A study based on four immunoassays: Hepatitis C virus antibody against different antigens may have unequal contributions to detection

Background All commercial Hepatitis C virus antibody (anti-HCV) assays use a combination of recombinant antigens to detect antibody response. Antibody responses to individual antigenic regions (core, NS3/4 and NS5) used in assays have not been investigated. Methods In this study, we quantified HCV viral load, tested anti-HCV with four commercial assays (Ortho-ELISA, Murex-ELISA, Architect-CMIA and Elecsys-ECLIA) in 682 plasma specimens. In antigenic region ELISA platform, microwells were coated with three antigens: core (c22-3), NS3/4 (c200) and NS5 individually. The signal-to-cutoff (S/Co) values of different assays, and antibody responses to individual antigens were compared. The specimens were divided into HCV RNA positive group, anti-HCV consistent group, and anti-HCV discrepant group. Results Anti-core and anti-NS3/4 were simultaneously detected in 99.2% of HCV RNA positive specimens and showed great consistency with total anti-HCV signals. Responses to the core region were more robust than those to the NS3/4 region in anti-HCV consistent group (p < 0.001). Anti-NS5 only occurred in companying with responses to the core and NS3/4 antigens, and failed to affect the final anti-HCV positive signals. In anti-HCV discrepant group, 39.0% of positive signals could not be traced back to any single antigenic region. Conclusion Antibody responses to the core and NS3/4 antigens were stronger, whereas responses to the NS5 antigen were the weakest, indicating that individual antigenic regions played different roles in total anti-HCV signals. This study provides an impetus for optimizing commercial anti-HCV assays.

Structural and allergenic properties of the Fatty Acid Binding Protein from shrimp Litopenaeus vannamei

Background: The shrimp Litopaneus vanammei is an important source of food allergens but its allergenic repertoire is poorly characterized. Cross reactivity between crustacean and mites has been characterized, with tropomyosin, the most relevant allergen involved. The aim of this study was the structural and immunological characterization of an allergen belonging to the Fatty Acid Binding Protein (FABP) family from L. vannamei (LvFABP). Methods: ELISA, skin prick test (SPT) and basophil activation assays were performed to determine IgE reactivity and allergenicity of LvFABP. LC-MS/MS and Circular Dichroism experiments were done for structural analysis. B-cell epitope mapping with overlapping peptides, and cross-inhibition studies using human sera were done to identify antigenic regions and cross-reactivity. Results: The recombinant LvFABP showed IgE reactivity in 27% of allergic patients tested and showed allergenic activity when tested for basophil activation and SPT in shrimp sensitized patients. CD-spectroscopy of LvFABP revealed that the protein is folded with a secondary structure composed of mainly β-strands and a smaller fraction of  helices. This is consistent with molecular modelling results, which exhibit a typical β barrel fold with two α-helices and ten β-strands. Epitope mapping identified two IgE binding antigenic regions and inhibition assays found high cross reactivity between LvFABP and Blo t 13, mediated by the antigenic region involving amino acids 53 to 73. Conclusions: Our results support LvFABP as an allergen with cross reactivity with the allergen Blo t 13. This new allergen could help to understand new mechanisms of sensitization to seafood such as shrimp.

In silico analysis of cross reactivity among phospholipases from Hymenoptera species

Background: Phospholipases are enzymes with the capacity to hydrolyze membrane lipids and have been characterized in several allergenic sources, such as hymenoptera species. However, cross-reactivity among phospholipases allergens are little understood. The objective of this study was to determine potential antigenic regions involved in cross-reactivity among allergens of phospholipases using an in silico approach. Methods: In total, 18 amino acids sequences belonging to phospholipase family derived from species of the order hymenoptera were retrieved from the UniProt database to perform phylogenetic analysis to determine the closest molecular relationship. Multialignment was done to identify conserved regions and matched with antigenic regions predicted by ElliPro server. 3D models were obtained from modeling by homology and were used to locate cross-reactive antigenic regions. Results: Phylogenetic analysis showed that the 18 phospholipases split into four monophyletic clades (named here as A, B, C and D). Phospholipases from A clade shared an amino acid sequences’ identity of 79%. Antigenic patches predicted by Ellipro were located in highly conserved regions, suggesting that they could be involved in cross-reactivity in this group (Ves v 1, Ves a 1 and Ves m 1). Conclusions: At this point, we advanced to the characterization of potential antigenic sites involved in cross-reactivity among phospholipases. Inhibition assays are needed to confirm our finding.

Identification and expression analysis of antigenic sites of hepatitis C virus genotype 3a NS3 and NS5A genes of local isolate

Background Hepatitis C virus, a silent killer, has infected 71 million people globally. The recombinant viral antigenic proteins might be used in the early diagnosis of HCV infection. The NS3 and NS5A genes of HCV function in HCV replication and influence host cellular factors that are involved in HCV pathogenesis. The current study was designed to select NS3 and NS5A antigenic sites, amplified, cloned, and expressed in order to find out better assays for diagnosis or drug and vaccine development. The antigenic sites within NS3 and NS5A genes were selected and confirmed through sequencing and were cloned. The antigenic recombinant proteins were expressed in bacterial strain E. coli BL21ply*, and the expression was confirmed by western blotting by using gene-specific and vector-specific antibodies. Results Specific antigenic regions within the NS3 and NS5A genes of the HCV 3A genotype were amplified. PCR results showed 328 bp and 747 bp antigenic regions, respectively. The regions were confirmed by DNA sequencing and cloned into a bacterial expression vector. Expression analysis showed 12 kDa and 28 kDa of NS3 and NS5A antigenic recombinant proteins, respectively. Taken together, these studies will help to analyze the genetic variability within the local HCV isolates as these antigenic recombinant proteins were quite important in the screening of HCV-infected patients. Conclusions This study might help to enhance the progress in the treatment of HCV infection through the modeling of HCV non-structural genes (NS3 and NS5A) from local isolate, and it might also present the viral genes as potential therapeutic targets.

In silico analysis of cross reactivity among phospholipases from Hymenoptera species

Background: Phospholipases are enzymes with the capacity to hydrolyze membrane lipids and have been characterized in several allergenic sources, such as hymenoptera species. However, cross-reactivity among phospholipases allergens are little understood. The objective of this study was to determine potential antigenic regions involved in cross-reactivity among allergens of phospholipases using an in silico approach. Methods: In total, 18 amino acids sequences belonging to phospholipase family derived from species of the order hymenoptera were retrieved from the UniProt database to perform phylogenetic analysis to determine the closest molecular relationship. Multialignment was done to identify conserved regions and matched with antigenic regions predicted by ElliPro server. 3D models were obtained from modeling by homology and were used to locate cross-reactive antigenic regions. Results: Phylogenetic analysis showed that the 18 phospholipases split into four monophyletic clades (named here as A, B, C and D). Phospholipases from A clade shared an amino acid sequences’ identity of 79%. Antigenic patches predicted by Ellipro were located in highly conserved regions, suggesting that they could be involved in cross-reactivity in this group (Ves v 1, Ves a 1 and Ves m 1). Conclusions: At this point, we advanced to the characterization of potential antigenic sites involved in cross-reactivity among phospholipases. Inhibition assays are needed to confirm our finding.

Whole Genome In-Silico Analysis of South African G1P[8] Rotavirus Strains before and after Vaccine Introduction over a Period of 14 Years

Rotavirus G1P[8] strains account for more than half of the group A rotavirus (RVA) infections in children under five years of age, globally. A total of 103 stool samples previously characterized as G1P[8] and collected seven years before and seven years after introducing the Rotarix® vaccine in South Africa were processed for whole-genome sequencing. All the strains analyzed had a Wa-like constellation (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1). South African pre- and post-vaccine G1 strains were clustered in G1 lineage-I and II while the majority (84.2%) of the P[8] strains were grouped in P[8] lineage-III. Several amino acid sites across ten gene segments with the exception of VP7 were under positive selective pressure. Except for the N147D substitution in the antigenic site of eight post-vaccine G1 strains when compared to both Rotarix® and pre-vaccine strains, most of the amino acid substitutions in the antigenic regions of post-vaccine G1P[8] strains were already present during the pre-vaccine period. Therefore, Rotarix® did not appear to have an impact on the amino acid differences in the antigenic regions of South African post-vaccine G1P[8] strains. However, continued whole-genome surveillance of RVA strains to decipher genetic changes in the post-vaccine period remains imperative.

Computationally validated SARS-CoV-2 CTL and HTL Multi-Patch Vaccines designed by reverse epitomics approach, shows potential to cover large ethnically distributed human population worldwide

BackgroundThe SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a positive-sense single-stranded RNA coronavirus responsible for the ongoing 2019-2020 COVID-19 outbreak. The highly contagious COVID-19 disease has spread to 216 countries in less than six months. Though several vaccine candidates are being claimed, an effective vaccine is yet to come. In present study we have designed and theoretically validated novel Multi-Patch Vaccines against SARS-CoV-2.MethodologyA novel reverse epitomics approach, “overlapping-epitope-clusters-to-patches” method is utilized to identify multiple antigenic regions from the SARS-CoV-2 proteome. These antigenic regions are here termed as “Ag-Patch or Ag-Patches”, for Antigenic Patch or Patches. The identification of Ag-Patches is based on clusters of overlapping epitopes rising from a particular region of SARS-CoV-2 protein. Further, we have utilized the identified Ag-Patches to design Multi-Patch Vaccines (MPVs), proposing a novel methodology for vaccine design and development. The designed MPVs were analyzed for immunologically crucial parameters, physiochemical properties and cDNA constructs.ResultsWe identified 73 CTL (Cytotoxic T-Lymphocyte), 49 HTL (Helper T-Lymphocyte) novel Ag-Patches from the proteome of SARS-CoV-2. The identified Ag-Patches utilized to design MPVs cover 768 (518 CTL and 250 HTL) overlapping epitopes targeting different HLA alleles. Such large number of epitope coverage is not possible for multi-epitope vaccines. The large number of epitopes covered implies large number of HLA alleles targeted, and hence large ethnically distributed human population coverage. The MPVs:Toll-Like Receptor ectodomain complex shows stable nature with numerous hydrogen bond formation and acceptable root mean square deviation and fluctuation. Further, the cDNA analysis favors high expression of the MPVs constructs in human cell line.ConclusionHighly immunogenic novel Ag-Patches are identified from the entire proteome of SARS CoV-2 by a novel reverse epitomics approach. We conclude that the novel Multi-Patch Vaccines could be a highly potential novel approach to combat SARS-CoV-2, with greater effectiveness, high specificity and large human population coverage worldwide.Abstract FigureABSTRACT FIGURE:A Multi-Patch Vaccine design to combat SARS-CoV-2 and a method to prepare thereof.Multi-Patch Vaccine designing to combat SARS-CoV-2 infection by reverse epitomics approach, “Overlapping-epitope-clusters-to-patches” method.

Novel Protein-Based Pneumococcal Vaccines: Assessing the Use of Distinct Protein Fragments Instead of Full-Length Proteins as Vaccine Antigens

Non-serotype-specific protein-based pneumococcal vaccines have received extensive research focus due to the limitations of polysaccharide-based vaccines. Pneumococcal proteins (PnPs), universally expressed among serotypes, may induce broader immune responses, stimulating humoral and cellular immunity, while being easier to manufacture and less expensive. Such an approach has raised issues mainly associated with sequence/level of expression variability, chemical instability, as well as possible undesirable reactogenicity and autoimmune properties. A step forward employs the identification of highly-conserved antigenic regions within PnPs with the potential to retain the benefits of protein antigens. Besides, their low-cost and stable construction facilitates the combination of several antigenic regions or peptides that may impair different stages of pneumococcal disease offering even wider serotype coverage and more efficient protection. This review discusses the up-to-date progress on PnPs that are currently under clinical evaluation and the challenges for their licensure. Focus is given on the progress on the identification of antigenic regions/peptides within PnPs and their evaluation as vaccine candidates, accessing their potential to overcome the issues associated with full-length protein antigens. Particular mention is given of the use of newer delivery system technologies including conjugation to Toll-like receptors (TLRs) and reformulation into nanoparticles to enhance the poor immunogenicity of such antigens.