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Purpose: Guanarito mammarenavirus (GTOV) is a highly pathogenic virus that leads to Venezuelan hemorrhagic fever (VHF). Despite being a severe disease, there are currently no commercially available drugs or vaccines for its prevention. Methods: Here we computationally formulated a mRNA vaccine construct (VC) from the genome of GTOV to produce immunity against its infections. Two proteins, namely zinc-finger motif protein (NP_899220.1), and nucleocapsid protein (NP_899211.1) were screened as potential candidates for downstream analysis. Results: We determined the T and B cell epitopes of the candidate proteins. The resulting epitopes were analyzed, and the best epitopes were utilized in the formation of the peptide vaccine construct. The secondary and tertiary structures of the peptide construct were predicted and validated. Docking was conducted to check the binding energy of the designed peptide vaccine with the human immune receptors, namely TLR2 and TLR4. Our designed vaccine showed stable interactions with the HLA molecules, as verified through normal mode and MD simulation analysis. The immune simulation results indicated a positive immune response against the construct. A potentially stable mRNA vaccine was formulated by adding of sequences such as the Kozak, Goblin 5′ UTR, tPA-signal peptide, MITD, 3′ UTRs, and a poly(A) tail to the peptide vaccine construct. Lastly, the expression probability of the mRNA vaccine was confirmed in the expression system of E. coli strain K12. Conclusion: The designed vaccine showed the potential to elicit an immune response against the GTOV infection; however, experimental validation is recommended to verify the in-silico findings of this study. [ABSTRACT FROM AUTHOR]

Porcine deltacoronavirus (PDCoV) is a globally distributed swine enteropathogenic virus that emerged in the last decade. A recent report of PDCoV infection in Haitian children also highlights potential public health implications. In this study, two monoclonal antibodies (mAbs), 1C2 and 5H5, were generated and showed high specificity for the PDCoV S protein. Both mAbs displayed high-titer neutralizing capabilities, suggesting their potential for passive immunotherapy. Epitope mapping revealed that the mAbs likely recognized conformational epitopes in the S1 subunit domains A and B of the native S protein, thereby blocking the interaction between the S1 receptor-binding domain and the cellular receptor, which could inhibit viral entry into host cells. This study offers new biological tools for PDCoV detection and lays the groundwork for the future development of porcine-specific antibodies for the prevention and treatment of PDCoV in piglets. [ABSTRACT FROM AUTHOR]

Background: Epstein–Barr virus (EBV) is a significant global public health concern because of its association with various malignancies and autoimmune diseases. Over 90% of the global population is chronically infected with EBV, impacting numerous cancer-related cases annually. However, none of the effective prophylactic vaccines against EBV is approved at present. Methods: In this study, we developed a novel vaccine candidate based on epitope peptides from the receptor-binding domain of EBV-encoded gp350 glycoprotein to prevent EBV infection. These epitope peptides detected a binding capability with host cells were then fused by flexibility linkers and expressed in Escherichia coli to reduce the unnecessary glycan modifications to simulate their free-glycan status. The fused recombinant protein (L350) was displayed on the surface of ferritin-based nanoparticle. The immunogenicity of the L350–ferritin nanoparticle was evaluated in Balb/c mice, and the neutralizing titers of sera from immunized mice were detected by means of an infection blocking assay in an in vitro cell model. Results: All the five epitope peptides could bind to AKATA cells, and their fused recombinant protein (L350) was successfully presented on the surface of self-assembled ferritin nanoparticles. Sera from the L350–ferritin nanoparticle-immunized mice showed high titers of both L350 protein-specific and gp350D123 protein-specific antibodies, and sera from gp350D123 protein-immunized mice could also recognize L350 protein well. Most importantly, the L350–ferritin nanoparticle induced efficient neutralizing antibodies to block EBV-GFP infection in AKATA cells and also constructed a strong antigen-specific B-cell memory in immunized mice. Moreover, histopathological changes of main tissues from all vaccinated mice were not observed. Conclusion: These data indicate that the L350–ferritin nanoparticle vaccine candidate has considerable potential application in preventing EBV infection and provides a promising basis for developing prophylactic EBV vaccines. [ABSTRACT FROM AUTHOR]

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Simple Summary: This study performed a comprehensive meta-analysis of SARS-CoV-2 antibody and T cell epitopes. The goal was to identify candidate immunodominant antigen epitopes that are highly conserved and evolutionarily constrained. It found that immune responses were concentrated in certain regions of the virus proteins. Notably, B cell and CD4+ T cell responses were positively correlated with high viral variability, while CD8+ T cell responses showed a negative correlation. This study identified highly conserved and evolutionarily constrained SARS-CoV-2 epitopes, which could be crucial for broad-spectrum vaccine development. These findings provide important insights for future research and clinical applications. SARS-CoV-2 continues to be a major global health threat. In this study, we performed a comprehensive meta-analysis on the epitopes of SARS-CoV-2, revealing its immunological landscape. Furthermore, using Shannon entropy for sequence conservation analysis and structural network-based methods identified candidate epitopes that are highly conserved and evolutionarily constrained in SARS-CoV-2 and other zoonotic coronaviruses. Finally, the population coverage of T cell epitopes was analyzed. The results highlighted regions within each SARS-CoV-2 protein where the immunological activity of antibodies, CD4+, and CD8+ T cell responses was predominantly concentrated. Sequence-based correlation analysis found that epitopes recognized by B cells and CD4+ T cells showed a positive correlation with high viral variability, and these high variability regions were typically linked to robust immune responses. Conversely, epitopes recognized by CD8+ T cells exhibited a negative correlation with high variability. From a structural network degree perspective, no clear correlation was identified between B cell antibody epitopes and CD4+ T cell reactivity with the degree of residue network connectivity. However, a significant positive correlation was observed between CD8+ T cell reactivity and the degree of residue network connectivity. By integrating sequence Shannon entropy and structural network correlation analysis, we pinpointed highly conserved and evolutionarily constrained SARS-CoV-2 candidate epitopes. Furthermore, we utilized immunoinformatics to assess the conservation of SARS-CoV-2 within coronaviruses and the population coverage of these epitopes. Our analysis uncovered key immune responses linked to preventing viral infection and viral clearance, emphasized areas of interest for broad-spectrum SARS-CoV-2 vaccine development, and offered insights for future research and clinical applications. [ABSTRACT FROM AUTHOR]

Microvirin is a lectin molecule known to have monovalent interaction with glycoprotein gp120. A previously reported high-resolution structural analysis defines the mannobiose-binding cavity of Microvirin. Nonetheless, structure does not directly define the energetics of binding contributions of protein contact residues. To better understand the nature of the MVN-Env glycan interaction, we used mutagenesis to evaluate the residue contributions to the mannobiose binding site of MVN that are important for Env gp120 glycan binding. MVN binding site amino acid residues were individually replaced by alanine, and the resulting purified recombinant MVN variants were examined for gp120 interaction using competition Enzyme-Linked Immunosorbent Assay (ELISA), biosensor surface plasmon resonance, calorimetry, and virus neutralization assays. Our findings highlight the role of both uncharged polar and non-polar residues in forming a hydropathic recognition site for the monovalent glycan engagement of Microvirin, in marked contrast to the charged residues utilized in the two Cyanovirin-N (CVN) glycan-binding sites. [ABSTRACT FROM AUTHOR]

MHC B2 haplotype chickens have been reported to induce strong immune response against various avian pathogens. However, little is known about the CD8+T-cell epitope with MHC B2-restricted on subgroup J avian leukosis virus (ALV-J). In this study, we explored the ALV-J-induced cellular immune response in B2 haplotype chickens in vivo. We found that ALV-J infection significantly increased the proportion of CD8+T cells in chickens and up-regulated the expression of cytotoxic genes like Granzyme A and antiviral genes like IFIT5 at 14 days post-infection (dpi). We selected 32 candidate peptides based on the peptide-binding motif and further identified three MHC B2-restricted CD8+T epitopes on ALV-J, including Pol652−660, Gag374−382, and Gag403−411 which induced significant levels of chicken IFN-γ production in splenocytes from ALV-J infected chickens using the ELISpot assay. In addition, we also verified that the three identified epitopes stimulated memory splenocytes elevating TNF-α and IL-2 protein expression. Importantly, we found that the three positive peptides were highly conserved among ALV-A, ALV-B, ALV-E, ALV-J, and ALV-K. Taken together, we identified three MHC B2-restricted CD8+T cell epitopes on ALV-J, providing a foundation for developing effective T cell epitope vaccines targeting conserved internal viral proteins. [ABSTRACT FROM AUTHOR]

Bacteria from the genus Proteus are facultative human pathogens, primarily attacking the urinary tract and wounds. A total of 85 O serogroups have been identified so far among these bacilli. P. mirabilis Bprz 86 was isolated from the fistula of a patient in Łódź, Poland. Enzyme-Linked Immunosorbent Assay (ELISA) and Western blotting studies involving the P. mirabilis Bprz 86 lipopolysaccharide (LPS) and the strain-specific rabbit antiserum indicated that the strain, which does not belong to any of the O1–O85 serogroups, shares a common epitope with Proteus O17 antigens and is identical to another clinical P. mirabilis strain, Sm 120, isolated from the urine of a patient in the area. The O-specific polysaccharide (O antigen) was obtained from P. mirabilis Bprz 86 LPS through mild acid degradation, and the six-constituent structure of its repeating unit was determined using chemical analyses and 1D and 2D 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy. It includes (R)-3-hydroxybutanoyl, which, along with fucosamine and glucose residues, forms a fragment also present in the O17 antigens. Based on the obtained serological and chemical data, the two studied P. mirabilis isolates were proposed as candidates for a new successive O serogroup in the genus Proteus, O86. [ABSTRACT FROM AUTHOR]

Background: Bullous pemphigoid (BP) and mucous membrane pemphigoid (MMP) are rare autoimmune blistering disorders characterized by autoantibodies (autoAbs) targeting dermo-epidermal junction components such as BP180 and BP230. The differential diagnosis, based on both the time of appearance and the extension of cutaneous and/or mucosal lesions, is crucial to distinguish these diseases for improving therapy outcomes and delineating the correct prognosis; however, in some cases, it can be challenging. In addition, negative results obtained by commercially available enzyme-linked immunosorbent assays (ELISAs) with BP and MMP sera, especially from patients with ocular involvement, often delay diagnosis and treatment, leading to a greater risk of poor outcomes. Objectives: Our aim was to find potentially different reactivity profiles in BP and MMP and improve available approaches for diagnosis with focus on ocular MMP. Methods: Two cohorts of 90 BP and 90 MMP, recruited from different Italian clinical centers, were characterized also employing a novel ELISA based on the BP180 extracellular domain (ECD-BP180). Results: Immunoglobulin G (IgG) reactivity to BP180 and BP230 in MMP sera was significantly reduced in comparison with BP, mostly affecting BP230 and E-1080 (53% and 36% in BP vs. 11% and 3% in MMP, respectively, p < 0.0001). The combined sensitivity of BP180-NC16A and ECD-BP180 ELISAs was greater compared to BP180-NC16A and BP230 ELISAs both in BP (97% and 92%, respectively) and in MMP (42% and 31%, respectively). The present study shows that MMP patients with ocular involvement rarely reacted to BP180 by IgG in contrast with patients with oral and/or cutaneous involvement (p = 0.0245 and p = 0.0377, respectively), suggesting that an oral and/or cutaneous MMP positive to BP180 hardly evolves to ocular MMP. Of note, one-third of ocular MMP showed immunoglobulin A (IgA) reactivity to ECD-BP180 by immunoblotting. Conclusions: The present study provides several hints to perform a correct and timely diagnosis in BP and MMP, which is crucial for improving therapy outcomes and delineating the correct prognosis. [ABSTRACT FROM AUTHOR]

Peanuts are one of the most common allergenic foods in the world and can cause severe allergic reactions. Ara h 2 is the most allergenic components of peanut allergens. Although critical amino acids at the epitopes were identified, the effect of their physicochemical properties remained unclear. The critical amino acids in epitopes of Ara h 2 (epitope 1: 26ELQGDRRCQSQLER39 and epitope 2: 62RDPYSPSQDPYSPS75). The critical amino acids were substituted by amino acid with different physicochemical properties. Then, the potential allergenicity of those peptides was assessed by IgE binding and cell model. Changing the properties of amino acids by mutation affects potential sensitisation. The charge distribution and molecular weight of critical amino acids in the epitope affected the IgE binding capacity significantly. The conclusion based on peptide might be different from that on whole protein, and our study provided direction of amino acids mutation in desensitisation therapy. [ABSTRACT FROM AUTHOR]

Leptospirosis is a neglected zoonosis caused by a pathogenic spirochete, Leptospira interrogans. The mode of infection in humans is through an abrasion in human skin or the conjunctiva and mucous membrane. Infected patients usually show different symptoms resembling bacterial or viral infections such as the flu. Hence, diagnosing leptospirosis in the early stage is complex, and can be easily confused with other infections. A strategical pathway was developed to analyze the hypothetical proteins in L. interrogans and unveil their potential as diagnostic markers. Subcellular localization tools such as PSORTb, CELLO, SOSUI-GramN, and ProtCompB were used to segregate the outer membrane and surface proteins from the overall pool of hypothetical proteins. The shortlisted proteins were checked for their virulency, and antigenicity through tools such as VirulentPred, and VaxiJen, respectively. Proteins with the highest scores were fed into ElliPro which predicted both linear and discontinuous epitopes in each protein. Proteins with many epitopes were further analyzed with BepiPred 3.0, which provided the epitope probability for each protein's amino acid. Epitope probability of the potential proteins was compared with the standard diagnostic marker, LipL32. The comparison revealed that a protein (UniProt ID D4YW28) has better immunogenic potential than the gold standard marker, LipL32. In conclusion, this protein can be used as a diagnostic marker for the detection of leptospirosis and it will also serve as a better vaccine candidate. [ABSTRACT FROM AUTHOR]

Vaccines have historically played a pivotal role in controlling epidemics. Effective vaccines for viruses causing significant human disease, e.g. , Ebola, Lassa fever, or Crimean Congo hemorrhagic fever virus, would be invaluable to public health strategies and counter-measure development missions. Here, we propose coverage metrics to quantify vaccine-induced CD8+ T cell-mediated immune protection, as well as metrics to characterize immuno-dominant epitopes, in light of human genetic heterogeneity and viral evolution. Proof-of-principle of our approach and methods are demonstrated for Ebola virus, SARS-CoV-2, and Burkholderia pseudomallei (vaccine) proteins. [ABSTRACT FROM AUTHOR]

Simple Summary: African swine fever has widely spread in China in the last couple of years and posed a significant impact on pig industry. Currently, there is no safe and effective commercial vaccine available and the prevention and control of the disease mainly rely on the early detection and stamping out strategy. Therefore, it is important to develop a specific and easy for use method for diagnosis of this disease. In this study, we generated two specific monoclonal antibodies against the capsid protein p17 of African swine fever virus particles. Both monoclonal antibodies have been successfully applied in different immune assays including enzyme-linked immunosorbent assay, Western blotting assay and immunofluorescence assay. Moreover, both p17 monoclonal antibodies were found to recognize a new antigenic epitope of 72–78 amino acids of p17 protein, which it is identical across all genotype I and II strains of African swine fever virus. Based on this epitope peptide, an indirect ELISA has been established to effectively detect antibodies during virus infection, and it will significantly promote the serological diagnosis and contribute to the disease prevention and control. African swine fever (ASF) has widely spread around the world in the last 100 years since its discovery. The African swine fever virus (ASFV) particles are made of more than 150 proteins, with the p17 protein encoded by the D117L gene serving as one of the major capsid proteins and playing a crucial role in the virus's morphogenesis and immune evasion. Thus, monoclonal antibody (mAb) targeting p17 is important for the research and detection of ASFV infection. Here, we produced two specific mAbs against p17, designated as 1G2 and 6G3, respectively, and both have been successfully used in enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunofluorescence assay. Moreover, we found that both 1G2 and 6G3 mAbs recognize a novel epitope of 72–78 amino acids of p17 protein, highly conserved across all genotype I and II strains. Based on this epitope, an indirect ELISA has been established to effectively detect antibodies during ASFV infection, and it exhibits high consistency with commercial ASFV ELISA kits. In summary, the production of the specific p17 mAbs and the identification of the recognized epitope will significantly promote the serological diagnosis of ASFV. [ABSTRACT FROM AUTHOR]

Background: Echinococcosis is a zoonotic infectious disease that poses a significant threat to the health of individuals living in rural regions. While vaccination represents a potential strategy for disease prevention, there is currently no effective vaccine available for humans to prevent cystic echinococcosis (CE). This study aimed to design a novel multi-epitope vaccine (MEV) against Echinococcus granulosus for human use, employing immunoinformatics methods. Methods: We identified core epitopes from two key antigens, EgA31 and EgG1Y162, and integrated them into the immunoglobulin variable region of CTLA-4 (CTLA-4lgV) to create the CVE31-162 vaccine construct. The secondary and tertiary structures of the CVE31-162 were established using bioinformatics methods. The interaction between the CVE31-162 and B7 molecules was assessed through molecular dynamics simulations. Finally, both in vitro and in vivo experiments were conducted to validate the effectiveness of the CVE31-162 against the immunological effects of Echinococcus granulosus. Results: Bioinformatics analysis indicated that CVE31-162 exhibits favorable antigenicity, stability, and non-allergenicity. Furthermore, CVE31-162 demonstrated a stable three-dimensional structural model. Molecular docking (MD) and molecular dynamics simulations (MDS) revealed a strong binding affinity between CVE31-162 and B7 molecules. Immune simulation results suggested that the vaccine elicits robust humoral and cell-mediated immune responses. Both in vitro and in vivo experiments demonstrated that immunized mice exhibited significantly elevated levels of antigen-specific antibodies and enhanced lymphocyte proliferation compared to the control group. Conclusions: CVE31-162, which is based on the interaction between CTLA-4 and B7, represents a promising multi-epitope vaccine for Echinococcus granulosus. [ABSTRACT FROM AUTHOR]

Sarcoplasmic-calcium-binding protein (SCP) was isolated, purified and identified from Penaeus vannamei. Meanwhile, its physicochemical properties (immunoactivity, thermal stability, pH stability and digestive stability), secondary structures and epitopes were studied. The results showed that the purified protein, which was obtained by successive steps of crude protein extraction, ammonium sulfate fractionation and anion exchange chromatography and exhibited a molecular mass of 21.6 kDa, was identified as SCP with a peptide coverage of 93.26%. SCP had strong immunoactivity, which decreased during heat treatment (≥ 65 ℃) and under strongly acidic and alkaline conditions. SCP had strong stability against intestinal fluid digestion, but poor stability against gastric fluid digestion. The secondary structure of SCP consisted of 26% α-helix, 16.9% β-sheet, 17.5% β-turn, and 39.6% random coil. Eight epitopes in SCP were predicted and identified using bioinformatics tools combined with immunological techniques.

Abstract Porcine deltacoronavirus (PDCoV) is a globally distributed swine enteropathogenic virus that emerged in the last decade. A recent report of PDCoV infection in Haitian children also highlights potential public health implications. In this study, two monoclonal antibodies (mAbs), 1C2 and 5H5, were generated and showed high specificity for the PDCoV S protein. Both mAbs displayed high-titer neutralizing capabilities, suggesting their potential for passive immunotherapy. Epitope mapping revealed that the mAbs likely recognized conformational epitopes in the S1 subunit domains A and B of the native S protein, thereby blocking the interaction between the S1 receptor-binding domain and the cellular receptor, which could inhibit viral entry into host cells. This study offers new biological tools for PDCoV detection and lays the groundwork for the future development of porcine-specific antibodies for the prevention and treatment of PDCoV in piglets.

Abstract MHC B2 haplotype chickens have been reported to induce strong immune response against various avian pathogens. However, little is known about the CD8+T-cell epitope with MHC B2-restricted on subgroup J avian leukosis virus (ALV-J). In this study, we explored the ALV-J-induced cellular immune response in B2 haplotype chickens in vivo. We found that ALV-J infection significantly increased the proportion of CD8+T cells in chickens and up-regulated the expression of cytotoxic genes like Granzyme A and antiviral genes like IFIT5 at 14 days post-infection (dpi). We selected 32 candidate peptides based on the peptide-binding motif and further identified three MHC B2-restricted CD8+T epitopes on ALV-J, including Pol652−660, Gag374−382, and Gag403−411 which induced significant levels of chicken IFN-γ production in splenocytes from ALV-J infected chickens using the ELISpot assay. In addition, we also verified that the three identified epitopes stimulated memory splenocytes elevating TNF-α and IL-2 protein expression. Importantly, we found that the three positive peptides were highly conserved among ALV-A, ALV-B, ALV-E, ALV-J, and ALV-K. Taken together, we identified three MHC B2-restricted CD8+T cell epitopes on ALV-J, providing a foundation for developing effective T cell epitope vaccines targeting conserved internal viral proteins.

Abstract The outbreak and spread of African swine fever virus (ASFV) have caused considerable economic losses to the pig industry worldwide. Currently, to promote the development of effective ASF vaccines, especially subunit vaccines, more antigenic protein targets are urgently needed. In this work, six transmembrane proteins (I329L, E146L, C257L, EP153R, I177L, and F165R) were expressed in mammalian cell lines and screened with pig anti-ASFV serum. It was found that the E146L protein was an immunodominant protein antigen among the six selected proteins. Moreover, the E146L protein induced antibody responses in all immunized pigs. To gain insight into the antigenic characteristics of the E146L protein, three monoclonal antibodies (mAbs; 12H12, 15G1, and 15H10) were generated by immunizing BALB/c mice with the purified E146L protein. The epitopes of the mAbs were further finely mapped through a peptide fusion protein expression strategy. Finally, the epitopes of the mAbs were identified as 48PDESSIAYMRFRN61 of the mAb 12H12, 138TLTGLQRII146 of the mAb 15G1, and 30GWSPFKYSKGNT41 of the mAb 15H10. Furthermore, the epitope of mAb 15H10 was validated as the immunodominant epitope with ASFV-infected pig sera. The chemically synthesized mAb 15H10 epitope peptide (EP1) exhibited the most extensive immunoreactivity with artificially or naturally ASFV-infected pig sera. The epitope 15H10 is located on the surface of the E146L protein and is highly conserved. These findings provide insight into the structure and function of the E146L protein of ASFV.

Cryptococcus neoformans is the highest‐ranked fungal pathogen in the Fungal Priority Pathogens List (FPPL) released by the World Health Organization (WHO). In this study, through in silico simulations, a multi‐epitope vaccine against Cryptococcus neoformans was developed using the mannoprotein antigen (MP88) as a vaccine candidate. Following the retrieval of the MP88 protein sequences, these were used to predict antigenic B‐cell and T‐cell epitopes via the bepipred tool and the artificial neural network, respectively. Conserved B‐cell epitopes AYSTPA, AYSTPAS, PASSNCK, and DSAYPP were identified as the most promising B‐cell epitopes. While YMAADQFCL, VSYEEWMNY, and FQQRYTGTF were identified as the best candidates for CD8+ T‐cell epitopes; and YARLLSLNA, ISYGTAMAV, and INQTSYARL were identified as the most promising CD4+ T‐cell epitopes. The vaccine construct was modeled along with adjuvant and peptide linkers and the expasy protparam tool was used to predict the physiochemical properties. According to this, the construct vaccine was predicted to be antigenic, nontoxic, nonallergenic, soluble, stable, hydrophilic, and thermostable. Furthermore, the three‐dimensional structure was also used in docking analyses with Toll‐like receptor (TLR4). Finally, the cDNA of vaccine was successfully cloned into the E. coli pET‐28a (+) expression vector. The results presented here could contribute towards the design of an effective vaccine against Cryptococcus neoformans.

Introduction: VHHs (VH of heavy-chain-only antibodies) represent a unique alternative to Q7 conventional antibodies because of their smaller size, comparable binding affinity and biophysical properties. Method: In this study, we systematically analyzed VHH NGS sequences from 22 Alpacas and structure data from public database. Results: VHHs in Alpaca can be grouped into five main types with multiple distinct sequence and structure features. Based on the existence of hallmark residues in FR2 region, VHHs can be classified into two groups: nonclassical VHHs (without hallmark residues) and classical VHHs (with hallmark residues). Based on VHH hallmark residues at 42 position (IMGT numbering, FR2 region) and number of cysteines, we found that Alpaca classical VHHs can be further separated into three main types: F_C2 VHHs with F (phenylalanine) at position 42 and having 2 cysteines within sequences, Y_C2 VHHs with Y (tyrosine) at position 42 and having 2 cysteines, and F_C4 with F at position 42 and having 4 cysteines. Non-classical VHHs can be further separated into 2 types based on germlines mapped: N_V3 for VHHs mapped to V3 germlines and N_V4 for V4 germlines. Based on whether FR2 residues are involved in binding, two kinds of paratopes can be identified. Different types of VHHs showed distinct associations with these two paratopes and displayed significant differences in paratope size, residue usage and other structure features. Discussion: Such results will have significant implications in VHH discovery, engine e ring, and design for innovative therapeutics. [ABSTRACT FROM AUTHOR]

The outbreak and spread of African swine fever virus (ASFV) have caused considerable economic losses to the pig industry worldwide. Currently, to promote the development of effective ASF vaccines, especially subunit vaccines, more antigenic protein targets are urgently needed. In this work, six transmembrane proteins (I329L, E146L, C257L, EP153R, I177L, and F165R) were expressed in mammalian cell lines and screened with pig anti-ASFV serum. It was found that the E146L protein was an immunodominant protein antigen among the six selected proteins. Moreover, the E146L protein induced antibody responses in all immunized pigs. To gain insight into the antigenic characteristics of the E146L protein, three monoclonal antibodies (mAbs; 12H12, 15G1, and 15H10) were generated by immunizing BALB/c mice with the purified E146L protein. The epitopes of the mAbs were further finely mapped through a peptide fusion protein expression strategy. Finally, the epitopes of the mAbs were identified as 48PDESSIAYMRFRN61 of the mAb 12H12, 138TLTGLQRII146 of the mAb 15G1, and 30GWSPFKYSKGNT41 of the mAb 15H10. Furthermore, the epitope of mAb 15H10 was validated as the immunodominant epitope with ASFV-infected pig sera. The chemically synthesized mAb 15H10 epitope peptide (EP1) exhibited the most extensive immunoreactivity with artificially or naturally ASFV-infected pig sera. The epitope 15H10 is located on the surface of the E146L protein and is highly conserved. These findings provide insight into the structure and function of the E146L protein of ASFV. [ABSTRACT FROM AUTHOR]

The most fruitful prevention and treatment tools for the COVID-19 pandemic have proven to be vaccines and therapeutic antibodies, which have reduced the spread of the disease to manageable proportions. The search for the most effective antibodies against the widest set of COV-2 variants has required a long time and substantial resources. It would be desirable to have a tool that will enable us to understand the structural basis on which mutants escape at least some of the epitope-bound antibodies, a tool that may substantially reduce the time and resources invested in this effort. In this work, we applied a computational-based tool (employed previously by us to understand COV-2 spike binding to its cognate cell receptor) to the study of the effect of Delta and Omicron mutations on the escape tendencies. Our binding energy predictions agree extremely well with the experimentally observed escape tendencies. They have also allowed us to set forth a structural explanation for the results that could be used for the screening of antibodies. Lastly, our results explain the differences in molecular interactions that govern interaction of the spike variants with the receptor as opposed to those with antibodies. [ABSTRACT FROM AUTHOR]

Background: Newcastle disease virus (NDV) is a highly contagious and economically devastating pathogen affecting poultry worldwide, leading to significant losses in the poultry industry. Despite existing vaccines, outbreaks continue to occur, highlighting the need for more effective vaccination strategies. Developing a multi-epitopic peptide vaccine offers a promising approach to enhance protection against NDV. Objectives: Here, we aimed to design and evaluate a multi-epitopic vaccine against NDV using molecular dynamics (MD) simulation. Methodology: We retrieved NDV sequences for the fusion (F) protein and hemagglutinin–neuraminidase (HN) protein. Subsequently, B-cell and T-cell epitopes were predicted. The top potential epitopes were utilized to design the vaccine construct, which was subsequently docked against chicken TLR4 and MHC1 receptors to assess the immunological response. The resulting docked complex underwent a 1 microsecond (1000 ns) MD simulation. For experimental evaluation, the vaccine's efficacy was assessed in mice and chickens using a controlled study design, where animals were randomly divided into groups receiving either a local ND vaccine or the peptide vaccine or a control treatment. Results: The 40 amino acid peptide vaccine demonstrated strong binding affinity and stability within the TLR4 and MHC1 receptor–peptide complexes. The root mean square deviation of peptide vaccine and TLR4 receptor showed rapid stabilization after an initial repositioning. The root mean square fluctuation revealed relatively low fluctuations (below 3 Å) for the TLR4 receptor, while the peptide exhibited higher fluctuations. The overall binding energy of the peptide vaccine with TLR4 and MHC1 receptors amounted to −15.7 kcal·mol−1 and −36.8 kcal·mol−1, respectively. For experimental evaluations in mice and chicken, the peptide vaccine was synthesized using services of GeneScript Biotech® (Singapore) PTE Limited. Experimental evaluations showed a significant immune response in both mice and chickens, with the vaccine eliciting robust antibody production, as evidenced by increasing HI titers over time. Statistical analysis was performed using an independent t-test with Type-II error to compare the groups, calculating the p-values to determine the significance of the immune response between different groups. Conclusions: Multi-epitopic peptide vaccine has demonstrated a good immunological response in natural hosts. [ABSTRACT FROM AUTHOR]

Introduction: Modern histocompatibility algorithms depend on the comparison and analysis of high-resolution HLA protein sequences and structures, especially when considering epitope-based algorithms, which aim to model the interactions involved in antibody or T cell binding. HLA genotype imputation can be performed in the cases where only low/intermediate-resolution HLA genotype is available or if specific loci are missing, and by providing an individuals' race/ethnicity/ancestry information, imputation results can be more accurate. This study assesses the effect of imputing high-resolution genotypes on molecular mismatch scores under a variety of ancestry assumptions. Methods: We compared molecular matching scores from "ground-truth" highresolution genotypes against scores from genotypes which are imputed from low-resolution genotypes. Analysis was focused on a simulated patient-donor dataset and confirmed using two real-world datasets, and deviations were aggregated based on various ancestry assumptions. Results: We observed that using multiple imputation generally results in lower error in molecular matching scores compared to single imputation, and that using the correct ancestry assumptions can reduce error introduced during imputation. Discussion: We conclude that for epitope analysis, imputation is a valuable and low-risk strategy, as long as care is taken regarding epitope analysis context, ancestry assumptions, and (multiple) imputation strategy. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cells (MSCs) have immunomodulatory properties and are therefore considered promising tools in kidney transplantation. Although most studies have been conducted with autologous MSCs, using allogeneic MSCs as an off-the-shelf product is more feasible in clinical settings. However, allogeneic MSCs could potentially induce an immune response, which might eventually be directed towards the kidney allograft because of shared human leukocyte antigen (HLA) epitope mismatches between the kidney and MSC donor. In this study, we performed in-depth analyses of two cohorts (n = 20) that received third-party MSC therapy after kidney transplantation. While the Neptune Study from Leiden University Medical Center specifically selected MSC to avoid repeated HLA antigen mismatches between kidney and MSC donors, the study from the University of Liège did not perform specific MSC selection. The comparative analyses of amino acid mismatches between these cohorts showed that MSC selection to avoid repeated HLA mismatches at the split antigen level was not sufficient to prevent repeated mismatches at the amino acid level. However, repeated amino acid mismatches were not associated with the occurrence of donor-specific antibodies (DSAs). Thus, the clinical relevance of repeated amino acid mismatches seems to be limited with regard to the risk of DSA formation. Since DSA formation was limited (3 of 20 patients) in this study, larger studies are required to investigate the relevance of preventing repeated HLA mismatches in allogeneic MSC therapy in kidney transplantation. [ABSTRACT FROM AUTHOR]

Identifying epitopes, or the segments of a protein that bind to antibodies, is critical for the development of a variety of immunotherapeutics and diagnostics. In vaccine design, the intent is to identify the minimal epitope of an antigen that can elicit an immune response and avoid off-target effects. For prognostics and diagnostics, the epitope-antibody interaction is exploited to measure antigens associated with disease outcomes. Experimental methods such as X-ray crystallography, cryo-electron microscopy, and peptide arrays are used widely to map epitopes but vary in accuracy, throughput, cost, and feasibility. By comparing machine learning epitope mapping tools, we discuss the importance of data selection, feature design, and algorithm choice in determining the specificity and prediction accuracy of an algorithm. This review discusses limitations of current methods and the potential for machine learning to deepen interpretation and increase feasibility of these methods. We also propose how machine learning can be employed to refine epitope prediction to address the apparent promiscuity of polyreactive antibodies and the challenge of defining conformational epitopes. We highlight the impact of machine learning on our current understanding of epitopes and its potential to guide the design of therapeutic interventions with more predictable outcomes. [ABSTRACT FROM AUTHOR]

Genetic studies have identified the voltage‐gated sodium channel 1.7 (Nav1.7) as pain target. Due to the ineffectiveness of small molecules and monoclonal antibodies as therapeutics for pain, single‐domain antibodies (VHHs) are developed against the human Nav1.7 (hNav1.7) using a novel antigen presentation strategy. A 70 amino‐acid peptide from the hNav1.7 protein is identified as a target antigen. A recombinant version of this peptide is grafted into the complementarity determining region 3 (CDR3) loop of an inert VHH in order to maintain the native 3D conformation of the peptide. This antigen is used to isolate one VHH able to i) bind hNav1.7, ii) slow the deactivation of hNav1.7, iii) reduce the ability of eliciting action potentials in nociceptors, and iv) reverse hyperalgesia in in vivo rat and mouse models. This VHH exhibits the potential to be developed as a therapeutic capable of suppressing pain. This novel antigen presentation strategy can be applied to develop biologics against other difficult targets such as ion channels, transporters and GPCRs. [ABSTRACT FROM AUTHOR]

Aims/Introduction: Phage display method is a crucial tool to find novel clinically valuable diabetes‐associated autoantigens and identify known autoantigen epitopes that are associated with diabetes and could provide scientific support and guidance for the artificial construction and synthesis of Type I diabetes mellitus (T1DM) novel biomarkers. Materials and Methods: The phage display system was used for the "biopanning" of T1DM serum. Following the sequencing of the phage DNAs, the homologous sequences of the above fusion heptapeptide were further investigated by BLAST to track the origin of the polypeptide sequences. The antibody spectrum revealed new T1DM‐associated epitopes and antibodies. Results: A total of 1200 phage DNA were sequenced and 9 conserved polypeptide sequences were collected. It was confirmed that the zinc transporter and islet amyloid protease were among them. The conserved polypeptide sequence 8 and another three distinctive polypeptide sequences derived from Proteus were discovered. Furthermore, we expressed recombinant proteins with homologous polypeptide sequences for the human islet amyloid polypeptide (IAPP) and polypeptide precursor human zinc transporter 8 (ZNT8). Through clinical sample detection for the serum from T1DM (n = 100) and T2DM (n = 200) patients, results demonstrate the importance and relevance of these polypeptides in the recognition and classification of various forms of diabetes. Conclusion: Human pancreatic and concurrent bacterial‐derived protein antigens and their epitopes were identified in this research by the phage display system, which is crucial for distinguishing different types of diabetes. [ABSTRACT FROM AUTHOR]

Cytotoxic CD8+ T lymphocytes (CTLs) have been implicated in the severity of COVID-19. The TCR-pMHC ternary complex, formed by the T cell receptor (TCR) and peptide-MHC (major histocompatibility complex), constitutes the molecular basis of CTL responses against SARS-CoV-2. While numerous studies have been conducted on T cell immunity, the molecular mechanisms underlying CTLmediated immunity against SARS-CoV-2 infection have not been well elaborated. In this review, we described the association between HLA variants and different immune responses to SARS-CoV-2 infection, which may lead to varying COVID-19 outcomes. We also summarized the specific TCR repertoires triggered by certain SARS-CoV-2 CTL epitopes, which might explain the variations in disease outcomes among different patients. Importantly, we have highlighted the primary strategies used by SARS-CoV-2 variants to evade T-cell killing: disrupting peptide-MHC binding, TCR recognition, and antigen processing. This review provides valuable insights into the molecule mechanism of CTL responses during SARS-CoV-2 infection, aiding efforts to control the pandemic and prepare for future challenges. [ABSTRACT FROM AUTHOR]

Inhalation allergies caused by cats and dogs can lead to a range of discomforting symptoms, such as rhinitis and asthma, in humans. With the increasing popularity of and care provided to these companion animals, the allergens they produce pose a growing threat to susceptible patients' health. Allergens from cats and dogs have emerged as significant risk factors for triggering asthma and allergic rhinitis worldwide; however, there remains a lack of systematic measures aimed at assisting individuals in recognizing and preventing allergies caused by these animals. This review provides comprehensive insights into the classification of cat and dog allergens, along with their pathogenic mechanisms. This study also discusses implementation strategies for prevention and control measures, including physical methods, gene-editing technology, and immunological approaches, as well as potential strategies for enhancing allergen immunotherapy combined with immunoinformatics. Finally, it presents future prospects for the prevention and treatment of human allergies caused by cats and dogs. This review will improve knowledge regarding allergies to cats and dogs while providing insights into potential targets for the development of next-generation treatments. [ABSTRACT FROM AUTHOR]

Reliable and ultra-fast DNA and RNA sequencing have been achieved with the emergence of high-throughput sequencing technology. When combining the results of DNA and RNA sequencing for tumor cells of cancer patients, neoantigens that potentially stimulate the immune response of either CD4+ or CD8+ T cells can be identified. However, due to the abundance of somatic mutations and the high polymorphic nature of human leukocyte antigen (HLA) it is challenging to accurately predict the neoantigens. Moreover, comparing to HLA-I presented peptides, the HLA-II presented peptides are more variable in length, making the prediction of HLA-II loaded neoantigens even harder. A number of computational approaches have been proposed to address this issue but none of them considers the DNA origin of the neoantigens from the perspective of 3D genome. Here we investigate the DNA origins of the immune-positive and non-negative HLA-II neoantigens in the context of 3D genome and discovered that the chromatin 3D architecture plays an important role in more effective HLA-II neoantigen prediction. We believe that the 3D genome information will help to increase the precision of HLA-II neoantigen discovery and eventually benefit precision and personalized medicine in cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Despite its medical relevance, there is no commercial vaccine that protects the population at risk from multidrug-resistant (MDR) Klebsiella pneumoniae infections. The availability of massive omic data and novel algorithms may improve antigen selection to develop effective prophylactic strategies. Up to 133 exposed proteins in the core proteomes, between 516 and 8666 genome samples, of the six most relevant MDR clonal groups (CGs) carried conserved B-cell epitopes, suggesting minimized future evasion if utilized for vaccination. Antigens showed a range of epitopicity, functional constraints, and potential side effects. Eleven antigens, including three sugar porins, were represented in all MDR-CGs, constitutively expressed, and showed limited reactivity with gut microbiota. Some of these antigens had important interactomic interactions and may elicit adhesion-neutralizing antibodies. Synergistic bivalent to pentavalent combinations that address expression conditions, interactome location, virulence activities, and clone-specific proteins may overcome the limiting protection of univalent vaccines. The combination of five central antigens accounted for 41% of all non-redundant interacting partners of the antigen dataset. Specific antigen mixtures represented in a few or just one MDR-CG further reduced the chance of microbiota interference. Rational antigen selection schemes facilitate the design of high-coverage and "magic bullet" multivalent vaccines against recalcitrant K. pneumoniae lineages. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is the highest‐ranked fungal pathogen in the Fungal Priority Pathogens List (FPPL) released by the World Health Organization (WHO). In this study, through in silico simulations, a multi‐epitope vaccine against Cryptococcus neoformans was developed using the mannoprotein antigen (MP88) as a vaccine candidate. Following the retrieval of the MP88 protein sequences, these were used to predict antigenic B‐cell and T‐cell epitopes via the bepipred tool and the artificial neural network, respectively. Conserved B‐cell epitopes AYSTPA, AYSTPAS, PASSNCK, and DSAYPP were identified as the most promising B‐cell epitopes. While YMAADQFCL, VSYEEWMNY, and FQQRYTGTF were identified as the best candidates for CD8+ T‐cell epitopes; and YARLLSLNA, ISYGTAMAV, and INQTSYARL were identified as the most promising CD4+ T‐cell epitopes. The vaccine construct was modeled along with adjuvant and peptide linkers and the expasy protparam tool was used to predict the physiochemical properties. According to this, the construct vaccine was predicted to be antigenic, nontoxic, nonallergenic, soluble, stable, hydrophilic, and thermostable. Furthermore, the three‐dimensional structure was also used in docking analyses with Toll‐like receptor (TLR4). Finally, the cDNA of vaccine was successfully cloned into the E. coli pET‐28a (+) expression vector. The results presented here could contribute towards the design of an effective vaccine against Cryptococcus neoformans. [ABSTRACT FROM AUTHOR]

BACKGROUND: The Cancer Epitope Database and Analysis Resource (CEDAR) is a newly developed repository of cancer epitope data from peer-reviewed publications, which includes epitope-specific T cell, antibody, and MHC ligand assays. Here we focus on prostate cancer as our first cancer category to demonstrate the capabilities of CEDAR, and to shed light on the advances of epitope-related prostate cancer research. RESULTS: The meta-analysis focused on a subset of data describing epitopes from 8 prostate-specific (PS) antigens. A total of 460 epitopes were associated with these proteins, 187 T cell, 109B cell, and 271 MHC ligand epitopes. The number of epitopes was not correlated with the length of the protein; however, we found a significant positive correlation between the number of references per specific PS antigen and the number of reported epitopes. Forty-four different class I and 27 class II restrictions were found, with the most epitopes described for HLA-A*02:01 and HLA-DRB1*01:01. Cytokine assays were mostly limited to IFNg assays and a very limited number of tetramer assays were performed. Monoclonal and polyclonal B cell responses were balanced, with the highest number of epitopes studied in ELISA/Western blot assays. Additionally, epitopes were generically described as associated with prostate cancer, with little granularity specifying diseases state. We found that in vivo and tumor recognition assays were sparse, and the number of epitopes with annotated B/T cell receptor information were limited. Potential immunodominant regions were identified by the use of the ImmunomeBrowser tool. CONCLUSION: CEDAR provides a comprehensive repository of epitopes related to prostate-specific antigens. This inventory of epitope data with its wealth of searchable T cell, B cell and MHC ligand information provides a useful tool for the scientific community. At the same time, we identify significant knowledge gaps that could be addressed by experimental analysis.

BackgroundEpstein–Barr virus (EBV) is a significant global public health concern because of its association with various malignancies and autoimmune diseases. Over 90% of the global population is chronically infected with EBV, impacting numerous cancer-related cases annually. However, none of the effective prophylactic vaccines against EBV is approved at present.MethodsIn this study, we developed a novel vaccine candidate based on epitope peptides from the receptor-binding domain of EBV-encoded gp350 glycoprotein to prevent EBV infection. These epitope peptides detected a binding capability with host cells were then fused by flexibility linkers and expressed in Escherichia coli to reduce the unnecessary glycan modifications to simulate their free-glycan status. The fused recombinant protein (L350) was displayed on the surface of ferritin-based nanoparticle. The immunogenicity of the L350–ferritin nanoparticle was evaluated in Balb/c mice, and the neutralizing titers of sera from immunized mice were detected by means of an infection blocking assay in an in vitro cell model.ResultsAll the five epitope peptides could bind to AKATA cells, and their fused recombinant protein (L350) was successfully presented on the surface of self-assembled ferritin nanoparticles. Sera from the L350–ferritin nanoparticle-immunized mice showed high titers of both L350 protein-specific and gp350D123 protein-specific antibodies, and sera from gp350D123 protein-immunized mice could also recognize L350 protein well. Most importantly, the L350–ferritin nanoparticle induced efficient neutralizing antibodies to block EBV-GFP infection in AKATA cells and also constructed a strong antigen-specific B-cell memory in immunized mice. Moreover, histopathological changes of main tissues from all vaccinated mice were not observed.ConclusionThese data indicate that the L350–ferritin nanoparticle vaccine candidate has considerable potential application in preventing EBV infection and provides a promising basis for developing prophylactic EBV vaccines.

The surge in the clinical use of therapeutic antibodies has reshaped the landscape of pharmaceutical therapy for many diseases, including rare and challenging conditions. However, the administration of exogenous biologics could potentially trigger unwanted immune responses such as generation of anti-drug antibodies (ADAs). Real-world experiences have illuminated the clear correlation between the ADA occurrence and unsatisfactory therapeutic outcomes as well as immune-related adverse events. By retrospectively examining research involving immunogenicity analysis, we noticed the growing emphasis on elucidating the immunogenic epitope profiles of antibody-based therapeutics aiming for mechanistic understanding the immunogenicity generation and, ideally, mitigating the risks. As such, we have comprehensively summarized here the progress in both experimental and computational methodologies for the characterization of T and B cell epitopes of therapeutics. Furthermore, the successful practice of epitope-driven deimmunization of biotherapeutics is exceptionally highlighted in this article.

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 β-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

Abstract Acinetobacter baumannii is a gram-negative bacillus prevalent in nature, capable of thriving under various environmental conditions. As an opportunistic pathogen, it frequently causes nosocomial infections such as urinary tract infections, bacteremia, and pneumonia, contributing to increased morbidity and mortality in clinical settings. Consequently, developing novel vaccines against Acinetobacter baumannii is of utmost importance. In our study, we identified 10 highly conserved antigenic proteins from the NCBI and UniProt databases for epitope mapping. We subsequently screened and selected 8 CTL, HTL, and LBL epitopes, integrating them into three distinct vaccines constructed with adjuvants. Following comprehensive evaluations of immunological and physicochemical parameters, we conducted molecular docking and molecular dynamics simulations to assess the efficacy and stability of these vaccines. Our findings indicate that all three multi-epitope mRNA vaccines designed against Acinetobacter baumannii are promising; however, further animal studies are required to confirm their reliability and effectiveness.