In Silico Tools for Predicting Novel Epitopes.

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

Utilizing the Banana S-Adenosyl-L-Homocysteine Hydrolase Allergen to Identify Cross-Reactive IgE in Ryegrass-, Latex-, and Kiwifruit-Allergic Individuals.

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

Identification of a linear B-cell epitope on the "puff" loop of the Senecavirus A VP2 protein involved in receptor binding.

Senecavirus A (SVA) is an important emerging swine pathogen that causes vesicular lesions in swine and acute death in newborn piglets. VP2 plays a significant role in the production of antibodies, which can be used in development of diagnostic tools and vaccines. Herein, the aim of the current study was to identify B-cell epitopes (BCEs) of SVA for generation of epitope-based SVA marker vaccine. Three monoclonal antibodies (mAbs), named 2E4, 1B8, and 2C7, against the SVA VP2 protein were obtained, and two novel linear BCEs, 177SLGTYYR183 and 266SPYFNGL272, were identified by peptide scanning. The epitope 177SLGTYYR183 was recognized by the mAb 1B8 and was fully exposed on the VP2 surface, and alanine scanning analysis revealed that it contained a high continuity of key amino acids. Importantly, we confirmed that 177SLGTYYR183 locates on "the puff" region within the VP2 EF loop, and contains three key amino acid residues involved in receptor binding. Moreover, a single mutation, Y182A, blocked the interaction of the mutant virus with the mAb 1B8, indicating that this mutation is the pivotal point for antibody recognition. In summary, the BCEs that identified in this study could be used to develop diagnostic tools and an epitope-based SVA marker vaccine.

Recombinant linear multiple epitopes of σB protein protect Muscovy ducks against novel duck reovirus infection.

Infection by the novel duck reovirus (NDRV) in ducklings causes high mortality, which leads to substantial economic losses in the duck industry in China. To date, no commercial vaccine is available for this disease. In this study, linear B cell epitopes of the σB protein of the NDRV were predicted and recombinant multiple linear B cell epitopes (MLBEs) were constructed through linkers. The recombinant MLBEs were then expressed and purified. One-day-old Muscovy ducklings were immunized with different doses of MLBEs and challenged with 5 × 104 ELD50 of the virulent CHY strain of NDRV 14 days after immunization. The ducklings vaccinated with 20 and 40 µg of MLBE performed no clinical signs or gross or histopathological lesions, indicating 100% protection against infection. The viral load in the liver and spleens of these birds was significantly lower than that in the control group. Additionally, these ducklings exhibited positive seroconversion at 7 days after vaccination on enzyme-linked immunosorbent assay. These results indicate that MLBE of σB could be used as a candidate for developing vaccines against NDRV infection.

A recombinant chimeric antigen constructed with B-cell epitopes from Mycobacterium leprae hypothetical proteins is effective for the diagnosis of leprosy.

The diagnosis if leprosy is difficult, as it requires clinical expertise and sensitive laboratory tests. In this study, we develop a serological test for leprosy by using bioinformatics tools to identify specific B-cell epitopes from Mycobacterium leprae hypothetical proteins, which were used to construct a recombinant chimeric protein, M1. The synthetic peptides were obtained and showed good reactivity to detect leprosy patients, although the M1 chimera have showed sensitivity (Se) and specificity (Sp) values higher than 90.0% to diagnose both paucibacillary (PB) and multibacillary (MB) leprosy patients, but not those developing tegumentary or visceral leishmaniasis, tuberculosis, Chagas disease, malaria, histoplasmosis and aspergillosis, in ELISA experiments. Using sera from household contacts, values for Se and Sp were 100% and 65.3%, respectively. In conclusion, our proof-of-concept study has generated data that suggest that a new recombinant protein could be developed into a diagnostic antigen for leprosy.

Multi-epitope protein production and its application in the diagnosis of opisthorchiasis.

BACKGROUND: Opisthorchiasis and cholangiocarcinoma (CCA) continue to be public health concerns in many Southeast Asian countries. Although the prevalence of opisthorchiasis is declining, reported cases tend to have a light-intensity infection. Therefore, early detection by using sensitive methods is necessary. Several sensitive methods have been developed to detect opisthorchiasis. The immunological detection of antigenic proteins has been proposed as a sensitive method for examining opisthorchiasis. METHODS: The Opisthorchis viverrini antigenic proteins, including cathepsin B (OvCB), asparaginyl endopeptidase (OvAEP), and cathepsin F (OvCF), were used to construct multi-antigenic proteins. The protein sequences of OvCB, OvAEP, and OvCF, with a high probability of B cell epitopes, were selected using BepiPred 1.0 and the IEDB Analysis Resource. These protein fragments were combined to form OvCB_OvAEP_OvCF recombinant DNA, which was then used to produce a recombinant protein in Escherichia coli strain BL21(DE3). The potency of the recombinant protein as a diagnostic target for opisthorchiasis was assessed using immunoblotting and compared with that of the gold standard method, the modified formalin-ether concentration technique. RESULTS: The recombinant OvCB_OvAEP_OvCF protein showed strong reactivity with total immunoglobulin G (IgG) antibodies against light-intensity O. viverrini infections in the endemic areas. Consequently, a high sensitivity (100%) for diagnosing opisthorchiasis was reported. However, cross-reactivity with sera from other helminth and protozoan infections (including taeniasis, strongyloidiasis, giardiasis, E. coli infection, enterobiasis, and mixed infection of Echinostome spp. and Taenia spp.) and no reactivity with sera from patients with non-parasitic infections led to a reduced specificity of 78.4%. In addition, the false negative rate (FNR), false positive rate (FPR), positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy were 0%, 21.6%, 81.4%, 100%, and 88.9%, respectively. CONCLUSIONS: The high sensitivity of the recombinant OvCB_OvAEP_OvCF protein in detecting opisthorchiasis demonstrates its potential as an opisthorchiasis screening target. Nonetheless, research on reducing cross-reactivity should be undertaken by detecting other antibodies in other sample types, such as saliva, urine, and feces.

Establishment of an ELISA Based on a Recombinant Antigenic Protein Containing Multiple Prominent Epitopes for Detection of African Swine Fever Virus Antibodies.

African swine fever virus (ASFV) poses a significant threat to the global pig industry, necessitating accurate and efficient diagnostic methods for its infection. Previous studies have often focused on a limited number of epitopes from a few proteins for detecting antibodies against ASFV. Therefore, the current study aimed to use multiple B-cell epitopes in developing an indirect Enzyme-Linked Immunosorbent Assay (ELISA) for enhanced detection of ASFV antibodies. For the expression of recombinant protein, k3 derived from 27 multiple peptides of 11 ASFV proteins, such as p72, pA104R, pB602L, p12, p14.5, p49, pE248R, p30, p54, pp62, and pp220, was used. To confirm the expression of the recombinant protein, we used the Western blotting analysis. The purified recombinant K3 protein served as the antigen in our study, and we employed the indirect ELISA technique to detect anti-ASFV antibodies. The present finding showed that there was no cross-reactivity with antibodies targeting Foot-and-mouth disease virus (FMDV), Porcine circovirus type 2 (PCV2), Pseudorabies virus (PRV), Porcine reproductive and respiratory syndrome virus (PRRSV), and Classical swine fever virus (CSFV). Moreover, the current finding was sensitive enough to find anti-ASFV in serum samples that had been diluted up to 32 times. The test (k3-iELISA) showed diagnostic specificity and sensitivity of 98.41% and 97.40%, respectively. Moreover, during the present investigation, we compared the Ingenasa kit and the k3-iELISA to test clinical pig serum, and the results revealed that there was 99.00% agreement between the two tests, showing good detection capability of the k3-iELISA method. Hence, the current finding showed that the ELISA kit we developed can be used for the rapid detection of ASFV antibodies and used as an alternative during serological investigation of ASF in endemic areas.

Serodiagnosis of paucibacillary and multibacillary leprosy using a recombinant chimeric protein composed of specific B-cell epitopes derived from Mycobacterium leprae proteins.

Leprosy diagnosis is difficult due to the clinical similarity with other infectious diseases, and laboratory tests presents problems related to sensitivity and/or specificity. In this study, we used bioinformatics to assess Mycobacterium leprae proteins and formulated a chimeric protein that was tested as a diagnostic marker for the disease. The amino acid sequences from ML0008, ML0126, ML0308, ML1057, ML2028, ML2038, ML2498 proteins were evaluated, and the B-cell epitopes QASVAYPATSYADFRAHNHWWNGP, SLQRSISPNSYNTARVDP and QLLGQTADVAGAAKSGPVQPMGDRGSVSPVGQ were considered M. leprae-specific and used to construct the gene encoding the recombinant antigen. The gene was constructed, the recombinant protein was expressed, purified and tested in ELISA using 252 sera, which contained samples from multibacillary (MB) or paucibacillary (PB) leprosy patients, from their household contacts and healthy individuals, as well as from patients with Chagas disease, visceral and tegumentary leishmaniases (VL/TL), malaria, tuberculosis, and HIV. Sensitivity (Se) and specificity (Sp) for MB and PB samples compared to sera from both healthy subjects and individuals with cross-reactive diseases were 100%. The Se value for MB and PB samples compared to sera from household contacts was 100%, but Sp was 64%. In conclusion, data suggest that this protein could be considered in future studies for leprosy diagnosis.

Construction by artificial intelligence and immunovalidation of hypoallergenic mite allergen Der f 36 vaccine.

Background: The house dust mite (HDM) is widely recognized as the most prevalent allergen in allergic diseases. Allergen-specific immunotherapy (AIT) has been successfully implemented in clinical treatment for HDM. Hypoallergenic B-cell epitope-based vaccine designed by artificial intelligence (AI) represents a significant progression of recombinant hypoallergenic allergen derivatives. Method: The three-dimensional protein structure of Der f 36 was constructed using Alphafold2. AI-based tools were employed to predict B-cell epitopes, which were subsequently verified through IgE-reaction testing. Hypoallergenic Der f 36 was then synthesized, expressed, and purified. The reduced allergenicity was assessed by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and basophil activation test. T-cell response to hypoallergenic Der f 36 and Der f 36 was evaluated based on cytokine expression in the peripheral blood mononuclear cells (PBMCs) of patients. The immunogenicity was evaluated and compared through rabbit immunization with hypoallergenic Der f 36 and Der f 36, respectively. The inhibitory effect of the blocking IgG antibody on the specific IgE-binding activity and basophil activation of Der f 36 allergen was also examined. Results: The final selected non-allergic B-cell epitopes were 25-48, 57-67, 107-112, 142-151, and 176-184. Hypoallergenic Der f 36 showed significant reduction in IgE-binding activity. The competitive inhibition of IgE-binding to Der f 36 was investigated using the hypoallergenic Der f 36, and only 20% inhibition could be achieved, which is greatly reduced when compared with inhibition by Der f 36 (98%). The hypoallergenic Der f 36 exhibited a low basophil-stimulating ratio similar to that of the negative control, and it could induce an increasing level of IFN-γ but not Th2 cytokines IL-5 and IL-13 in PBMCs. The vaccine-specific rabbit blocking IgG antibodies could inhibit the patients' IgE binding and basophil stimulation activity of Derf 36. Conclusion: This study represents the first application of an AI strategy to facilitate the development of a B-cell epitope-based hypoallergenic Der f 36 vaccine, which may become a promising immunotherapy for HDM-allergic patients due to its reduced allergenicity and its high immunogenicity in inducing blocking of IgG.

Development and characterization of monoclonal antibodies against p37 protein of African swine fever virus.

African Swine Fever Virus (ASFV) is a highly contagious pathogen posing a serious threat to the global swine industry. Despite this, there is currently no effective vaccine against this virus. Within ASFV's core shell structure, p37, a product of polyprotein pp220, shares sequence similarity with SUMO-1 proteases. Localization studies show p37 in various nuclear regions during early infection, shifting to the cytoplasm later on. Research indicates active export of p37 from the nucleus, mediated by CRM1-dependent and -independent pathways. Hydrophobic amino acids in p37 are crucial for these pathways, highlighting their importance throughout the ASFV replication cycle. Additionally, p37 serves as the first nucleocytoplasmic shuttle protein encoded by ASFV, participating in the intranuclear material transport process during ASFV infection of host cells. In this study, we successfully screened five murine monoclonal antibodies targeting p37. Through the truncated expression method, we identified four dominant antigenic epitopes of p37 for the first time. Furthermore, utilizing alanine scanning technology, we determined the key amino acid residues for each epitope. This research not only provides essential information for a deeper understanding of the protein's function but also establishes a significant theoretical foundation for the design and development of ASFV vaccines.

Identification of Three Novel Linear B-Cell Epitopes in Non-Structural Protein 3 of Porcine Epidemic Diarrhea Virus Using Monoclonal Antibodies.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic swine coronavirus that causes diarrhea and high mortality in piglets, resulting in significant economic losses within the global swine industry. Nonstructural protein 3 (Nsp3) is the largest in coronavirus, playing critical roles in viral replication, such as the processing of polyproteins and the formation of replication-transcription complexes (RTCs). In this study, three monoclonal antibodies (mAbs), 7G4, 5A3, and 2D7, targeting PEDV Nsp3 were successfully generated, and three distinct linear B-cell epitopes were identified within these mAbs by using Western blotting analysis with 24 truncations of Nsp3. The epitope against 7G4 was located on amino acids 31-TISQDLLDVE-40, the epitope against 5A3 was found on amino acids 141-LGIVDDPAMG-150, and the epitope against 2D7 was situated on amino acids 282-FYDAAMAIDG-291. Intriguingly, the epitope 31-TISQDLLDVE-40 recognized by the mAb 7G4 appears to be a critical B-cell linear epitope due to its high antigenic index and exposed location on the surface of Nsp3 protein. In addition, bioinformatics analysis unveiled that these three epitopes were highly conserved in most genotypes of PEDV. These findings present the first characterization of three novel linear B-cell epitopes in the Nsp3 protein of PEDV and provide potential tools of mAbs for identifying host proteins that may facilitate viral infection.

An immunoinformatics and structural vaccinology approach to design a novel and potent multi-epitope base vaccine targeting Zika virus.

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

A method for predicting linear and conformational B-cell epitopes in an antigen from its primary sequence.

B-cell is an essential component of the immune system that plays a vital role in providing the immune response against any pathogenic infection by producing antibodies. Existing methods either predict linear or conformational B-cell epitopes in an antigen. In this study, a single method was developed for predicting both types (linear/conformational) of B-cell epitopes. The dataset used in this study contains 3875 B-cell epitopes and 3996 non-B-cell epitopes, where B-cell epitopes consist of both linear and conformational B-cell epitopes. Our primary analysis indicates that certain residues (like Asp, Glu, Lys, and Asn) are more prominent in B-cell epitopes. We developed machine-learning based methods using different types of sequence composition and achieved the highest AUROC of 0.80 using dipeptide composition. In addition, models were developed on selected features, but no further improvement was observed. Our similarity-based method implemented using BLAST shows a high probability of correct prediction with poor sensitivity. Finally, we developed a hybrid model that combines alignment-free (dipeptide based random forest model) and alignment-based (BLAST-based similarity) models. Our hybrid model attained a maximum AUROC of 0.83 with an MCC of 0.49 on the independent dataset. Our hybrid model performs better than existing methods on an independent dataset used in this study. All models were trained and tested on 80 % of the data using a cross-validation technique, and the final model was evaluated on 20 % of the data, called an independent or validation dataset. A webserver and standalone package named "CLBTope" has been developed for predicting, designing, and scanning B-cell epitopes in an antigen sequence available at (https://webs.iiitd.edu.in/raghava/clbtope/).

Evolutionary History and Genetic Variation of Zika Virus: Connection Between Thailand Zika Viruses and Global Outbreaks Strains.

Background: Zika virus (ZIKV) has significant potential to cause future outbreaks due to insufficient countermeasures. The evolution of ZIKV in Southeast Asian countries remains poorly understood. Materials and Methods: The phylogenetic, phylogeographic network, and recombination analyses of 366 ZIKV complete genome sequences identified between 1947 and 2021 were performed and the amino acid variation landscape was determined to reveal the evolutionary characteristics. Results: ZIKV falls into two major genogroups: GI and GII, segregated into further subgenogroups (GI-1 to GI-3) and (GII-1 to GII-3), respectively. Importantly, Thailand strains cluster with Southeast Asian outbreak strains (Singapore 2016, the Philippines 2012, Cambodia 2010) into GII-2 and form a lineage independent of French Polynesia and the Americas large outbreak strains. Thailand ZIKV strains shared their ancestral route to the strains from French Polynesia, which further connects to Brazil ZIKV through a short mutational branch. Both recombination and specific mutations may contribute to the emergence of new virus lineage in Thailand. Conclusion: This report provides insights into the evolutionary characteristics of ZIKV in Southeast Asia, which may be helpful for epidemiological investigation, vaccine development, and surveillance of the virus.

Identification of T cell and linear B cell epitopes on African horse sickness virus serotype 4 proteins VP1-1, VP2, VP4, VP7 and NS3.

The viral proteins VP1-1, VP2, VP4, VP7 and NS3, of African horse sickness virus serotype 4 (AHSV4), have previously been identified to contain CD8+ T cell epitopes. In this study, overlapping peptides spanning the entire sequences of these AHSV4 proteins were synthesized and used to map epitopes. Peripheral blood mononuclear cells (PBMC) isolated from five horses immunized with an attenuated AHSV4 were stimulated in vitro with the synthesized peptides. Various memory immune assays were used to identify the individual peptides that contain CD8+ T cell epitopes, CD4+ T cell epitopes and linear B cell epitopes. The newly discovered individual peptides of AHSV4 proteins VP1-1, VP4, VP7 and/or NS3 that contain CD8+ T cell, CD4+ T cell or linear B cell epitopes could contribute to the design and development of new generation AHS peptide-based vaccines and therapeutics.

Immunological depiction of synthetic B-cell epitopes of Mycobacterium tuberculosis.

Background: To combat the tuberculosis (TB) epidemic, the development of a better and faster diagnosis or more effective vaccine is essential. Pulmonary TB (PTB) is one of the major causes of morbidity and mortality. Early diagnosis of TB is difficult. Serological assays have been performed with several antigens of laboratory strains such as Mycobacterium tuberculosis H37Rv which have not been found to be highly sensitive. In the present study, various peptides were synthesized which were predicted on the basis of immunoreactivity and differential expression in clinical isolates of M. tuberculosis compared to their expression in a laboratory strain of M. tuberculosis. Therefore, the aim of this study was to compare the antibody levels in PTB and healthy controls against these peptides. Methods: An effort was made to evaluate antibody response to peptides derived from proteins Rv2588c, Rv0512, Rv0148, Rv0896, and Rv0635 of M. tuberculosis in PTB patients and healthy individuals through enzyme-linked immunosorbent assay. Five milliliters of venous blood samples was collected from each participant, and serum was separated and stored until use. Results: Antibody levels against these peptides, Rv2588c, Rv0512, Rv0148, Rv0896. and Rv0635 in 139 PTB patients and 52 healthy controls were evaluated. Higher immune response was observed in PTB patients when compared with healthy individuals. Strong immunoglobulin G responses with high percentage, considerable difference among patients and healthy controls was observed with P < 0.0001. Conclusion: In our study, we found significant statistical differences in antibody levels in PTB patients and healthy individuals against these peptides. These peptides are suggestive of being a potential new candidate (s) for early diagnosis of TB.

Mapping of conserved immunodominant epitope peptides in the outer membrane porin (Omp) L of prominent Enterobacteriaceae pathogens associated with gastrointestinal infections.

BACKGROUND: Members of Enterobacteriaceae such as Escherichia coli O 157:H7, Salmonella sp., Shigella sp., Klebsiella sp., and Citrobacter freundii are responsible for the outbreak of serious foodborne illness and other mucosal infections across the globe. The outer membrane proteins (OMPs) of Enterobacteriaceae are highly immunogenic in eliciting immune responses against pathogens. Moreover, the OMPs are highly conserved in the Enterobacteriaceae family. Sequence homology in the OMPs will ensure the presence of conserved immunodominant regions with predominant epitopes. The OmpL is such an immunogen that is highly conserved among the Enterobacteriaceae pathogens. In this study, we performed computational analysis on the outer membrane porin (Omp) L of prominent Enterobacteriaceae pathogens. RESULTS: Multiple sequence and structural alignment analysis have revealed that the OmpL protein is highly conserved among the selected Enterobacteriaceae pathogens. This amount of sequence and structural homology uncovered the conserved antibody binding B-cell epitopes in the OmpL protein. The B-cell epitopes predicted in the OmpL of Salmonella typhimurium are highly conserved among the other Enterobacteriaceae pathogens. CONCLUSION: In conclusion, these conserved B-cell epitopes will vouch for the generation of heterologous humoral immune response in conferring cross protection against the Enterobacteriaceae pathogens and control their outbreaks across the globe.

In silico discovery of epitopes of gag and env proteins for the development of a multi-epitope vaccine candidate against Maedi Visna Virus using reverse vaccinology approach.

Maedi Visna Virus (MVV) causes a chronic viral disease in sheep. Since there is no specific therapeutic drug that targets MVV, development of a vaccine against the MVV is inevitable. This study aimed to analyze the gag and env proteins as vaccine candidate proteins and to identify epitopes in these proteins. In addition, it was aimed to construct a multi-epitope vaccine candidate. According to the obtained results, the gag protein was detected to be more conserved and had a higher antigenicity value. Also, the number of alpha helix in the secondary structure was higher and transmembrane helices were not detected. Although many B cell and MHC-I/II epitopes were predicted, only 19 of them were detected to have the properties of antigenic, non-allergenic, non-toxic, soluble, and non-hemolytic. Of these epitopes, five were remarkable due to having the highest antigenicity value. However, the final multi-epitope vaccine was constructed with 19 epitopes. A strong affinity was shown between the final multi-epitope vaccine and TLR-2/4. In conclusion, the gag protein was a better antigen. However, both proteins had epitopes with high antigenicity value. Also, the final multi-epitope vaccine construct had a potential to be used as a peptide vaccine due to its immuno-informatics results.

B-Cell Epitopes-Based Chimeric Protein from SARS-CoV-2 N and S Proteins Is Recognized by Specific Antibodies in Serum and Urine Samples from Patients.

The impact of the COVID-19 pandemic caused by the SARS-CoV-2 virus underscored the crucial role of laboratorial tests as a strategy to control the disease, mainly to indicate the presence of specific antibodies in human samples from infected patients. Therefore, suitable recombinant antigens are relevant for the development of reliable tests, and so far, single recombinant proteins have been used. In this context, B-cell epitopes-based chimeric proteins can be an alternative to obtain tests with high accuracy through easier and cheaper production. The present study used bioinformatics tools to select specific B-cell epitopes from the spike (S) and the nucleocapsid (N) proteins from the SARS-CoV-2 virus, aiming to produce a novel recombinant chimeric antigen (N4S11-SC2). Eleven S and four N-derived B-cell epitopes were predicted and used to construct the N4S11-SC2 protein, which was analyzed in a recombinant format against serum and urine samples, by means of an in house-ELISA. Specific antibodies were detected in the serum and urine samples of COVID-19 patients, which were previously confirmed by qRT-PCR. Results showed that N4S11-SC2 presented 83.7% sensitivity and 100% specificity when using sera samples, and 91.1% sensitivity and 100% specificity using urine samples. Comparable findings were achieved with paired urine samples when compared to N and S recombinant proteins expressed in prokaryotic systems. However, better results were reached for N4S11-SC2 in comparison to the S recombinant protein when using paired serum samples. Anti-N4S11-SC2 antibodies were not clearly identified in Janssen Ad26.COV2.S COVID-19-vaccinated subjects, using serum or paired urine samples. In conclusion, this study presents a new chimeric recombinant antigen expressed in a prokaryotic system that could be considered as an alternative diagnostic marker for the SARS-CoV-2 infection, with the potential benefits to be used on serum or urine from infected patients.

Identification and genetic characteristics of tusavirus in fecal samples of patients with chronic diseases in Guangzhou, China.

Purpose: The Tunisian stool-associated parvovirus [Tusavirus (TuV)] is a novel member of the genus Protoparvovirus, which may be linked to diarrhea. Herein, we investigated the prevalence of TuV in different populations and analyzed its genetic and bioinformatic characteristics. Methods: This study was conducted in a tertiary hospital in Guangzhou (China) from February 2018 to July 2022. Demographic and clinical information and stool samples were collected from individuals who visited the hospital. ProtScale, SwissModel, Datamonkey, and other tools were used to analyze and predict the physicochemical parameters, tertiary structure, selection pressure, and B-cell epitopes of capsid viral protein 2 of TuV (VP2-TuV). Results: A total of 3,837 participants were enrolled, among which two stool samples from patients with chronic illnesses were tested positive for TuV DNA. However, no positive sample was detected among patients with diarrhea. Two near-complete genome sequences were amplified. The genetic analysis revealed the presence of diversity among TuVs isolated from distinct host species. Bioinformatics analysis revealed that VP2-TuV exhibited hydrophilic properties and lacked transmembrane domains and signal peptides. The secondary structure of VP2-TuV was composed mainly of random coils and ß-strands. Selective-pressure analysis of the VP2 region suggested that TuV primarily underwent negative selection during evolution. Negatively selected codon sites coincided with residues comprising of B-cell epitopes, suggesting minimal changes in the immunogenicity of TuV over time. Conclusion: TuV was detected in patients with chronic diseases but not in patients with diarrhea. The putative roles of TuV in the pathogenicity of human diseases and zoonotic viruses must be determined by additional studies.