An integrated comparative genomics, subtractive proteomics and immunoinformatics framework for the rational design of a Pan-Salmonella multi-epitope vaccine.

Salmonella infections pose a significant global public health concern due to the substantial expenses associated with monitoring, preventing, and treating the infection. In this study, we explored the core proteome of Salmonella to design a multi-epitope vaccine through Subtractive Proteomics and immunoinformatics approaches. A total of 2395 core proteins were curated from 30 different isolates of Salmonella (strain NZ CP014051 was taken as reference). Utilizing the subtractive proteomics approach on the Salmonella core proteome, Curlin major subunit A (CsgA) was selected as the vaccine candidate. csgA is a conserved gene that is related to biofilm formation. Immunodominant B and T cell epitopes from CsgA were predicted using numerous immunoinformatics tools. T lymphocyte epitopes had adequate population coverage and their corresponding MHC alleles showed significant binding scores after peptide-protein based molecular docking. Afterward, a multi-epitope vaccine was constructed with peptide linkers and Human Beta Defensin-2 (as an adjuvant). The vaccine could be highly antigenic, non-toxic, non-allergic, and have suitable physicochemical properties. Additionally, Molecular Dynamics Simulation and Immune Simulation demonstrated that the vaccine can bind with Toll Like Receptor 4 and elicit a robust immune response. Using in vitro, in vivo, and clinical trials, our findings could yield a Pan-Salmonella vaccine that might provide protection against various Salmonella species.

Immunogenicity of PvCyRPA, PvCelTOS and Pvs25 chimeric recombinant protein of Plasmodium vivax in murine model.

In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice's splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies.

An Immunoinformatic-Based In Silico Identification on the Creation of a Multiepitope-Based Vaccination Against the Nipah Virus.

The zoonotic viruses pose significant threats to public health. Nipah virus (NiV) is an emerging virus transmitted from bats to humans. The NiV causes severe encephalitis and acute respiratory distress syndrome, leading to high mortality rates, with fatality rates ranging from 40% to 75%. The first emergence of the disease was found in Malaysia in 1998-1999 and later in Bangladesh, Cambodia, Timor-Leste, Indonesia, Singapore, Papua New Guinea, Vietnam, Thailand, India, and other South and Southeast Asian nations. Currently, no specific vaccines or antiviral drugs are available. The potential advantages of epitope-based vaccines include their ability to elicit specific immune responses while minimizing potential side effects. The epitopes have been identified from the conserved region of viral proteins obtained from the UniProt database. The selection of conserved epitopes involves analyzing the genetic sequences of various viral strains. The present study identified two B cell epitopes, seven cytotoxic T lymphocyte (CTL) epitopes, and seven helper T lymphocyte (HTL) epitope interactions from the NiV proteomic inventory. The antigenic and physiological properties of retrieved protein were analyzed using online servers ToxinPred, VaxiJen v2.0, and AllerTOP. The final vaccine candidate has a total combined coverage range of 80.53%. The tertiary structure of the constructed vaccine was optimized, and its stability was confirmed with the help of molecular simulation. Molecular docking was performed to check the binding affinity and binding energy of the constructed vaccine with TLR-3 and TLR-5. Codon optimization was performed in the constructed vaccine within the Escherichia coli K12 strain, to eliminate the danger of codon bias. However, these findings must require further validation to assess their effectiveness and safety. The development of vaccines and therapeutic approaches for virus infection is an ongoing area of research, and it may take time before effective interventions are available for clinical use.

Phylogenetic Tracing of Evolutionarily Conserved Zonula Occludens Toxin Reveals a "High Value" Vaccine Candidate Specific for Treating Multi-Strain Pseudomonas aeruginosa Infections.

Extensively drug-resistant Pseudomonas aeruginosa infections are emerging as a significant threat associated with adverse patient outcomes. Due to this organism's inherent properties of developing antibiotic resistance, we sought to investigate alternative strategies such as identifying "high value" antigens for immunotherapy-based purposes. Through extensive database mining, we discovered that numerous Gram-negative bacterial (GNB) genomes, many of which are known multidrug-resistant (MDR) pathogens, including P. aeruginosa, horizontally acquired the evolutionarily conserved gene encoding Zonula occludens toxin (Zot) with a substantial degree of homology. The toxin's genomic footprint among so many different GNB stresses its evolutionary importance. By employing in silico techniques such as proteomic-based phylogenetic tracing, in conjunction with comparative structural modeling, we discovered a highly conserved intermembrane associated stretch of 70 amino acids shared among all the GNB strains analyzed. The characterization of our newly identified antigen reveals it to be a "high value" vaccine candidate specific for P. aeruginosa. This newly identified antigen harbors multiple non-overlapping B- and T-cell epitopes exhibiting very high binding affinities and can adopt identical tertiary structures among the least genetically homologous P. aeruginosa strains. Taken together, using proteomic-driven reverse vaccinology techniques, we identified multiple "high value" vaccine candidates capable of eliciting a polarized immune response against all the P. aeruginosa genetic variants tested.

Development of an enzyme-linked immunosorbent assay using a monoclonal antibody to a dominant epitope in non-structural protein 4 of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) is one of the most severe swine diseases causing great economic losses for the international swine industry. Non-structural protein 4 (NSP4) is critical to the life cycle of PRRSV and contains dominant B cell epitopes. This study prepared a monoclonal antibody against Nsp4, and 2D11, which contained the sequence 138KQGGGIVTRPSGQFCN153, was confirmed as the epitope. A 2D11-based double antibody sandwich enzyme-linked immunosorbent assay (dasELISA) was next developed with a cut value of 0.1987. A total of 1354 pig serum samples were detected by dasELISA and compared to a commercial ELISA kit (N-coated iELISA), resulting in a positive coincidence rate of 98.8% and negative coincidence rate of 96.9%. A total of 119 sera were positive by dasELISA while negative by iELISA. Higher positive rates by dasELISA were found in pig farms where PRRSV antibody levels varied widely. These results indicated that the dasELISA was a useful tool to detect PRRSV antibody in clinical samples.

IGH Complementarity Determining Region-3-Cytomegalovirus Protein Chemical Complementarity Linked to Better Overall Survival Probabilities for Glioblastoma.

Cytomegalovirus (CMV) has long been thought to have an association with glioblastoma multiforme (GBM), although the exact role of CMV and any subsequent implications for treatment have yet to be fully understood. This study addressed whether IGH complementarity determining region-3 (CDR3)-CMV protein chemical complementarity, with IGH CDR3s representing both tumor resident and blood-sourced IGH recombinations, was associated with overall survival (OS) distinctions. IGH recombination sequencing reads were obtained from (a) the Clinical Proteomic Tumor Analysis Consortium, tumor RNAseq files; and (b) the cancer genome atlas, blood exome-derived files. The Adaptive Match web tool was used to calculate chemical complementarity scores (CSs) based on hydrophobic interactions, and those scores were used to group GBM cases and assess survival probabilities. We found a higher OS probability for cases whose hydrophobic IGH CDR3-CMV protein chemical complementarity scores (Hydro CSs) were in the upper 50th percentile for several CMV proteins, including UL99 and UL123, as well as for CSs based on known B cell epitopes representing these proteins. We also identified multiple immune signature genes, including CD79A and TNFRSF17, for which higher RNA expression was associated with higher Hydro CSs. Results were consistent with the idea that stronger immunoglobulin responses to CMV are associated with better OS probabilities for GBM.

Identification of two novel B cell epitopes on E184L protein of African swine fever virus using monoclonal antibodies.

African swine fever virus (ASFV) is a large double-stranded DNA virus with a complex structural architecture and encodes more than 150 proteins, where many are with unknown functions. E184L has been reported as one of the immunogenic ASFV proteins that may contribute to ASFV pathogenesis and immune evasion. However, the antigenic epitopes of E184L are not yet characterized. In this study, recombinant E184L protein was expressed in prokaryotic expression system and four monoclonal antibodies (mAbs), designated as 1A10, 2D2, 3H6, and 4C10 were generated. All four mAbs reacted specifically with ASFV infected cells. To identify the epitopes of the mAbs, a series of overlapped peptides of E184L were designed and expressed as maltose binding fusion proteins. Accordingly, the expressed fusion proteins were probed with each E184L mAb separately by using Western blot. Following a fine mapping, the minimal linear epitope recognized by mAb 1A10 was identified as 119IQRQGFL125, and mAbs 2D2, 3H6, and 4C10 recognized a region located between 153DPTEFF158. Alignment of amino acids of E184L revealed that the two linear epitopes are highly conserved among different ASFV isolates. Furthermore, the potential application of the two epitopes in ASFV diagnosis was assessed through epitope-based ELISA using 24 ASFV positive and 18 negative pig serum and the method were able to distinguish positive and negative samples, indicating the two epitopes are dominant antigenic sites. To our knowledge, this is the first study to characterize the B cell epitopes of the antigenic E184L protein of ASFV, offering valuable tools for future research, as well as laying a foundation for serological diagnosis and epitope-based marker vaccine development.

Construction of multi-epitope vaccine against the Rhipicephalus microplus tick: an immunoinformatics approach.

Rhipicephalus microplus, known as the hard tick, is a vector for the parasites Babesia spp. and Anaplasma marginale, both of which can cause significant financial losses to the livestock industry. There is currently no effective vaccine for R. microplus tick infestations, despite the identification of numerous prospective tick vaccine candidates. As a result, the current research set out to develop an immunoinformatics-based strategy using existing methods for designing a multi-epitope based vaccination that is not only effective but also safe and capable of eliciting cellular and humoral immune responses. First, R. microplus proteins Bm86, Subolesin, and Bm95 were used to anticipate and link B and T-cell epitopes (HTL and CTL) to one another. Antigenicity testing, allergenicity assessment, and toxicity screening were just a few of the many immunoinformatics techniques used to identify potent epitopes. Multi-epitope vaccine design was chosen based on the antigenic score 0.935 that is promising vaccine candidate. Molecular docking was used to determine the nature of the interaction between TLR2 and the vaccine construct. Finally, molecular dynamic simulation was used to assess the stability and compactness of the resulting vaccination based on docking scores. The developed vaccine was shown to be stable, have immunogenic qualities, be soluble, and to have high expression by in silico cloning. These findings suggest that experimental investigation of the multi-epitope based vaccine designed in the current study will produce achievable vaccine candidates against R. microplus ticks, enabling more effective control of infestations.

A highly neutralizing human monoclonal antibody targeting a novel linear epitope on staphylococcal enterotoxin B.

Staphylococcal Enterotoxin B (SEB), produced by Staphylococcus aureus (S. aureus), is a powerful superantigen that induces severe immune disruption and toxic shock syndrome (TSS) upon binding to MHC-II and TCR. Despite its significant impact on the pathogenesis of S. aureus, there are currently no specific therapeutic interventions available to counteract the mechanism of action exerted by this toxin. In this study, we have identified a human monoclonal antibody, named Hm0487, that specifically targets SEB by single-cell sequencing using PBMCs isolated from volunteers enrolled in a phase I clinical trial of the five-antigen S. aureus vaccine. X-ray crystallography studies revealed that Hm0487 exhibits high affinity for a linear B cell epitope in SEB (SEB138-147), which is located distantly from the site involved in the formation of the MHC-SEB-TCR ternary complex. Furthermore, in vitro studies demonstrated that Hm0487 significantly impacts the interaction of SEB with both receptors and the binding to immune cells, probably due to an allosteric effect on SEB rather than competing with receptors for binding sites. Moreover, both in vitro and in vivo studies validated that Hm0487 displayed efficient neutralizing efficacy in models of lethal shock and sepsis induced by either SEB or bacterial challenge. Our findings unveil an alternative mechanism for neutralizing the pathogenesis of SEB by Hm0487, and this antibody provides a novel strategy for mitigating both SEB-induced toxicity and S. aureus infection.

In Silico Tools for Predicting Novel Epitopes.

Identifying antigens within a pathogen is a critical task to develop effective vaccines and diagnostic methods, as well as understanding the evolution and adaptation to host immune responses. Historically, antigenicity was studied with experiments that evaluate the immune response against selected fragments of pathogens. Using this approach, the scientific community has gathered abundant information regarding which pathogenic fragments are immunogenic. The systematic collection of this data has enabled unraveling many of the fundamental rules underlying the properties defining epitopes and immunogenicity, and has resulted in the creation of a large panel of immunologically relevant predictive (in silico) tools. The development and application of such tools have proven to accelerate the identification of novel epitopes within biomedical applications reducing experimental costs. This chapter introduces some basic concepts about MHC presentation, T cell and B cell epitopes, the experimental efforts to determine those, and focuses on state-of-the-art methods for epitope prediction, highlighting their strengths and limitations, and catering instructions for their rational use.

Systems approach to design multi-epitopic peptide vaccine candidate against fowl adenovirus structural proteins for Gallus gallus domesticus.

Introduction: Fowl adenovirus (FAdV) is a significant pathogen in poultry, causing various diseases such as hepatitis-hydropericardium, inclusion body hepatitis, and gizzard erosion. Different serotypes of FAdV are associated with specific conditions, highlighting the need for targeted prevention strategies. Given the rising prevalence of FAdV-related diseases globally, effective vaccination and biosecurity measures are crucial. In this study, we explore the potential of structural proteins to design a multi-epitope vaccine targeting FAdV. Methods: We employed an in silico approach to design the multi-epitope vaccine. Essential viral structural proteins, including hexon, penton, and fiber protein, were selected as vaccine targets. T-cell and B-cell epitopes binding to MHC-I and MHC-II molecules were predicted using computational methods. Molecular docking studies were conducted to validate the interaction of the multi-epitope vaccine candidate with chicken Toll-like receptors 2 and 5. Results: Our in silico methodology successfully identified potential T-cell and B-cell epitopes within the selected viral structural proteins. Molecular docking studies revealed strong interactions between the multi-epitope vaccine candidate and chicken Toll-like receptors 2 and 5, indicating the structural integrity and immunogenic potential of the designed vaccine. Discussion: The designed multi-epitope vaccine presents a promising approach for combating FAdV infections in chickens. By targeting essential viral structural proteins, the vaccine is expected to induce a robust immunological response. The in silico methodology utilized in this study provides a rapid and cost-effective means of vaccine design, offering insights into potential vaccine candidates before experimental validation. Future studies should focus on in vitro and in vivo evaluations to further assess the efficacy and safety of the proposed vaccine.

Formulation of next-generation polyvalent vaccine candidates against three important poxviruses by targeting DNA-dependent RNA polymerase using an integrated immunoinformatics and molecular modeling approach.

BACKGROUND: Poxviruses comprise a group of large double-stranded DNA viruses and are known to cause diseases in humans, livestock animals, and other animal species. The Mpox virus (MPXV; formerly Monkeypox), variola virus (VARV), and volepox virus (VPXV) are among the prevalent poxviruses of the Orthopoxviridae genera. The ongoing Mpox infectious disease pandemic caused by the Mpox virus has had a major impact on public health across the globe. To date, only limited repurposed antivirals and vaccines are available for the effective treatment of Mpox and other poxviruses that cause contagious diseases. METHODS: The present study was conducted with the primary goal of formulating multi-epitope vaccines against three evolutionary closed poxviruses i.e., MPXV, VARV, and VPXV using an integrated immunoinformatics and molecular modeling approach. DNA-dependent RNA polymerase (DdRp), a potential vaccine target of poxviruses, has been used to determine immunodominant B and T-cell epitopes followed by interactions analysis with Toll-like receptor 2 at the atomic level. RESULTS: Three multi-epitope vaccine constructs, namely DdRp_MPXV (V1), DdRp_VARV (V2), and DdRp_VPXV (V3) were designed. These vaccine constructs were found to be antigenic, non-allergenic, non-toxic, and soluble with desired physicochemical properties. Protein-protein docking and interaction profiling analysis depicts a strong binding pattern between the targeted immune receptor TLR2 and the structural models of the designed vaccine constructs, and manifested a number of biochemical bonds (hydrogen bonds, salt bridges, and non-bonded contacts). State-of-the-art all-atoms molecular dynamics simulations revealed highly stable interactions of vaccine constructs with TLR2 at the atomic level throughout the simulations on 300 nanoseconds. Additionally, the outcome of the immune simulation analysis suggested that designed vaccines have the potential to induce protective immunity against targeted poxviruses. CONCLUSIONS: Taken together, formulated next-generation polyvalent vaccines were found to have good efficacy against closely related poxviruses (MPXV, VARV, and VPXV) as demonstrated by our extensive immunoinformatics and molecular modeling evaluations; however, further experimental investigations are still needed.

Computational design of novel chimeric multiepitope vaccine against bacterial and viral disease in tilapia (Oreochromis sp.).

Regarding several infectious diseases in fish, multiple vaccinations are not favorable. The chimeric multiepitope vaccine (CMEV) harboring several antigens for multi-disease prevention would enhance vaccine efficiency in terms of multiple disease prevention. Herein, the immunogens of tilapia's seven pathogens including E. tarda, F. columnare, F. noatunensis, S. iniae, S. agalactiae, A. hydrophila, and TiLV were used for CMEV design. After shuffling and annotating the B-cell epitopes, 5,040 CMEV primary protein structures were obtained. Secondary and tertiary protein structures were predicted by AlphaFold2 creating 25,200 CMEV. Proper amino acid alignment in the secondary structures was achieved by the Ramachandran plot. In silico determination of physiochemical and other properties including allergenicity, antigenicity, glycosylation, and conformational B-cell epitopes were determined. The selected CMEV (OSLM0467, OSLM2629, and OSLM4294) showed a predicted molecular weight (MW) of 70 kDa, with feasible sites of N- and O-glycosylation, and a number of potentially conformational B-cell epitope residues. Molecular docking, codon optimization, and in-silico cloning were tested to evaluate the possibility of protein expression. Those CMEVs will further elucidate in vitro and in vivo to evaluate the efficacy and specific immune response. This research will highlight the new era of vaccines designed based on in silico structural vaccine design.

Immunoinformatics approach for designing a multiepitope subunit vaccine against porcine epidemic diarrhea virus genotype IIA spike protein.

Background: Porcine epidemic diarrhea (PED), caused by the porcine epidemic diarrhea virus (PEDV), is associated with high mortality and morbidity rates, especially in neonatal pigs. This has resulted in significant economic losses for the pig industry. PEDV genotype II-based vaccines were found to confer better immunity against both heterologous and homologous challenges; specifically, spike (S) proteins, which are known to play a significant role during infection, are ideal for vaccine development. Aim: This study aims to design a multi-epitope subunit vaccine targeting the S protein of the PEDV GIIa strain using an immunoinformatics approach. Methods: Various bioinformatics tools were used to predict HTL, CTL, and B-cell epitopes. The epitopes were connected using appropriate linkers and conjugated with the CTB adjuvant and M-ligand. The final multiepitope vaccine construct (fMEVc) was then docked to toll-like receptor 4 (TLR4). The stability of the fMEVc-TLR4 complex was then simulated using GROMACS. C-immsim was then used to predict the in vitro immune response of the fMEVc. Results: Six epitopes were predicted to induce antibody production, ten epitopes were predicted to induce CTL responses, and four epitopes were predicted to induce HTL responses. The assembled epitopes conjugated with the CTB adjuvant and M-ligand, fMEVc, is antigenic, non-allergenic, stable, and soluble. The construct showed a favorable binding affinity for TLR4, and the protein complex was shown to be stable through molecular dynamics simulations. A robust immune response was induced after immunization, as demonstrated through immune stimulation. Conclusion: In conclusion, the multi-epitope subunit vaccine construct for PEDV designed in this study exhibits promising antigenicity, stability, and immunogenicity, eliciting robust immune responses and suggesting its potential as a candidate for further vaccine development.

Imprinting of serum neutralizing antibodies by Wuhan-1 mRNA vaccines.

Immune imprinting is a phenomenon in which prior antigenic experiences influence responses to subsequent infection or vaccination1,2. The effects of immune imprinting on serum antibody responses after boosting with variant-matched SARS-CoV-2 vaccines remain uncertain. Here we characterized the serum antibody responses after mRNA vaccine boosting of mice and human clinical trial participants. In mice, a single dose of a preclinical version of mRNA-1273 vaccine encoding Wuhan-1 spike protein minimally imprinted serum responses elicited by Omicron boosters, enabling generation of type-specific antibodies. However, imprinting was observed in mice receiving an Omicron booster after two priming doses of mRNA-1273, an effect that was mitigated by a second booster dose of Omicron vaccine. In both SARS-CoV-2-infected and uninfected humans who received two Omicron-matched boosters after two or more doses of the prototype mRNA-1273 vaccine, spike-binding and neutralizing serum antibodies cross-reacted with Omicron variants as well as more distantly related sarbecoviruses. Because serum neutralizing responses against Omicron strains and other sarbecoviruses were abrogated after pre-clearing with Wuhan-1 spike protein, antibodies induced by XBB.1.5 boosting in humans focus on conserved epitopes targeted by the antecedent mRNA-1273 primary series. Thus, the antibody response to Omicron-based boosters in humans is imprinted by immunizations with historical mRNA-1273 vaccines, but this outcome may be beneficial as it drives expansion of cross-neutralizing antibodies that inhibit infection of emerging SARS-CoV-2 variants and distantly related sarbecoviruses.

A Structural In Silico Analysis of the Immunogenicity of L-Asparaginase from Penicillium cerradense.

L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with L-asparaginase have been observed, mainly caused by immunogenicity and allergenicity. Some strategies have been adopted, such as searching for new microorganisms that produce the enzyme and applying protein engineering. Therefore, this work aimed to elucidate the molecular structure and predict the immunogenic profile of L-asparaginase from Penicillium cerradense, recently revealed as a new fungus of the genus Penicillium and producer of the enzyme, as a motivation to search for alternatives to bacterial L-asparaginase. In the evolutionary relationship, L-asparaginase from P. cerradense closely matches Aspergillus species. Using in silico tools, we characterized the enzyme as a protein fragment of 378 amino acids (39 kDa), including a signal peptide containing 17 amino acids, and the isoelectric point at 5.13. The oligomeric state was predicted to be a homotetramer. Also, this L-asparaginase presented a similar immunogenicity response (T- and B-cell epitopes) compared to Escherichia coli and Dickeya chrysanthemi enzymes. These results suggest a potentially useful L-asparaginase, with insights that can drive strategies to improve enzyme production.

SEMA 2.0: web-platform for B-cell conformational epitopes prediction using artificial intelligence.

Prediction of conformational B-cell epitopes is a crucial task in vaccine design and development. In this work, we have developed SEMA 2.0, a user-friendly web platform that enables the research community to tackle the B-cell epitopes prediction problem using state-of-the-art protein language models. SEMA 2.0 offers comprehensive research tools for sequence- and structure-based conformational B-cell epitopes prediction, accurate identification of N-glycosylation sites, and a distinctive module for comparing the structures of antigen B-cell epitopes enhancing our ability to analyze and understand its immunogenic properties. SEMA 2.0 website https://sema.airi.net is free and open to all users and there is no login requirement. Source code is available at https://github.com/AIRI-Institute/SEMAi.

Design of multi-epitope chimeric vaccine against Monkeypox virus and SARS-CoV-2: A vaccinomics perspective.

The current era we experience is full with pandemic infectious agents that no longer threatens the major local source but the whole globe. Almost the most emerging infectious agents are severe acute respiratory syndrome coronavirus-2 (SARS CoV-2), followed by monkeypox virus (MPXV). Since no approved antiviral drugs nor licensed active vaccines are yet available, we aimed to utilize immunoinformatics approach to design chimeric vaccine against the two mentioned viruses. This is the first study to deal with design divalent vaccine against SARS-CoV-2 and MPXV. ORF8, E and M proteins from Omicron SARS-CoV-2 and gp182 from MPXV were used as the protein precursor from which multi-epitopes (inducing B-cell, helper T cells, cytotoxic T cells and interferon-É£) chimeric vaccine was contrived. The structure of the vaccine construct was predicted, validated, and docked to toll-like receptor-2 (TLR-2). Moreover, its sequence was also used to examine the immune simulation profile and was then inserted into the pET-28a plasmid for in silico cloning. The vaccine construct was probable antigen (0.543) and safe (non-allergen) with strong binding energy to TLR-2 (-1169.8 kcal/mol) and found to have significant immune simulation profile. In conclusion, the designed chimeric vaccine was potent and safe against SARS-CoV-2 and MPXV, which deserves further consideration.

Bioinformatics approach for structure modeling, vaccine design, and molecular docking of Brucella candidate proteins BvrR, OMP25, and OMP31.

Brucellosis is a zoonotic disease with significant economic and healthcare costs. Despite the eradication efforts, the disease persists. Vaccines prevent disease in animals while antibiotics cure humans with limitations. This study aims to design vaccines and drugs for brucellosis in animals and humans, using protein modeling, epitope prediction, and molecular docking of the target proteins (BvrR, OMP25, and OMP31). Tertiary structure models of three target proteins were constructed and assessed using RMSD, TM-score, C-score, Z-score, and ERRAT. The best models selected from AlphaFold and I-TASSER due to their superior performance according to CASP 12 - CASP 15 were chosen for further analysis. The motif analysis of best models using MotifFinder revealed two, five, and five protein binding motifs, however, the Motif Scan identified seven, six, and eight Post-Translational Modification sites (PTMs) in the BvrR, OMP25, and OMP31 proteins, respectively. Dominant B cell epitopes were predicted at (44-63, 85-93, 126-137, 193-205, and 208-237), (26-46, 52-71, 98-114, 142-155, and 183-200), and (29-45, 58-82, 119-142, 177-198, and 222-251) for the three target proteins. Additionally, cytotoxic T lymphocyte epitopes were detected at (173-181, 189-197, and 202-210), (61-69, 91-99, 159-167, and 181-189), and (3-11, 24-32, 167-175, and 216-224), while T helper lymphocyte epitopes were displayed at (39-53, 57-65, 150-158, 163-171), (79-87, 95-108, 115-123, 128-142, and 189-197), and (39-47, 109-123, 216-224, and 245-253), for the respective target protein. Furthermore, structure-based virtual screening of the ZINC and DrugBank databases using the docking MOE program was followed by ADMET analysis. The best five compounds of the ZINC database revealed docking scores ranged from (- 16.8744 to - 15.1922), (- 16.0424 to - 14.1645), and (- 14.7566 to - 13.3222) for the BvrR, OMP25, and OMP31, respectively. These compounds had good ADMET parameters and no cytotoxicity, while DrugBank compounds didn't meet Lipinski's rule criteria. Therefore, the five selected compounds from the ZINC20 databases may fulfill the pharmacokinetics and could be considered lead molecules for potentially inhibiting Brucella's proteins.

A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle.

Introduction: Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Methods and results: Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. Discussion and conclusion: The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against B. bovis.