the shortlisted potent uORF epitopes are resistant to recurrent mutations. Additionally, four N-protein (expressed by canonical ORF) epitopes are found to be potent. Thus, SARS-CoV-2 uORF B-cell and T-cell epitopes identified here along with canonical ORF epitopes may aid in the design of a promising epitope-based polyvalent vaccine (when connected through appropriate linkers) against SARS-CoV-2. Such a vaccine can act as a bulwark against SARS-CoV-2, especially in the scenario of emergence of variants with recurring mutations in the spike protein., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Uttamrao, Sathyaseelan, Patro and Rathinavelan.)

Background: The increase in global mortality rates from SARS-COV2 (COVID-19) infection has been alarming thereby necessitating the continual search for viable therapeutic interventions. Due to minimal microbial components, subunit (peptide-based) vaccines have demonstrated improved efficacies in stimulating immunogenic responses by host B- and T-cells., Methods: Integrative immunoinformatics algorithms were used to determine linear and discontinuous B-cell epitopes from the S-glycoprotein sequence. End-point selection of the most potential B-cell epitope was based on highly essential physicochemical attributes. NetCTL-I and NetMHC-II algorithms were used to predict probable MHC-I and II T-cell epitopes for globally frequent HLA-A∗O2:01, HLA-B∗35:01, HLA-B∗51:01 and HLA-DRB1∗15:02 molecules. Highly probable T-cell epitopes were selected based on their high propensities for C-terminal cleavage, transport protein (TAP) processing and MHC-I/II binding., Results: Preferential epitope binding sites were further identified on the HLA molecules using a blind peptide-docking method. Phylogenetic analysis revealed close relativity between SARS-CoV-2 and SARS-CoV S-protein. LALHRSYLTPGDSSSGWTAGAA 242→263 was the most probable B-cell epitope with optimal physicochemical attributes. MHC-I antigenic presentation pathway was highly favourable for YLQPRTFLL 269-277 (HLA-A∗02:01), LPPAYTNSF 24-32 (HLA-B∗35:01) and IPTNFTISV7 14-721 (HLA-B∗51:01). Also, LTDEMIAQYTSALLA 865-881 exhibited the highest binding affinity to HLA-DR B1∗15:01 with core interactions mediated by IAQYTSALL 870-878 . COVID-19 YLQPRTFLL 269-277 was preferentially bound to a previously undefined site on HLA-A∗02:01 suggestive of a novel site for MHC-I-mediated T-cell stimulation., Conclusion: This study implemented combinatorial immunoinformatics methods to model B- and T-cell epitopes with high potentials to trigger immunogenic responses to the S protein of SARS-CoV-2., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2021 Chang Gung University. Published by Elsevier B.V. All rights reserved.)