Computer-aided designing of a novel multi­epitope DNA vaccine against severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne viral disease caused by the SFTS virus (Dabie bandavirus), which has become a substantial risk to public health. No specific treatment is available now, that calls for an effective vaccine. Given this, we aimed to develop a multi-epitope DNA vaccine through the help of bioinformatics. The final DNA vaccine was inserted into a special plasmid vector pVAX1, consisting of CD8+ T cell epitopes, CD4+ T cell epitopes and B cell epitopes (six epitopes each) screened from four genome-encoded proteins--nuclear protein (NP), glycoprotein (GP), RNA-dependent RNA polymerase (RdRp), as well as nonstructural protein (NSs). To ascertain if the predicted structure would be stable and successful in preventing infection, an immunological simulation was run on it. In conclusion, we designed a multi-epitope DNA vaccine that is expected to be effective against Dabie bandavirus, but in vivo trials are needed to verify this claim.

Chimeric Porcine Parvovirus VP2 Virus-like Particles with Epitopes of South African Serotype 2 Foot-and-Mouth Disease Virus Elicits Specific Humoral and Cellular Responses in Mice.

Southern Africa Territories 2 (SAT2) foot-and-mouth disease (FMD) has crossed long-standing regional boundaries in recent years and entered the Middle East. However, the existing vaccines offer poor cross-protection against the circulating strains in the field. Therefore, there is an urgent need for an alternative design approach for vaccines in anticipation of a pandemic of SAT2 Foot-and-mouth disease virus (FMDV). The porcine parvovirus (PPV) VP2 protein can embed exogenous epitopes into the four loops on its surface, assemble into virus-like particles (VLPs), and induce antibodies and cytokines to PPV and the exogenous epitope. In this study, chimeric porcine parvovirus VP2 VLPs (chimeric PPV-SAT2-VLPs) expressing the T-and/or B-cell epitopes of the structural protein VP1 of FMDV SAT2 were produced using the recombinant pFastBac™ Dual vector of baculoviruses in Sf9 and HF cells We used the Bac-to-Bac system to construct the recombinant baculoviruses. The VP2-VLP--SAT2 chimeras displayed chimeric T-cell epitope (amino acids 21-40 of VP1) and/or the B-cell epitope (amino acids 135-174) of SAT FMDV VP1 by substitution of the corresponding regions at the N terminus (amino acids 2-23) and/or loop 2 and/or loop 4 of the PPV VP2 protein, respectively. In mice, the chimeric PPV-SAT2-VLPs induced specific antibodies against PPV and the VP1 protein of SAT2 FMDV. The VP2-VLP-SAT2 chimeras induced specific antibodies to PPV and the VP1 protein specific epitopes of FMDV SAT2. In this study, as a proof-of-concept, successfully generated chimeric PPV-VP2 VLPs expressing epitopes of the structural protein VP1 of FMDV SAT2 that has a potential to prevent FMDV SAT2 and PPV infection in pigs.

Designing and Developing Serological Test for the Diagnosis of Human Fascioliasis Using a New Recombinant Multi-epitope.

PURPOSE: Fascioliasis is a common parasitic disease in humans and herbivores which is caused by Fasciola hepatica and Fasciola gigantica and has a worldwide distribution. Serological tests such as the enzyme-linked immunosorbent assay (ELISA) technique play a prominent role in the fast diagnosis of the disease. However, there are diagnostic limitations, including cross-reactivity with other worms, which decline the specificity of the results. This study aimed to evaluate the structure of a recombinant multi-epitope antigen produced from linear and conformational B-cell epitopes of three parasitic proteins with sera of individuals with fasciolosis, healthy controls, and those with other diseases to gain accurate sensitivity and specificity. METHODS: After designing the multi-epitope structure of cathepsin L1, FhTP16.5, and SAP-2 antigens and then synthesizing, cloning, and expressing, the extracted purified protein was evaluated by indirect ELISA to detect IgG antibodies against Fasciola hepatica parasite among the sera of 39 serum samples of Fasciola hepatica, 35 healthy individual samples, and 20 samples of other types of parasitic diseases. The synthesized multi-epitope produced from cathepsin L1, FhTP16.5, and SAP-2 antigens was evaluated using the indirect ELISA. RESULTS: The analysis of the samples mentioned for IgG antibody diagnosis against Fasciola hepatica showed 97.43% (95% confidence interval, 94.23-100%) sensitivity and 100% (95% confidence interval, 97-100%) specificity. CONCLUSION: The recombinant B-cell multi-epitope with high antigenic potency may increase the specificity of epitopic peptides and ultimately help improve and develop indirect ELISA commercial kits for the diagnosis of fascioliasis in humans.

Decrypting the multi-genome data for chimeric vaccine designing against the antibiotic resistant Yersinia pestis.

Yersinia pestis, the causative agent of plague, is a gram-negative bacterium that can be fatal if not treated properly. Three types of plague are currently known: bubonic, septicemic, and pneumonic plague, among which the fatality rate of septicemic and pneumonic plague is very high. Bubonic plague can be treated, but only if antibiotics are used at the initial stage of the infection. But unfortunately, Y. pestis has also shown resistance to certain antibiotics such as kanamycin, minocycline, tetracycline, streptomycin, sulfonamides, spectinomycin, and chloramphenicol. Despite tremendous progress in vaccine development against Y. pestis, there is no proper FDA-approved vaccine available to protect people from its infections. Therefore, effective broad-spectrum vaccine development against Y. pestis is indispensable. In this study, vaccinomics-assisted immunoinformatics techniques were used to find possible vaccine candidates by utilizing the core proteome prepared from 58 complete genomes of Y. pestis. Human non-homologous, pathogen-essential, virulent, and extracellular and membrane proteins are potential vaccine targets. Two antigenic proteins were prioritized for the prediction of lead epitopes by utilizing reverse vaccinology approaches. Four vaccine designs were formulated using the selected B- and T-cell epitopes coupled with appropriate linkers and adjuvant sequences capable of inducing potent immune responses. The HLA allele population coverage of the T-cell epitopes selected for vaccine construction was also analyzed. The V2 constructs were top-ranked and selected for further analysis on the basis of immunological, physicochemical, and immune-receptor docking interactions and scores. Docking and molecular dynamic simulations confirmed the stability of construct V2 interactions with the host immune receptors. Immune simulation analysis anticipated the strong immune profile of the prioritized construct. In silico restriction cloning ensured the feasible cloning ability of the V2 construct in the expression system of E. coli strain K12. It is anticipated that the designed vaccine construct may be safe, effective, and able to elicit strong immune responses against Y. pestis infections and may, therefore, merit investigation using in vitro and in vivo assays.

Immunoinformatics design of novel multi-epitope vaccine against Trueperella Pyogenes using collagen adhesion protein, fimbriae, and pyolysin.

Trueperella pyogenes (T. pyogenes) is an opportunistic pathogen that causes infertility, mastitis, and metritis in animals. T. pyogenes is also a zoonotic disease and is considered an economic loss agent in the livestock industry. Therefore, vaccine development is necessary. Using an immunoinformatics approach, this study aimed to construct a multi-epitope vaccine against T. pyogenes. The collagen adhesion protein, fimbriae, and pyolysin (PLO) sequences were initially retrieved. The HTL, CTL, and B cell epitopes were predicted. The vaccine was designed by binding these epitopes with linkers. To increase vaccine immunogenicity, profilin was added to the N-terminal of the vaccine construct. The antigenic features and safety of the vaccine model were investigated. Docking, molecular dynamics simulation of the vaccine with immune receptors, and immunological simulation were used to evaluate the vaccine's efficacy. The vaccine's sequence was then optimized for cloning. The vaccine construct was designed based on 18 epitopes of T. pyogenes. The computational tools validated the vaccine as non-allergenic, non-toxic, hydrophilic, and stable at different temperatures with acceptable antigenic features. The vaccine model had good affinity and stability to bovine TLR2, 4, and 5 as well as stimulation of IgM, IgG, IL-2, IFN-γ, and Th1 responses. This vaccine also increased long-lived memory cells, dendritic cells, and macrophage population. In addition, codon optimization was done and cloned in the E. coli K12 expression vector (pET-28a). For the first time, this study introduced a novel multi-epitope vaccine candidate based on collagen adhesion protein, fimbriae, and PLO of T. pyogenes. It is expected this vaccine stimulates an effective immune response to prevent T. pyogenes infection.

Leveraging the immunoinformatics approach for designing the SARS-CoV-2 omicron-specific antigenic cassette of mRNA vaccine.

Emergence of SARS-CoV-2 Omicron variant has presented a significant challenge to global health, demanding rapid development of mRNA-based vaccines. The mRNA-guided vaccine platforms offer various advantages over traditional vaccine platforms. The mRNA by nature is a short-lived molecule that guides the cells to manufacture antigenic proteins. In the present work, we have created an omicron spike antigenic protein sequence characterized by base composition analysis, modeling, and docking with the ACE-2 receptor. Further, we predicted the B-cell and T-cell epitopes followed by antigenicity, toxicity, and allergenicity. Finally, the protein was reverse translated, codon-optimized, and encoding mRNA sequence was checked for its stability by predicting the secondary structures. A comprehensive examination of in-silico data revealed 628.2 as a potent antigenic candidate that was finally used in Gemcovac®-OM, a heterologous booster mRNA vaccine for COVID-19.

A novel conserved B-cell epitope in pB602L of African swine fever virus.

African swine fever virus (ASFV) is a complex DNA virus and the only member of the Asfarviridae family. It causes high mortality and severe economic losses in pigs. The ASFV pB602L protein plays a key role in virus assembly and functions as a molecular chaperone of the major capsid protein p72. In addition, pB602L is an important target for the development of diagnostic tools for African swine fever (ASF) because it is a highly immunogenic antigen against ASFV. In this study, we expressed and purified ASFV pB602L and validated its immunogenicity in serum from naturally infected pigs with ASFV. Furthermore, we successfully generated an IgG2a κ subclass monoclonal antibody (mAb 7E7) against pB602L using hybridoma technology. Using western blot and immunofluorescence assays, mAb 7E7 specifically recognized the ASFV Pig/HLJ/2018/strain and eukaryotic recombinant ASFV pB602L protein in vitro. The 474SKENLTPDE482 epitope in the ASFV pB602L C-terminus was identified as the minimal linear epitope for mAb 7E7 binding, with dozens of truncated pB602l fragments characterized by western blot assay. We also showed that this antigenic epitope sequence has a high conservation and antigenic index. Our study contributes to improved vaccine and antiviral development and provides new insights into the serologic diagnosis of ASF. KEY POINTS: • We developed a monoclonal antibody against ASFV pB602L, which can specifically recognize the ASFV Pig/HLJ/2018/ strain. • This study found one novel conserved B-cell epitope 474SKENLTPDE482. • In the 3D structure, 474SKENLTPDE482 is exposed on the surface of ASFV pB602L, forming a curved linear structure.

Novel toxin-based mRNA vaccine against Clostridium perfringens using in silico approaches.

Clostridium perfringens is a bacterium that causes gastrointestinal diseases in humans and animals. The several powerful toxins such as alpha toxin (CPA), beta toxin (CPB), enterotoxin (CPE), Epsilon toxin (ETX), and theta toxin, play a major role in its pathogenesis. Traditional vaccine development methods are time-consuming and costly. In silico approaches offer an alternative strategy for designing vaccines by analyzing biological data and predicting immunogenic peptides. In this study, computational tools were utilized to design a RNA vaccine targeting C. perfringens toxins. Toxin protein sequences were retrieved and their linear B-cell, MHCI, and MHCII binding epitopes were predicted. Allergenicity, toxigenicity, and IFN-γ induction were assessed to select non-allergenic, non-toxic, and IFN-γ-inducing epitopes. Molecular docking was performed to identify epitopes that fit within the binding cleft of MHC alleles. A final peptide vaccine construct was designed with selected epitopes separated by a linker sequence. The antigenicity and physicochemical properties of the vaccine were evaluated. Immune response simulation showed enhanced secondary and tertiary immune responses, increased levels of immunoglobulins, cytotoxic T lymphocytes, helper T lymphocytes, macrophage activity, and elevated levels IFN-γ and interleukin-2. Docking analysis was done to assess interactions between the vaccine structure and Toll-like receptors. Codon optimization was performed, and a final RNA vaccine construct was designed. The secondary structure of the RNA vaccine was predicted and validated. Overall, this study demonstrates the potential of in silico approaches for designing an RNA vaccine against C. perfringens toxins, contributing to improved prevention and control of associated diseases.

Development of a monoclonal antibody specifically recognizing a linear epitope on the capsid protein of the emerging Group III Getah virus.

Getah virus (GETV) is an emerging mosquito-borne alphavirus that can infect horses, pigs and other animals. Given the public health threat posed by GETV, research on its pathogenesis, diagnosis and prevention is urgently needed. In the current study, prokaryotic expression systems were used to express the capsid protein of GETV. This protein was then used to immunize BALB/c mice in order to generate monoclonal antibodies (mAbs). Subsequently, hybridoma cells secreting a mAb (2B11-4) against the capsid protein were obtained using the hybridoma technique. A B cell linear epitope, 18-PAYRPWR-24, located at the capsid protein's N-terminal region was identified using western blotting analysis with the produced mAb, 2B11-4. Sequence alignment indicated that this epitope was highly conserved in group III (GIII) strains of GETV, but varied among the other genotypes. Western blotting showed that mAb 2B11-4 could discriminate Group III GETVs from other genotypes. This study describes the preparation of a mAb against the GETV capsid protein and the identification of the specific localization of B-cell epitopes, and will contribute towards a better understanding of the biological importance of the GETV capsid protein. It will also pave the way for developing immunological detection methods and genotype diagnosis for GETVs.

Identification of a novel linear B-cell epitope on the p30 protein of African swine fever virus using monoclonal antibodies.

The outbreak of African Swine Fever (ASF) has caused huge economic losses to the pig industry. There are no safe and effective vaccines or diagnostics available. The p30 protein serves as a key target for the detection of ASFV antibodies and is an essential antigenic protein for early serological diagnosis. Here, the p30 protein was purified after being expressed in E. coli and its immunogenicity was verified in sera from pigs naturally infected with ASFV. Furthermore, a monoclonal antibody (McAb) designated as McAb 1B4G2-4 (subtype IgG1/kappa-type) was produced and it was verified to specifically recognize the ASFV Pig/HLJ/2018/strain and eukaryotic recombinant ASFV p30 protein. The epitope identified by McAb 1B4G2-4, defining the unique B-cell epitope 164HNFIQTI170, was located using peptide scanning. Comparing amino acid (aa) sequence revealed that this epitope is conserved in all reference ASFV strains from different regions of China, including the highly pathogenic strain Georgia 2007/1 (NC_044959.2) that is widely distributed. It is also exposed to the surface of the p30 protein, suggesting that it could be an important B-cell epitope. Our study may serve as a basis for the development of serological diagnostic methods and subunit vaccines.

Development of a novel multi-epitope oral DNA vaccine for rabies based on a food-borne microbial vector.

Rabies has been with humans for a long time, and its special transmission route and almost 100 % lethality rate made it once a nightmare for humans. In this study, by predicting the rabies virus glycoprotein outer membrane region and nucleoprotein B-cell antigenic epitopes, the coding sequence of the predicted highly antigenic polypeptide region obtained was assembled using the eukaryotic expression vector pcDNA3.1(-), and then E. coli was used as the delivery vector. The immunogenicity and protective properties of the vaccine were verified by in vivo and in vitro experiments, which demonstrated that the vaccine could produce antibodies in mice and prolong the survival time of mice exposed to the strong virus without any side effects. This study demonstrated that the preparation of an oral rabies DNA vaccine using food-borne microorganisms as a transport vehicle is feasible and could be a new strategy to eradicate rabies starting with wild animals.

Screening and identification of B cell epitope within the major capsid protein L1 of HPV 52, using monoclonal antibodies.

The L1 protein of Human papillomavirus (HPV), the main capsid protein, induces the formation of neutralizing antibodies. In this study, HPV52 L1 protein was induced to be expressed. Monoclonal antibody (mAb) 6A7 against L1 protein were screened by cell fusion techniques. Western Blot and immunofluorescence assay (IFA) demonstrated the specificity of the mAb. The L1 protein was truncated for prokaryotic expression (N1∼N7) and Dot-ELISA showed that 6A7 recognized N3 (aa 200-350). The immunodominant regions were truncated again for expression, with 6A7 recognizing N6 (aa 251-305). The N6 proteins were further truncated and then were constructed an four-segment eukaryotic expression vector. IFA showed that 6A7 could recognize amino acid 262-279. Amino acid 262-279 was selected to be truncated into short peptides P1 and P2. Finally, Peptide-ELISA and Dot-ELISA showed that the epitope regions of mAb 6A7 were amino acid 262-273. The mAbs with defined epitopes can lay the foundation for the analysis of antigenic epitope characteristics and promote the development of epitope peptide vaccines.

Brewpitopes: a pipeline to refine B-cell epitope predictions during public health emergencies.

The application of B-cell epitope identification to develop therapeutic antibodies and vaccine candidates is well established. However, the validation of epitopes is time-consuming and resource-intensive. To alleviate this, in recent years, multiple computational predictors have been developed in the immunoinformatics community. Brewpitopes is a pipeline that curates bioinformatic B-cell epitope predictions obtained by integrating different state-of-the-art tools. We used additional computational predictors to account for subcellular location, glycosylation status, and surface accessibility of the predicted epitopes. The implementation of these sets of rational filters optimizes in vivo antibody recognition properties of the candidate epitopes. To validate Brewpitopes, we performed a proteome-wide analysis of SARS-CoV-2 with a particular focus on S protein and its variants of concern. In the S protein, we obtained a fivefold enrichment in terms of predicted neutralization versus the epitopes identified by individual tools. We analyzed epitope landscape changes caused by mutations in the S protein of new viral variants that were linked to observed immune escape evidence in specific strains. In addition, we identified a set of epitopes with neutralizing potential in four SARS-CoV-2 proteins (R1AB, R1A, AP3A, and ORF9C). These epitopes and antigenic proteins are conserved targets for viral neutralization studies. In summary, Brewpitopes is a powerful pipeline that refines B-cell epitope bioinformatic predictions during public health emergencies in a high-throughput capacity to facilitate the optimization of experimental validation of therapeutic antibodies and candidate vaccines.

Identification of key residues of B cell epitopes in hemagglutinin of H6 influenza A virus.

IMPORTANCE: Since the escape immunity of influenza A viruses (IAVs) is mainly caused by the continuous antigenic variations in HA, the identification of key antigenic epitopes is crucial for better understanding of the escape immunity and vaccine development for IAVs. The antigenic sites of several HA subtypes, including H1, H3, H5, and H9, have been well characterized, whereas those of H6 subtype are poorly understood. Here, we mapped nine key residues of antigenic epitopes in H6 through escape mutants using a panel of MAbs. Moreover, MAbs 4C2 and 6E3, targeting 140 and 89 residues, respectively, could protect mice against lethal challenge of MA E-Teal/417. These key residues of antigenic epitopes identified here provide the molecular targets for further elucidating the antigenic evolution of H6 and better preparing the vaccine against H6 IAV.

Genetic variation of E6 and E7 genes of human papillomavirus type 16 from central China.

BACKGROUND: Persistent high-risk human papillomavirus (HR-HPV) infection is an important factor in the development of cervical cancer, and human papillomavirus type 16 (HPV-16) is the most common HR-HPV type worldwide. The oncogenic potential of HPV-16 is closely related to viral sequence variation. METHODS: In order to clarify the variant characteristics of HPV-16 E6 and E7 genes in central China, E6 and E7 sequences of 205 HPV-16 positive samples were amplified by polymerase chain reaction. PCR products of E6 and E7 genes were further sequenced and subjected to variation analysis, phylogenetic analysis, selective pressure analysis and B-cell epitope prediction. RESULTS: Twenty-six single nucleotide variants were observed in E6 sequence, including 21 non-synonymous and 5 synonymous variants. Twelve single nucleotide variants were identified in E7 sequence, including 6 non-synonymous and 6 synonymous variants. Four new variants were found. Furthermore, nucleotide variation A647G (N29S) in E7 was significantly related to the higher risk of HSIL and cervical cancer. Phylogenetic analysis showed that the E6 and E7 sequences were all distributed in A lineage. No positively selected site was found in HPV-16 E6 and E7 sequences. Non-conservative substitutions in E6, H31Y, D32N, D32E, I34M, L35V, E36Q, L45P, N65S and K75T, affected multiple B-cell epitopes. However, the variation of E7 gene had little impact on the corresponding B-cell epitopes (score < 0.85). CONCLUSION: HPV-16 E6 and E7 sequences variation data may contribute to HR-HPV prevention and vaccine development in Jingzhou, central China.

BCEDB: a linear B-cell epitopes database for SARS-CoV-2.

The 2019 Novel Coronavirus (SARS-CoV-2) has infected millions of people worldwide and caused millions of deaths. The virus has gone numerous mutations to replicate faster, which can overwhelm the immune system of the host. Linear B-cell epitopes are becoming promising in prevention of various deadly infectious diseases, breaking the general idea of their low immunogenicity and partial protection. However, there is still no public repository to host the linear B-cell epitopes for facilitating the development vaccines against SARS-CoV-2. Therefore, we developed BCEDB, a linear B-cell epitopes database specifically designed for hosting, exploring and visualizing linear B-cell epitopes and their features. The database provides a comprehensive repository of computationally predicted linear B-cell epitopes from Spike protein; a systematic annotation of epitopes including sequence, antigenicity score, genomic locations of epitopes, mutations in different virus lineages, mutation sites on the 3D structure of Spike protein and a genome browser to visualize them in an interactive manner. It represents a valuable resource for peptide-based vaccine development. Database URL: http://www.oncoimmunobank.cn/bcedbindex.

A novel vaccine candidate against A. baumannii based on a new OmpW family protein (OmpW2); structural characterization, antigenicity and epitope investigation, and in-vivo analysis.

A. baumannii is an MDR pathogen whose SARS-CoV-2 has recently increased its mortality rate in hospitalized patients. So, the virulence factors investigation and design of a vaccine against this bacterium seem to be critical. In this regard, the OmpW2 protein was structurally characterized by this study, and its B-T cell epitopes were mapped by bioinformatic tools. In-vivo analyses were employed to verify the immunogenicity of this protein and its selected epitopes. The results indicated that OmpW2 is a conserved virulent antigen, not toxic for the host, and not similar to the human or mouse proteome. A putative interaction between OmpW2 and a Fe-S-cluster redox enzyme was detected. Based on the results, OmpW2 belongs to the OmpW superfamily and eight beta sheets have been predicted in its tight beta-barrel structure. Several exposed epitopes were detected in the OmpW2 sequence and structure, and a sub-unit potential vaccine was generated based on the epitopes. The ELISA results indicated that after the second booster vaccination of BALB/c mice with the whole OmpW2 protein or its sub-unit fragment, the IgG titer significantly raised (p < 0.05). The mortality rate and the bacterial burden in the lung, liver, kidney, and spleen in both passive and active immunized mice were significantly decreased (p ≤ 0.001). In-vivo experiments confirmed that the OmpW2 whole protein and its sub-unit fragment induce the host immune system and can be applied to design a commercial vaccine or diagnostic kit.

Structure/epitope analysis and IgE binding activities of three cyclophilin family proteins from Dermatophagoides pteronyssinus.

Cyclophilins (CyPs) are involved in basic cellular functions and a wide variety of pathophysiological processes. Many CyPs have been identified as the aetiological agent and influence on the immune system. In the present study, the physicochemical and immunologic characteristics of three proteins of CyPs family (CyPA, CyPB and CyPE) were analyzed. The results indicated that CyPE showed a closer evolutionary relationship with allergenic CyPA. The structure and antigenicity of CyPE was significantly similar with CyPA. B-cell epitopes of CyPE and CyPA were predicted via multiple immunoinformatics tools. Three consensus B-cell epitopes of CyPE and CyPAs were finally determined. To verify results of in silico analysis, three proteins of CyPs family (CyPA, CyPE and CyPB) were cloned and expressed from Dermatophagoides pteronyssinus. ELISA results indicated that the positive reaction rates of the three proteins to patient serum are CyPA (21.4%), CyPE (7.1%), and CyPB (0%), illustrating that the IgE activity was exhibited in CypA and CypE excluding CyPB. Structure and immunoinformatics analysis demonstrated that the RNA-binding motif of CyPE could reduce the immunogenicity of PPIase domain of CyPE. The reason that CyPB has no IgE activity might be the structure mutation of CyPB on B-cell epitopes.

Genetic diversity and in silico analysis of Plasmodium knowlesi Serine Repeat Antigen (SERA) 3 antigen 2 in Malaysia.

Plasmodium knowlesi is the leading cause of malaria in Malaysia. Serine Repeat Antigens (SERAs) have an essential role in the parasite life cycle. However, genetic characterization on P. knowlesi SERA3 Ag2 (PkSERA3 Ag2) is lacking. In the present study, nucleotide diversity, natural selection, and haplotypes of PkSERA3 Ag2 in clinical samples from Peninsular Malaysia and Malaysian Borneo were investigated. A total of 50 P. knowlesi clinical samples were collected from Peninsular Malaysia and Malaysian Borneo. The PkSERA3 Ag2 gene was amplified using PCR, and subsequently cloned and sequenced. Genetic diversity, haplotype, natural selection as well as genetic structure and differentiation of PkSERA3 Ag2 were analysed. In addition, in silico analyses were performed to identify repeat motifs, B-cell epitopes, and antigenicity indices of the protein. Analysis of 114 PkSERA3 Ag2 sequences revealed high nucleotide diversity of the gene in Malaysia. A codon-based Z-test indicated that the gene underwent purifying selection. Haplotype and population structure analyses identified two distinct PkSERA3 Ag2 clusters (K = 2, ΔK = 721.14) but no clear genetic distinction between PkSERA3 Ag2 from Peninsular Malaysia and Malaysian Borneo. FST index indicated moderate differentiation of the gene. In silico analyses revealed unique repeat motifs among PkSERA3 Ag2 isolates. Moreover, the amino acid sequence of PkSERA3 Ag2 exhibited potential B-cell epitopes and possessed high antigenicity indices. These findings enhance the understanding of PkSERA3 Ag2 gene as well as its antigenic properties. Further validation is necessary to ascertain the utility of PkSERA3 Ag2 as a serological marker for P. knowlesi infection.

Deep serological profiling of the Trypanosoma cruzi TSSA antigen reveals different epitopes and modes of recognition by Chagas disease patients.

BACKGROUND: Trypanosoma cruzi, the agent of Chagas disease, displays a highly structured population, with multiple strains that can be grouped into 6-7 evolutionary lineages showing variable eco-epidemiological traits and likely also distinct disease-associated features. Previous works have shown that antibody responses to 'isoforms' of the polymorphic parasite antigen TSSA enable robust and sensitive identification of the infecting strain with near lineage-level resolution. To optimize the serotyping performance of this molecule, we herein used a combination of immunosignaturing approaches based on peptide microarrays and serum samples from Chagas disease patients to establish a deep linear B-cell epitope profiling of TSSA. METHODS/PRINCIPLE FINDINGS: Our assays revealed variations in the seroprevalence of TSSA isoforms among Chagas disease populations from different settings, hence strongly supporting the differential distribution of parasite lineages in domestic cycles across the Americas. Alanine scanning mutagenesis and the use of peptides of different lengths allowed us to identify key residues involved in antibody pairing and the presence of three discrete B-cell linear epitopes in TSSAII, the isoform with highest seroprevalence in human infections. Comprehensive screening of parasite genomic repositories led to the discovery of 9 novel T. cruzi TSSA variants and one TSSA sequence from the phylogenetically related bat parasite T. cruzi marinkellei. Further residue permutation analyses enabled the identification of diagnostically relevant or non-relevant substitutions among TSSA natural polymorphisms. Interestingly, T. cruzi marinkellei TSSA displayed specific serorecognition by one chronic Chagas disease patient from Colombia, which warrant further investigations on the diagnostic impact of such atypical TSSA. CONCLUSIONS/SIGNIFICANCE: Overall, our findings shed new light into TSSA evolution, epitope landscape and modes of recognition by Chagas disease patients; and have practical implications for the design and/or evaluation of T. cruzi serotyping strategies.