HLA A*24:02-restricted T cell receptors cross-recognize bacterial and preproinsulin peptides in type 1 diabetes.

CD8+ T cells destroy insulin-producing pancreatic ß cells in type 1 diabetes through HLA class I-restricted presentation of self-antigens. Combinatorial peptide library screening was used to produce a preferred peptide recognition landscape for a patient-derived T cell receptor (TCR) that recognized the preproinsulin-derived (PPI-derived) peptide sequence LWMRLLPLL in the context of disease risk allele HLA A*24:02. Data were used to generate a strong superagonist peptide, enabling production of an autoimmune HLA A*24:02-peptide-TCR structure by crystal seeding. TCR binding to the PPI epitope was strongly focused on peptide residues Arg4 and Leu5, with more flexibility at other positions, allowing the TCR to strongly engage many peptides derived from pathogenic bacteria. We confirmed an epitope from Klebsiella that was recognized by PPI-reactive T cells from 3 of 3 HLA A*24:02+ patients. Remarkably, the same epitope selected T cells from 7 of 8 HLA A*24+ healthy donors that cross-reacted with PPI, leading to recognition and killing of HLA A*24:02+ cells expressing PPI. These data provide a mechanism by which molecular mimicry between pathogen and self-antigens could have resulted in the breaking of self-tolerance to initiate disease.

Designing a multi-epitope subunit vaccine against Orf virus using molecular docking and molecular dynamics.

Orf virus (ORFV) is an acute contact, epitheliotropic, zoonotic, and double-stranded DNA virus that causes significant economic losses in the livestock industry. The objective of this study is to design an immunoinformatics-based multi-epitope subunit vaccine against ORFV. Various immunodominant cytotoxic T lymphocytes (CTL), helper T lymphocytes (HTL), and B-cell epitopes from the B2L, F1L, and 080 protein of ORFV were selected and linked by short connectors to construct a multi-epitope subunit vaccine. Immunogenicity was enhanced by adding an adjuvant ß-defensin to the N-terminal of the vaccine using the EAAAK linker. The vaccine exhibited a significant degree of antigenicity and solubility, without allergenicity or toxicity. The 3D formation of the vaccine was subsequently anticipated, improved, and verified. The optimized model exhibited a lower Z-score of -4.33, indicating higher quality. Molecular docking results demonstrated that the vaccine strongly binds to TLR2 and TLR4. Molecular dynamics results indicated that the docked vaccine-TLR complexes were stable. Immune simulation analyses further confirmed that the vaccine can induce a marked increase in IgG and IgM antibody titers, and elevated levels of IFN-γ and IL-2. Finally, the optimized DNA sequence of the vaccine was cloned into the vector pET28a (+) for high expression in the E.coli expression system. Overall, the designed multi-epitope subunit vaccine is highly stable and can induce robust humoral and cellular immunity, making it a promising vaccine candidate against ORFV.

Prediction of promiscuous multiepitope-based peptide vaccine against RdRp of rotavirus using immunoinformatics studies.

Rotavirus, a dsRNA virus in the Reoviridae family, shows a segmented genome. The VP1 gene encodes the RNA-dependent RNA polymerase (RdRp). This study aims to develop a multiepitope-based vaccine targeting RdRp using immunoinformatic approaches. In this study, 100 available nucleotide sequences of VP1-Rotavirus belonging to different strains across the world were retrieved from NCBI database. The selected sequences were aligned, and a global consensus sequence was developed by using CLC work bench. The study involved immunoinformatic approaches and molecular docking studies to reveal the promiscuous epitopes that can be eventually used as active vaccine candidates for Rotavirus. In total, 27 highly immunogenic, antigenic, and non-allergenic T-cell and B-cell epitopes were predicted for the Multiepitope vaccine (MEV) against rotavirus. It was also observed that MEV can prove to be effective worldwide due to its high population coverage, demonstrating the consistency of this vaccine. Moreover, there is a high docking interaction and immunological response with a binding score of -50.2 kcal/mol, suggesting the vaccine's efficacy. Toll-like receptors (TLRs) also suggest that the vaccine is physiologically and immunologically effective. Collectively, our data point to an effective MEV against rotavirus that can effectively reduce viral infections and improve the health status worldwide.

Systematic analysis of SARS-CoV-2 Omicron subvariants' impact on B and T cell epitopes.

INTRODUCTION: Epitopes are specific structures in antigens that are recognized by the immune system. They are widely used in the context of immunology-related applications, such as vaccine development, drug design, and diagnosis / treatment / prevention of disease. The SARS-CoV-2 virus has represented the main point of interest within the viral and genomic surveillance community in the last four years. Its ability to mutate and acquire new characteristics while it reorganizes into new variants has been analyzed from many perspectives. Understanding how epitopes are impacted by mutations that accumulate on the protein level cannot be underrated. METHODS: With a focus on Omicron-named SARS-CoV-2 lineages, including the last WHO-designated Variants of Interest, we propose a workflow for data retrieval, integration, and analysis pipeline for conducting a database-wide study on the impact of lineages' characterizing mutations on all T cell and B cell linear epitopes collected in the Immune Epitope Database (IEDB) for SARS-CoV-2. RESULTS: Our workflow allows us to showcase novel qualitative and quantitative results on 1) coverage of viral proteins by deposited epitopes; 2) distribution of epitopes that are mutated across Omicron variants; 3) distribution of Omicron characterizing mutations across epitopes. Results are discussed based on the type of epitope, the response frequency of the assays, and the sample size. Our proposed workflow can be reproduced at any point in time, given updated variant characterizations and epitopes from IEDB, thereby guaranteeing to observe a quantitative landscape of mutations' impact on demand. CONCLUSION: A big data-driven analysis such as the one provided here can inform the next genomic surveillance policies in combatting SARS-CoV-2 and future epidemic viruses.

TgVax452, an epitope-based candidate vaccine targeting Toxoplasma gondii tachyzoite-specific SAG1-related sequence (SRS) proteins: immunoinformatics, structural simulations and experimental evidence-based approaches.

BACKGROUND: The highly expressed surface antigen 1 (SAG1)-related sequence (SRS) proteins of T. gondii tachyzoites, as a widespread zoonotic parasite, are critical for host cell invasion and represent promising vaccine targets. In this study, we employed a computer-aided multi-method approach for in silico design and evaluation of TgVax452, an epitope-based candidate vaccine against T. gondii tachyzoite-specific SRS proteins. METHODS: Using immunoinformatics web-based tools, structural modeling, and static/dynamic molecular simulations, we identified and screened B- and T-cell immunodominant epitopes and predicted TgVax452's antigenicity, stability, safety, adjuvanticity, and physico-chemical properties. RESULTS: The designed protein possessed 452 residues, a MW of 44.07 kDa, an alkaline pI (6.7), good stability (33.20), solubility (0.498), and antigenicity (0.9639) with no allergenicity. Comprehensive molecular dynamic (MD) simulation analyses confirmed the stable interaction (average potential energy: 3.3799 × 106 KJ/mol) between the TLR4 agonist residues (RS09 peptide) of the TgVax452 in interaction with human TLR4, potentially activating innate immune responses. Also, a dramatic increase was observed in specific antibodies (IgM and IgG), cytokines (IFN-γ), and lymphocyte responses, based on C-ImmSim outputs. Finally, we optimized TgVax452's codon adaptation and mRNA secondary structure for efficient expression in E. coli BL21 expression machinery. CONCLUSION: Our findings suggest that TgVax452 is a promising candidate vaccine against T. gondii tachyzoite-specific SRS proteins and requires further experimental studies for its potential use in preclinical trials.

Predicting T cell receptor functionality against mutant epitopes.

Cancer cells and pathogens can evade T cell receptors (TCRs) via mutations in immunogenic epitopes. TCR cross-reactivity (i.e., recognition of multiple epitopes with sequence similarities) can counteract such escape but may cause severe side effects in cell-based immunotherapies through targeting self-antigens. To predict the effect of epitope point mutations on T cell functionality, we here present the random forest-based model Predicting T Cell Epitope-Specific Activation against Mutant Versions (P-TEAM). P-TEAM was trained and tested on three datasets with TCR responses to single-amino-acid mutations of the model epitope SIINFEKL, the tumor neo-epitope VPSVWRSSL, and the human cytomegalovirus antigen NLVPMVATV, totaling 9,690 unique TCR-epitope interactions. P-TEAM was able to accurately classify T cell reactivities and quantitatively predict T cell functionalities for unobserved single-point mutations and unseen TCRs. Overall, P-TEAM provides an effective computational tool to study T cell responses against mutated epitopes.

Development of Hypoallergenic Derivatives of Cra a 1 with B Cell Epitope Deletion and T Cell Epitope Retention.

Tropomyosin was reported as an important allergen in Crassostrea angulata and designated as Cra a 1. The localization of the T cell epitopes and the reduction of the immunoreactivity of Cra a 1 are still lacking. In this study, four T cell epitopes were identified by using wild-type Cra a 1 (wtCra a 1)-immunized mouse splenocytes cultured with synthetic peptides. The immunoreactivity was maintained after chemical denaturation treatment, indicating that the linear epitope is an immunodominant epitope of wtCra a 1. Furthermore, the hypoallergenic derivative (mCra a 1) was developed by the deletion of linear B cell epitopes and retention of T cell epitopes. mCra a 1 could stimulate CD4+T cell proliferation and upregulate interleukin-10 secretion. Overall, basophil activation by mCra a 1 was low, but its ability to induce T cell proliferation was retained, suggesting that mCra a 1 may serve as a viable candidate for treating oyster allergy.

Reverse vaccinology approaches to design a potent multiepitope vaccine against the HIV whole genome: immunoinformatic, bioinformatics, and molecular dynamics approaches.

Substantial advances have been made in the development of promising HIV vaccines to eliminate HIV-1 infection. For the first time, one hundred of the most submitted HIV subtypes and CRFs were retrieved from the LANL database, and the consensus sequences of the eleven HIV proteins were obtained to design vaccines for human and mouse hosts. By using various servers and filters, highly qualified B-cell epitopes, as well as HTL and CD8 + epitopes that were common between mouse and human alleles and were also located in the conserved domains of HIV proteins, were considered in the vaccine constructs. With 90% coverage worldwide, the human vaccine model covers a diverse allelic population, making it widely available. Codon optimization and in silico cloning in prokaryotic and eukaryotic vectors guarantee high expression of the vaccine models in human and E. coli hosts. Molecular dynamics confirmed the stable interaction of the vaccine constructs with TLR3, TLR4, and TLR9, leading to a substantial immunogenic response to the designed vaccine. Vaccine models effectively target the humoral and cellular immune systems in humans and mice; however, experimental validation is needed to confirm these findings in silico.

In silico designing of multi-epitope vaccine against canine parvovirus using reverse vaccinology.

Canine parvovirus (CPV-2) is a highly contagious virus affecting dogs worldwide, posing a significant threat. The VP2 protein stands out as the predominant and highly immunogenic structural component of CPV-2. Soon after its emergence, CPV-2 was replaced by variants known as CPV-2a, 2b and 2c, marked by changes in amino acid residue 426 of VP2. Additional amino acid alterations have been identified within VP2, with certain modifications serving as signatures of emerging variants. In Brazil, CPV-2 outbreaks persist with diverse VP2 profiles. Vaccination is the main preventive measure against the virus. However, the emergence of substitutions presents challenges to conventional vaccine methods. Commercial vaccines are formulated with strains that usually do not match those currently circulating in the field. To address this, the study aimed to investigate CPV-2 variants in Brazil, predict epitopes, and design an in silico vaccine tailored to local variants employing reverse vaccinology. The methodology involved data collection, genetic sequence analysis, and amino acid comparison between field strains and vaccines, followed by the prediction of B and T cell epitope regions. The predicted epitopes were evaluated for antigenicity, allergenicity and toxicity. The final vaccine construct consisted of selected epitopes linked to an adjuvant and optimized for expression in Escherichia coli. Structural predictions confirmed the stability and antigenicity of the vaccine, while molecular docking demonstrated interaction with the canine toll-like receptor 4. Molecular dynamics simulations indicated a stable complex formation. In silico immune simulations demonstrated a progressive immune response post-vaccination, including increased antibody production and T-helper cell activity. The multi-epitope vaccine design targeted prevalent CPV-2 variants in Brazil and potentially other regions globally. However, experimental validation is essential to confirm our in silico findings.

Structural proteomics guided annotation of vaccine targets and designing of multi-epitopes vaccine to instigate adaptive immune response against Francisella tularensis.

Francisella tularensis can cause severe disease in humans via the respiratory or cutaneous routes and a case fatality ratio of up to 10 % is reported due to lack of proper antibiotic treatment, while F. novicida causes disease in severely immunocompromised individuals. Efforts are needed to develop effective vaccine candidates against Francisella species. Thus, in this study, a systematic computational work frame was used to deeply investigate the whole proteome of Francisella novicida containing 1728 proteins to develop vaccine against F. tularensis and related species. Whole-proteome analysis revealed that four proteins including (A0Q492) (A0Q7Y4), (A0Q4N4), and (A0Q5D9) are the suitable vaccine targets after the removal of homologous, paralogous and prediction of subcellular localization. These proteins were used to predict the T cell, B cell, and HTL epitopes which were joined together through suitable linkers to construct a multi-epitopes vaccine (MEVC). The MEVC was found to be highly immunogenic and non-allergenic while the physiochemical properties revealed the feasible expression and purification. Moreover, the molecular interaction of MEVC with TLR2, molecular simulation, and binding free energy analyses further validated the immune potential of the construct. According to Jcat analysis, the refined sequence demonstrates GC contents of 41.48 % and a CAI value of 1. The in-silico cloning and optimization process ensured compatibility with host codon usage, thereby facilitating efficient expression. Computational immune simulation studies underscored the capacity of MEVC to induce both primary and secondary immune responses. The conservation analysis further revealed that the selected epitopes exhibit 100 % conservation across different species and thus provides wider protection against Francisella.

Molecular characterization of canine circovirus based on the Capsid gene in Thailand.

BACKGROUND: Canine circovirus (CanineCV) is a single-stranded circular DNA virus that infects domestic and wild canids in many countries. CanineCV is associated with gastroenteritis and diarrhea, respiratory disease, and generalized vasculitis leading to a fatal event. The Capsid protein (Cap) is a structural protein of the virus which has high genetic variability and plays a role in the canine immune response. In this study, we cloned the full-length CanineCV Capsid gene (Cap). In-silico analyses were used to explore the genomic and amino acid variability and natural selection acting on the Cap gene. The immune relevance for T-cell and B-cell epitopes was predicted by the immunoinformatic approach. RESULTS: According to the Cap gene, our results showed that CanineCV was separated into five phylogenetic groups. The obtained CanineCV strain from this study was grouped with the previously discovered Thai strain (MG737385), as supported by a haplotype network. Entropy analyses revealed high nucleotide and amino acid variability of the Capsid region. Selection pressure analysis revealed four codons at positions 24, 50, 103, and 111 in the Cap protein evolved under diversifying selection. Prediction of B-cell epitopes exhibited four consensus sequences based on physiochemical properties, and eleven peptide sequences were predicted as T-cell epitopes. In addition, the positive selection sites were located within T-cell and B-cell epitopes, suggesting the role of the host immune system as a driving force in virus evolution. CONCLUSIONS: Our study provides knowledge of CanineCV genetic diversity, virus evolution, and potential epitopes for host cell immune response.

Structural insights into immune escape at killer T cell epitope by SARS-CoV-2 Spike Y453F variants.

CD8+ T cell immunity, mediated by human leukocyte antigen (HLA) and T cell receptor (TCR), plays a critical role in conferring immune memory and protection against viral pathogens. The emergence of SARS-CoV-2 variants poses a serious challenge to the efficacy of current vaccines. Whereas numerous SARS-CoV-2 mutations associated with immune escape from CD8+ T cells have been documented, the molecular effects of most mutations on epitope-specific TCR recognition remain largely unexplored. Here, we studied an HLA-A24-restricted NYN epitope (Spike448-456) that elicits broad CD8+ T cell responses in COVID-19 patients characterized by a common TCR repertoire. Four natural mutations, N450K, L452Q, L452R, and Y453F, arose within the NYN epitope and have been transmitted in certain viral lineages. Our findings indicate that these mutations have minimal impact on the epitope's presentation by cell surface HLA, yet they diminish the affinities of their respective peptide-HLA complexes (pHLAs) for NYN peptide-specific TCRs, particularly L452R and Y453F. Furthermore, we determined the crystal structure of HLA-A24 loaded with the Y453F peptide (NYNYLFRLF), and subsequently a ternary structure of the public TCRNYN-I complexed to the original NYN-HLA-A24 (NYNYLYRLF). Our structural analysis unveiled that despite competent presentation by HLA, the mutant Y453F peptide failed to establish a stable TCR-pHLA ternary complex due to reduced peptide: TCR contacts. This study supports the idea that cellular immunity restriction is an important driving force behind viral evolution.

Designing multi-epitope vaccine against human cytomegalovirus integrating pan-genome and reverse vaccinology pipelines.

Human cytomegalovirus (HCMV) is accountable for high morbidity in neonates and immunosuppressed individuals. Due to the high genetic variability of HCMV, current prophylactic measures are insufficient. In this study, we employed a pan-genome and reverse vaccinology approach to screen the target for efficient vaccine candidates. Four proteins, envelope glycoprotein M, UL41A, US23, and US28, were shortlisted based on cellular localization, high solubility, antigenicity, and immunogenicity. A total of 29 B-cell and 44 T-cell highly immunogenic and antigenic epitopes with high global population coverage were finalized using immunoinformatics tools and algorithms. Further, the epitopes that were overlapping among the finalized B-cell and T-cell epitopes were linked with suitable linkers to form various combinations of multi-epitopic vaccine constructs. Among 16 vaccine constructs, Vc12 was selected based on physicochemical and structural properties. The docking and molecular simulations of VC12 were performed, which showed its high binding affinity (-23.35 kcal/mol) towards TLR4 due to intermolecular hydrogen bonds, salt bridges, and hydrophobic interactions, and there were only minimal fluctuations. Furthermore, Vc12 eliciting a good response was checked for its expression in Escherichia coli through in silico cloning and codon optimization, suggesting it to be a potent vaccine candidate.

Designing a Conserved Immunogenic Peptide Construct from the Nucleocapsid Protein of Puumala orthohantavirus.

Puumala orthohantavirus (PUUV) is an emerging zoonotic virus endemic to Europe and Russia that causes nephropathia epidemica, a mild form of hemorrhagic fever with renal syndrome (HFRS). There are limited options for treatment and diagnosis of orthohantavirus infection, making the search for potential immunogenic candidates crucial. In the present work, various bioinformatics tools were employed to design conserved immunogenic peptides containing multiple epitopes of PUUV nucleocapsid protein. Eleven conserved peptides (90% conservancy) of the PUUV nucleocapsid protein were identified. Three conserved peptides containing multiple T and B cell epitopes were selected using a consensus epitope prediction algorithm. Molecular docking using the HPEP dock server demonstrated strong binding interactions between the epitopes and HLA molecules (ten alleles for each class I and II HLA). Moreover, an analysis of population coverage using the IEDB database revealed that the identified peptides have over 90% average population coverage across six continents. Molecular docking and simulation analysis reveal a stable interaction with peptide constructs of chosen immunogenic peptides and Toll-like receptor-4. These computational analyses demonstrate selected peptides' immunogenic potential, which needs to be validated in different experimental systems.

Mutations in glioblastoma proteins do not disrupt epitope presentation and recognition, maintaining a specific CD8 T cell immune response potential.

Antigen-specific cytotoxic CD8 T cells are extremely effective in controlling tumor growth and have been the focus of immunotherapy approaches. We leverage in silico tools to investigate whether the occurrence of mutations in proteins previously described as immunogenic and highly expressed by glioblastoma multiforme (GBM), such as Epidermal Growth Factor Receptor (EGFR), Isocitrate Dehydrogenase 1 (IDH1), Phosphatase and Tensin homolog (PTEN) and Tumor Protein 53 (TP53), may be contributing to the differential presentation of immunogenic epitopes. We recovered Class I MHC binding information from wild-type and mutated proteins using the Immune Epitope Database (IEDB). After that, we built peptide-MHC (pMHC-I) models in HLA-arena, followed by hierarchical clustering analysis based on electrostatic surface features from each complex. We identified point mutations that are determinants for the presentation of a set of peptides from TP53 protein. We point to structural features in the pMHC-I complexes of wild-type and mutated peptides, which may play a role in the recognition of CD8 T cells. To further explore these features, we performed 100 ns molecular dynamics simulations for the peptide pairs (wt/mut) selected. In pursuit of novel therapeutic targets for GBM treatment, we selected peptides where our predictive results indicated that mutations would not disrupt epitope presentation, thereby maintaining a specific CD8 T cell immune response. These peptides hold potential for future GBM interventions, including peptide-based or mRNA vaccine development applications.

Design of a novel multi-epitope vaccine against Marburg virus using immunoinformatics studies.

Marburg virus (MARV) is a highly contagious and virulent agent belonging to Filoviridae family. MARV causes severe hemorrhagic fever in humans and non-human primates. Owing to its highly virulent nature, preventive approaches are promising for its control. There is currently no approved drug or vaccine against MARV, and management mainly involves supportive care to treat symptoms and prevent complications. Our aim was to design a novel multi-epitope vaccine (MEV) against MARV using immunoinformatics studies. In this study, various proteins (VP35, VP40 and glycoprotein precursor) were used and potential epitopes were selected. CTL and HTL epitopes covered 79.44% and 70.55% of the global population, respectively. The designed MEV construct was stable and expressed in Escherichia coli (E. coli) host. The physicochemical properties were also acceptable. MARV MEV candidate could predict comprehensive immune responses such as those of humoral and cellular in silico. Additionally, efficient interaction to toll-like receptor 3 (TLR3) and its agonist (ß-defensin) was predicted. There is a need for validation of these results using further in vitro and in vivo studies.

Genetic variability of human papillomavirus type 18 based on E6, E7 and L1 genes in central China.

BACKGROUND: High-risk human papillomavirus (HR-HPV) infection is an important factor for the development of cervical cancer. HPV18 is the second most common HR-HPV after HPV16. METHODS: In this study, MEGA11 software was used to analyze the variation and phylogenetic tree of HPV18 E6-E7 and L1 genes. The selective pressure to E6, E7 and L1 genes was estimated using pamlX. In addition, the B cell epitopes of L1 amino acid sequences and T cell epitopes of E6-E7 amino acid sequences in HPV18 were predicted by ABCpred server and IEDB website, respectively. RESULTS: A total of 9 single nucleotide variants were found in E6-E7 sequences, of which 2 were nonsynonymous variants and 7 were synonymous variants. Twenty single nucleotide variants were identified in L1 sequence, including 11 nonsynonymous variants and 9 synonymous variants. Phylogenetic analysis showed that E6-E7 and L1 sequences were all distributed in A lineage. In HPV18 E6, E7 and L1 sequences, no positively selected site was found. The nonconservative substitution R545C in L1 affected hypothetical B cell epitope. Two nonconservative substitutions, S82A in E6, and R53Q in E7, impacted multiple hypothetical T cell epitopes. CONCLUSION: The sequence variation data of HPV18 may lay a foundation for the virus diagnosis, further study of cervical cancer and vaccine design in central China.

Chimeric lipoproteins for leptospirosis vaccine: immunogenicity and protective potential.

Leptospirosis, a neglected zoonotic disease, is caused by pathogenic spirochetes belonging to the genus Leptospira and has one of the highest morbidity and mortality rates worldwide. Vaccination stands out as one of the most effective preventive measures for susceptible populations. Within the outer membrane of Leptospira spp., we find the LIC12287, LIC11711, and LIC13259 lipoproteins. These are of interest due to their surface location and potential immunogenicity. Thorough examination revealed the conservation of these proteins among pathogenic Leptospira spp.; we mapped the distribution of T- and B-cell epitopes along their sequences and assessed the 3D structures of each protein. This information aided in selecting immunodominant regions for the development of a chimeric protein. Through gene synthesis, we successfully constructed a chimeric protein, which was subsequently expressed, purified, and characterized. Hamsters were immunized with the chimeric lipoprotein, formulated with adjuvants aluminum hydroxide, EMULSIGEN®-D, Sigma Adjuvant System®, and Montanide™ ISA206VG. Another group was vaccinated with an inactivated Escherichia coli bacterin expressing the chimeric protein. Following vaccination, hamsters were challenged with a virulent L. interrogans strain. Our evaluation of the humoral immune response revealed the production of IgG antibodies, detectable 28 days after the second dose, in contrast to pre-immune samples and control groups. This demonstrates the potential of the chimeric protein to elicit a robust humoral immune response; however, no protection against challenge was achieved. While this study provides valuable insights into the subject, further research is warranted to identify protective antigens that could be utilized in the development of a leptospirosis vaccine. KEY POINTS: • Several T- and B-cell epitopes were identified in all the three proteins. • Four different adjuvants were used in vaccine formulations. • Immunization stimulated significant levels of IgG2/3 in vaccinated animals.

Design of multi-epitope vaccine against porcine rotavirus using computational biology and molecular dynamics simulation approaches.

Porcine Rotavirus (PoRV) is a significant pathogen affecting swine-rearing regions globally, presenting a substantial threat to the economic development of the livestock sector. At present, no specific pharmaceuticals are available for this disease, and treatment options remain exceedingly limited. This study seeks to design a multi-epitope peptide vaccine for PoRV employing bioinformatics approaches to robustly activate T-cell and B-cell immune responses. Two antigenic proteins, VP7 and VP8*, were selected from PoRV, and potential immunogenic T-cell and B-cell epitopes were predicted using immunoinformatic tools. These epitopes were further screened according to non-toxicity, antigenicity, non-allergenicity, and immunogenicity criteria. The selected epitopes were linked with linkers to form a novel multi-epitope vaccine construct, with the PADRE sequence (AKFVAAWTLKAAA) and RS09 peptide attached at the N-terminus of the designed peptide chain to enhance the vaccine's antigenicity. Protein-protein docking of the vaccine constructs with toll-like receptors (TLR3 and TLR4) was conducted using computational methods, with the lowest energy docking results selected as the optimal predictive model. Subsequently, molecular dynamics (MD) simulation methods were employed to assess the stability of the protein vaccine constructs and TLR3 and TLR4 receptors. The results indicated that the vaccine-TLR3 and vaccine-TLR4 docking models remained stable throughout the simulation period. Additionally, the C-IMMSIM tool was utilized to determine the immunogenic triggering capability of the vaccine protein, demonstrating that the constructed vaccine protein could induce both cell-mediated and humoral immune responses, thereby playing a role in eliciting host immune responses. In conclusion, this study successfully constructed a multi-epitope vaccine against PoRV and validated the stability and efficacy of the vaccine through computational analysis. However, as the study is purely computational, experimental evaluation is required to validate the safety and immunogenicity of the newly constructed vaccine protein.

A long-term stable cold-chain-friendly HIV mRNA vaccine encoding multi-epitope viral protease cleavage site immunogens inducing immunogen-specific protective T cell immunity.

The lack of success in clinical trials for HIV vaccines highlights the need to explore novel strategies for vaccine development. Research on highly exposed seronegative (HESN) HIV-resistant Kenyan female sex workers revealed naturally protective immunity is correlated with a focused immune response mediated by virus-specific CD8 T cells. Further studies indicated that the immune response is unconventionally focused on highly conserved sequences around HIV viral protease cleavage sites (VPCS). Thus, taking an unconventional approach to HIV vaccine development, we designed lipid nanoparticles loaded with mRNA that encodes multi-epitopes of VPCS (MEVPCS-mRNA LNP), a strategic design to boost antigen presentation by dendritic cells, promoting effective cellular immunity. Furthermore, we developed a novel cold-chain compatible mRNA LNP formulation, ensuring long-term stability and compatibility with cold-chain storage/transport, widening accessibility of mRNA LNP vaccine in low-income countries. The in-vivo mouse study demonstrated that the vaccinated group generated VPCS-specific CD8 memory T cells, both systemically and at mucosal sites of viral entry. The MEVPCS-mRNA LNP vaccine-induced CD8 T cell immunity closely resembled that of the HESN group and displayed a polyfunctional profile. Notably, it induced minimal to no activation of CD4 T cells. This proof-of-concept study underscores the potential of the MEVPCS-mRNA LNP vaccine in eliciting CD8 T cell memory specific to the highly conserved multiple VPCS, consequently having a broad coverage in human populations and limiting viral escape mutation. The MEVPCS-mRNA LNP vaccine holds promise as a candidate for an effective prophylactic HIV vaccine.