Enhanced Assessment of Cross-Reactive Antigenic Determinants within the Spike Protein.

Despite successful vaccination efforts, the emergence of new SARS-CoV-2 variants poses ongoing challenges to control COVID-19. Understanding humoral responses regarding SARS-CoV-2 infections and their impact is crucial for developing future vaccines that are effective worldwide. Here, we identified 41 immunodominant linear B-cell epitopes in its spike glycoprotein with an SPOT synthesis peptide array probed with a pool of serum from hospitalized COVID-19 patients. The bioinformatics showed a restricted set of epitopes unique to SARS-CoV-2 compared to other coronavirus family members. Potential crosstalk was also detected with Dengue virus (DENV), which was confirmed by screening individuals infected with DENV before the COVID-19 pandemic in a commercial ELISA for anti-SARS-CoV-2 antibodies. A high-resolution evaluation of antibody reactivity against peptides representing epitopes in the spike protein identified ten sequences in the NTD, RBD, and S2 domains. Functionally, antibody-dependent enhancement (ADE) in SARS-CoV-2 infections of monocytes was observed in vitro with pre-pandemic Dengue-positive sera. A significant increase in viral load was measured compared to that of the controls, with no detectable neutralization or considerable cell death, suggesting its role in viral entry. Cross-reactivity against peptides from spike proteins was observed for the pre-pandemic sera. This study highlights the importance of identifying specific epitopes generated during the humoral response to a pathogenic infection to understand the potential interplay of previous and future infections on diseases and their impact on vaccinations and immunodiagnostics.

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.

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.

B-Cell Epitope Prediction for Antipeptide Paratopes with the HAPTIC2/HEPTAD User Toolkit (HUT).

B-cell epitope prediction is key to developing peptide-based vaccines and immunodiagnostics along with antibodies for prophylactic, therapeutic and/or diagnostic use. This entails estimating paratope binding affinity for variable-length peptidic sequences subject to constraints on both paratope accessibility and antigen conformational flexibility, as described herein for the HAPTIC2/HEPTAD User Toolkit (HUT). HUT comprises the Heuristic Affinity Prediction Tool for Immune Complexes 2 (HAPTIC2), the HAPTIC2-like Epitope Prediction Tool for Antigen with Disulfide (HEPTAD) and the HAPTIC2/HEPTAD Input Preprocessor (HIP). HIP enables tagging of residues (e.g., in hydrophobic blobs, ordered regions and glycosylation motifs) for exclusion from downstream analyses by HAPTIC2 and HEPTAD. HAPTIC2 estimates paratope binding affinity for disulfide-free disordered peptidic antigens (by analogy between flexible-ligand docking and protein folding), from terms attributed to compaction (in view of sequence length, charge and temperature-dependent polyproline-II helical propensity), collapse (disfavored by residue bulkiness) and contact (with glycine and proline regarded as polar residues that hydrogen bond with paratopes). HEPTAD analyzes antigen sequences that each contain two cysteine residues for which the impact of disulfide pairing is estimated as a correction to the free-energy penalty of compaction. All of HUT is freely accessible online ( https://freeshell.de/~badong/hut.htm ).

Identification and Validation of Peptides Specifying SARS-CoV-2 B-Cell Epitopes Eliciting Neutralizing Antibodies.

Vaccination is an effective means of inducing immune protection to prevent transmissible diseases. During the Covid-19 pandemic, immunizations using traditional and novel vaccine platforms such as the inactivated SARSCo-V-2 vaccine, adenoviral-vectored, and nucleic acid-based mRNA vaccines have been relatively successful in controlling the rates of infection and hospitalizations. Nevertheless, the danger posed by the emergence of SARS-CoV-2 variants would set the stage for the design of next generation vaccines. To overcome the lack of efficacy of current vaccines against emerging SARS-CoV-2 variants, new vaccines must be able to overcome the reduced effectiveness of the current vaccines. Since the current Covid-19 vaccines are dependent on the whole S-protein of Wuhan strain as the antigen, mutations have rendered the current Covid-19 vaccines less effective against variants of concern (VoCs). Instead of using the whole S-protein, peptide-based epitopes could be predicted using immunoinformatic approaches, simulation of the 3D structures, overlapping peptides covering the whole length of the S-protein or peptide arrays based on synthetic peptide combinatorial libraries comprising peptides recognizable by monoclonal antibodies. B-cell epitopes were predicted, and immunogenicity of peptides was validated in mice by immunizing mice with peptides conjugated to keyhole limpet hemocyanin (KLH) mixed with Montanide 51 as an adjuvant. The immunogenicity of epitopes that could elicit peptide specific IgGs was determined by peptide-based ELISA. Neutralizing activities were determined by cPass and pseudovirus-based neutralization assays.

Newly developed peptide-ELISA successfully detected anti-IgG antibodies against Maedi-Visna virus in sheep.

Maedi Visna Virus (MVV) is a retrovirus that can infect sheep. There is still no effective therapy or vaccine against this virus and timely diagnosis is important to combat the complications of the disease. In this study, we aimed to develop an ELISA using peptides derived from gag protein as antigen. For this purpose, B cell epitopes of gag protein were predicted and a docking analysis with the B cell receptor was performed to select peptides to be used in ELISA. After three soluble epitopes with the highest antigenicity were produced as peptides, the immunogenicity of each peptide was determined by ELISA using sheep serum samples categorized as MVV positive (n=24) and negative (n=13). Subsequently, in house ELISA using above mentioned immunogenic peptides as antigen was used to investigate MVV seroprevalence in sheep (n=88). According to the results, among three peptides, two of them strongly reacted with MVV positive serum samples and the mean absorbance values detected among positive and negative serum samples were statistically significant, indicating that these peptides were immunogenic (P=0.016 and P=0.038). The third peptide also reacted with positive serum samples but the mean absorbance value was not statistically significant and this peptide was considered non-immunogenic (P=0.175). The immunogenic two peptides showed the same high sensitivity and specificity values of 91.60 and 92.80 according to the commercial kit. Moreover, MVV seroprevalence detected by peptide-ELISAs using CKQGSKE and CRPQGKAGHKG peptides as antigen was 3.40 % and 4.5 %, respectively. As a result, it was shown that these peptides can be successfully used for serological diagnosis of MVV.

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 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.

Putative new combination vaccine candidates identified by reverse vaccinology and genomic approaches to control enteric pathogens.

OBJECTIVES: The pathogenic microorganisms that cause intestinal diseases can significantly jeopardize people's health. Currently, there are no authorized treatments or vaccinations available to combat the germs responsible for intestinal disease. METHODS: Using immunoinformatics, we developed a potent multi-epitope Combination (combo) vaccine versus Salmonella and enterohemorrhagic E. coli. The B and T cell epitopes were identified by performing a conservancy assessment, population coverage analysis, physicochemical attributes assessment, and secondary and tertiary structure assessment of the chosen antigenic polypeptide. The selection process for vaccine development included using several bioinformatics tools and approaches to finally choose two linear B-cell epitopes, five CTL epitopes, and two HTL epitopes. RESULTS: The vaccine had strong immunogenicity, cytokine production, immunological properties, non-toxicity, non-allergenicity, stability, and potential efficacy against infections. Disulfide bonding, codon modification, and computational cloning were also used to enhance the stability and efficacy of expression in the host E. coli. The vaccine's structure has a strong affinity for the TLR4 ligand and is very durable, as shown by molecular docking and molecular modeling. The results of the immunological simulation demonstrated that both B and T cells had a heightened response to the vaccination component. CONCLUSIONS: The comprehensive in silico analysis reveals that the proposed vaccine will likely elicit a robust immune response against pathogenic bacteria that cause intestinal diseases. Therefore, it is a promising option for further experimental testing.

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.

Reverse vaccinology-based multi-epitope vaccine design against Indian group A rotavirus targeting VP7, VP4, and VP6 proteins.

Rotavirus, a primary contributor to severe cases of infantile gastroenteritis on a global scale, results in significant morbidity and mortality in the under-five population, particularly in middle to low-income countries, including India. WHO-approved live-attenuated vaccines are linked to a heightened susceptibility to intussusception and exhibit low efficacy, primarily attributed to the high genetic diversity of rotavirus, varying over time and across different geographic regions. Herein, molecular data on Indian rotavirus A (RVA) has been reviewed through phylogenetic analysis, revealing G1P[8] to be the prevalent strain of RVA in India. The conserved capsid protein sequences of VP7, VP4 and VP6 were used to examine helper T lymphocyte, cytotoxic T lymphocyte and linear B-cell epitopes. Twenty epitopes were identified after evaluation of factors such as antigenicity, non-allergenicity, non-toxicity, and stability. These epitopes were then interconnected using suitable linkers and an N-terminal beta defensin adjuvant. The in silico designed vaccine exhibited structural stability and interactions with integrins (αvß3 and αIIbß3) and toll-like receptors (TLR2 and TLR4) indicated by docking and normal mode analyses. The immune simulation profile of the designed RVA multiepitope vaccine exhibited its potential to trigger humoral as well as cell-mediated immunity, indicating that it is a promising immunogen. These computational findings indicate potential efficacy of the designed vaccine against rotavirus infection.

Analysis of Virus-Specific B Cell Epitopes Reveals Extensive Antigen Degradation Prior to Recognition.

B cell epitopes must be visible for recognition by cognate B cells and/or antibodies. Here, we studied that premise for known linear B cell epitopes that were collected from the Immune Epitope Database as being recognized by humans during microbial infections. We found that the majority of such known B cell epitopes are virus-specific linear B cell epitopes (87.96%), and most are located in antigens that remain enclosed in host cells and/or virus particles, preventing antibody recognition (18,832 out of 29,225 epitopes). Moreover, we estimated that only a minority (32.72%) of the virus-specific linear B cell epitopes that are found in exposed viral regions (e.g., the ectodomains of envelope proteins) are solvent accessible on intact antigens. Hence, we conclude that ample degradation/processing of viral particles and/or infected cells must occur prior to B cell recognition, thus shaping the B cell epitope repertoire.

Development of a subunit vaccine against the cholangiocarcinoma causing Opisthorchis viverrini: a computational approach.

Opisthorchis viverrini is the etiological agent of the disease opisthorchiasis and related cholangiocarcinoma (CCA). It infects fish-eating mammals and more than 10 million people in Southeast Asia suffered from opisthorchiasis with a high fatality rate. The only effective drug against this parasite is Praziquantel, which has significant side effects. Due to the lack of appropriate treatment options and the high death rate, there is a dire need to develop novel therapies against this pathogen. In this study, we designed a multi-epitope chimeric vaccine design against O. viverrini by using immunoinformatics approaches. Non-allergenic and immunogenic MHC-1, MHC-2, and B cell epitopes of three candidate proteins thioredoxin peroxidase (Ov-TPx-1), cathepsin F1 (Ov-CF-1) and calreticulin (Ov-CALR) of O. viverrini, were predicted to construct a potent multiepitope vaccine. The coverage of the HLA-alleles of these selected epitopes was determined globally. Four vaccine constructs made by different adjuvants and linkers were evaluated in the context of their physicochemical properties, antigenicity, and allergenicity. Protein-protein docking and MD simulation found that vaccines 3 was more stable and had a higher binding affinity for TLR2 and TLR4 immune receptors. In-silico restriction cloning of vaccine model led to the formation of plasmid constructs for expression in a suitable host. Finally, the immune simulation showed strong immunological reactions to the engineered vaccine. These findings suggest that the final vaccine construct has the potential to be validated by in vivo and in vitro experiments to confirm its efficacy against the CCA causing O. viverrini.

A novel vaccine construct against Zika virus fever: insights from epitope-based vaccine discovery through molecular modeling and immunoinformatics approaches.

The Zika virus (ZIKV) is an emerging virus associated with the Flaviviridae family that mainly causes infection in pregnant women and leads to several abnormalities during pregnancy. This virus has unique properties that may lead to pathological diseases. As the virus has the ability to evade immune response, a crucial effort is required to deal with ZIKV. Vaccines are a safe means to control different pathogenic infectious diseases. In the current research, a multi-epitope-based vaccination against ZIKV is being designed using in silico methods. For the epitope prediction and prioritization phase, ZIKV polyprotein (YP_002790881.1) and flavivirus polyprotein (>YP_009428568.1) were targeted. The predicted B-cell epitopes were used for MHC-I and MHC-II epitope prediction. Afterward, several immunoinformatics filters were applied and nine (REDLWCGSL, MQDLWLLRR, YKKSGITEV, TYTDRRWCF, RDAFPDSNS, KPSLGLINR, ELIGRARVS, AITQGKREE, and EARRSRRAV) epitopes were found to be probably antigenic in nature, non-allergenic, non-toxic, and water soluble without any toxins. Selected epitopes were joined using a particular GPGPG linker to create the base vaccination for epitopes, and an extra EAAAK linker was used to link the adjuvant. A total of 312 amino acids with a molecular weight (MW) of 31.62762 and an instability value of 34.06 were computed in the physicochemical characteristic analysis, indicating that the vaccine design is stable. The molecular docking analysis predicted a binding energy of -329.46 (kcal/mol) for TLR-3 and -358.54 (kcal/mol) for TLR-2. Moreover, the molecular dynamics simulation analysis predicted that the vaccine and receptor molecules have stable binding interactions in a dynamic environment. The C-immune simulation analysis predicted that the vaccine has the ability to generate both humoral and cellular immune responses. Based on the design, the vaccine construct has the best efficacy to evoke immune response in theory, but experimental analysis is required to validate the in silico base approach and ensure its safety.

In vitro identification of neutralizing epitopes of Rhipicephalus microplus serpin 17 (RmS-17).

BACKGROUND: Rhipicephalus microplus poses a significant problem for livestock worldwide and is primarily controlled with synthetic acaricides. The continuous use of acaricides results in the selection of resistance and causes environmental harm. Vaccination presents an alternative solution to this problem, although searching for the suitable antigen is still a work in progress. Salivary proteins hold promise for inclusion in vaccine formulation due to their roles in modulating host responses, assisting blood feeding and pathogen transmission. Serpins are a class of proteinase inhibitors and are among the molecules found in tick saliva that modulate host blood coagulation, inflammation, and adaptive immune responses. Previous studies have demonstrated the potential of R. microplus serpin 17 (RmS-17) to interfere with the host's defenses, and antibodies have been shown to neutralize its effects. This makes RmS-17 an putative target for vaccine development. METHODS: Epitope mapping of RmS-17 was achieved using in silico approach combining linear B-cell epitope and antigenicity predictor. In addition, epitope mapping using overlapping peptides in an ELISA screening was used. The serpin tridimensional structure and the epitopes spatial location within the molecule were determined. Peptides were synthetized based on the predictions and used for the production of rabbit anti-sera. Purified IgG's were used to assess the antibodies capacity to neutralize RmS-17. RESULTS: Through in silico mapping, nine potential B cell epitope regions were screened, with p1RmS-17 and p2RmS-17 selected for the experiment based on antigen prediction. In the ELISA screening using overlapping peptides, eight antibody-binding regions were identified, and p3RmS-17 and p4RmS-17 were chosen. Antibodies raised against p3RmS-17 and p4RmS-17 partially neutralized RmS-17 activity. CONCLUSION: It was found that antibodies against a single epitope are sufficient to partially neutralize RmS-17 activity. These findings support the possibility of using an epitope-based vaccine for immunization against R. microplus.

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.

Predicting immune response targets in orthoflaviviruses through sequence homology and computational analysis.

CONTEXT: Flaviviruses cause severe encephalitic or hemorrhagic diseases in humans. Its members, Kyasanur forest disease virus (KFDV) and Alkhumra hemorrhagic fever virus (ALKV), cause hemorrhagic fever and are prevalent in India and Saudi Arabia, respectively, while the tick-borne encephalitis virus (TBEV) causes a dangerous encephalitic infection in Europe and Asia. However, little information is available about the targets of immune responses for these deadly viruses. Here, we predict potential antigenic peptide epitopes of viral envelope protein for inducing a cell-mediated and humoral immune response. METHODS: Using the Immune Epitope Database and Analysis Resource (IEDB-AR), we identified 13 MHC-I and two MHC-II dominant conserved epitopes in KFDV and ALKV and six MHC-I and three MHC-II epitopes in TBEV envelope proteins. Parallelly, we also predicted B-cell linear and discontinuous envelope protein epitopes for these viruses. Interestingly, the epitopes are conserved in all three viral envelope proteins. Further, the discontinuous epitopes are structurally compared with the available DENV, ZIKV, WNV, TBEV, and LIV envelope protein antibody structures. Overall structural comparison analyses highlight (i) lateral ridge epitope in the ED-III domain of E protein, and (ii) envelope dimer epitope (EDE) could be targeted for developing potent vaccine candidates as well as therapeutic antibody production. Moreover, existing structural and biochemical functions of the same epitopes in homologous viruses are predicted to have a reduced antibody-dependent enhancement (ADE) effect on flaviviral infection.

Immunoinformatics and structural aided approach to develop multi-epitope based subunit vaccine against Mycobacterium tuberculosis.

Tuberculosis is a highly contagious disease caused by Mycobacterium tuberculosis (Mtb), which is one of the prominent reasons for the death of millions worldwide. The bacterium has a substantially higher mortality rate than other bacterial diseases, and the rapid rise of drug-resistant strains only makes the situation more concerning. Currently, the only licensed vaccine BCG (Bacillus Calmette-Guérin) is ineffective in preventing adult pulmonary tuberculosis prophylaxis and latent tuberculosis re-activation. Therefore, there is a pressing need to find novel and safe vaccines that provide robust immune defense and have various applications. Vaccines that combine epitopes from multiple candidate proteins have been shown to boost immunity against Mtb infection. This study applies an immunoinformatic strategy to generate an adequate multi-epitope immunization against Mtb employing five antigenic proteins. Potential B-cell, cytotoxic T lymphocyte, and helper T lymphocyte epitopes were speculated from the intended proteins and coupled with 50 s ribosomal L7/L12 adjuvant, and the vaccine was constructed. The vaccine's physicochemical profile demonstrates antigenic, soluble, and non-allergic. In the meantime, docking, molecular dynamics simulations, and essential dynamics analysis revealed that the multi-epitope vaccine structure interacted strongly with Toll-like receptors (TLR2 and TLR3). MM-PBSA analysis was performed to ascertain the system's intermolecular binding free energies accurately. The immune simulation was applied to the vaccine to forecast its immunogenic profile. Finally, in silico cloning was used to validate the vaccine's efficacy. The immunoinformatics analysis suggests the multi-epitope vaccine could induce specific immune responses, making it a potential candidate against Mtb. However, validation through the in-vivo study of the developed vaccine is essential to assess its efficacy and immunogenicity profile, which will assure active protection against Mtb.

Exploring the nuclear proteins, viral capsid protein, and early antigen protein using immunoinformatic and molecular modeling approaches to design a vaccine candidate against Epstein Barr virus.

The available Epstein Barr virus vaccine has tirelessly harnessed the gp350 glycoprotein as its target epitope, but the result has not been preventive. Right here, we designed a global multi-epitope vaccine for EBV; with special attention to making sure all strains and preventive antigens are covered. Using a robust computational vaccine design approach, our proposed vaccine is armed with 6-16 mers linear B-cell epitopes, 4-9 mer CTL epitopes, and 8-15 mer HTL epitopes which are verified to induce interleukin 4, 10 & IFN-gamma. We employed deep computational mining coupled with expert intelligence in designing the vaccine, using human Beta defensin-3-which has been reported to induce the same TLRs as EBV-as the adjuvant. The tendency of the vaccine to cause autoimmune disorder is quenched by the assurance that the construct contains no EBNA-1 homolog. The protein vaccine construct exhibited excellent physicochemical attributes such as Aliphatic index 59.55 and GRAVY - 0.710; and a ProsaWeb Z score of - 3.04. Further computational analysis revealed the vaccine docked favorably with EBV indicted TLR 1, 2, 4 & 9 with satisfactory interaction patterns. With global coverage of 85.75% and the stable molecular dynamics result obtained for the best two interactions, we are optimistic that our nontoxic, non-allergenic multi-epitope vaccine will help to ameliorate the EBV-associated diseases-which include various malignancies, tumors, and cancers-preventively.