Epitopes Research Articles

Detection of Serum IgG Specific for Brachyspira pilosicoli and "Brachyspira canis" in Dogs.

Brachyspira pilosicoli (B. pilosicoli) is a pathogen in pigs, poultry, and humans causing colitis, diarrhea, and poor growth rates. Its role as a canine pathogen is controversial, and the seroprevalence of specific IgG antibodies against B. pilosicoli in dogs is unknown. A further, not yet officially recognized Brachyspira species in dogs is "Brachyspira canis" ("B. canis"), which is proposed to be apathogenic. This study evaluates enzyme-linked immunosorbent assays (ELISAs) measuring serum IgG antibodies specific for B. pilosicoli or "B. canis" and investigates levels of specific IgG antibodies against B. pilosicoli and "B. canis" in a cohort of clinical patients presented at an animal referral clinic. These ELISAs use detergent-extracted antigens from B. pilosicoli and "B. canis". To increase analytic specificity, we precipitated the antigens with trichloroacetic acid (TCA) to isolate and concentrate the respective protein fraction. Our results indicate that a large number of serum IgG antibodies bind to shared epitopes of detergent-extracted antigens of the two spirochaetes. Our data also suggest that dogs might not only carry B. pilosicoli but also have "B. canis"-specific serum IgG antibodies.

Mammalian cell display with automated oligo design and library assembly allows for rapid residue level conformational epitope mapping

Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens.

Designing an mRNA Vaccine against P. jirovecii Involved in Fatal Pneumonia Infections via Comparative Proteomics and Reverse Vaccinology Approaches.

Pneumocystis jirovecii is the most emerging life-threating health problem that causes acute and fatal pneumonia infection. It is rare and more contagious for patients with leukemia and immune-deficiency disorders. Until now there is no treatment available for this infection therefore, it is needed to develop any treatment against this pathogen. In this work, we used comparative proteomics, robust immune-informatics, and reverse vaccinology to create an mRNA vaccine against Pneumocystis jirovecii by targeting outer and transmembrane proteins. Using a comparative subtractive proteomic analysis of two Pneumocystis jirovecii proteomes, a distinct non-redundant Pneumocystis jirovecii (strain SE8) proteome was chosen. Seven Pneumocystis jirovecii transmembrane proteins were chosen from this proteome based on hydrophilicity, essentiality, virulence, antigenicity, pathway interaction, protein-protein network analysis, and allergenicity. The reverse vaccinology approach was used to predict the immunogenic and antigenic epitopes of major histocompatibility complex (MHC) I, II and B-cells from the selected proteins on the basis of their antigenicity, toxicity and allergenicity. These immunogenic epitopes were linked together to construct the mRNA-based vaccine. To enhance the immunogenicity, suitable adjuvant, linkers (GPGPG, KK, and CYY), and PRDRE sequences were used. Through predictive modeling and confirmation via the Ramachandran plot, we assessed secondary and 3D structures. The adjuvant RpfE was incorporated to enhance the vaccine construct's immunogenicity (GRAVY index: -0.271, instability index: 39.53, antigenicity: 1.0428). The physiochemical profiling of vaccine construct was predicted it an antigenic, efficient, and potential vaccine. Notably, strong interactions were observed between the vaccine construct and TLR-3/TLR-4 (-1301.7 kcal/mol-1 and -1374.7 kcal/mol-1). The results predicted that mRNA-based vaccines trigger a cellular and humoral immune response, making the vaccine potential candidate against Pneumocystis jirovecii and it is more suitable for in-vitro analysis and validation to prove its effectiveness.

Comparative analysis of the RVA VP7 and VP4 antigenic epitopes circulating in Iran and the Rotarix and RotaTeq vaccines

Analyzing the lineages and detecting antigenic variation in immunogenic motifs of Group A Rotavirus (RVA) variants is crucial because it can impact vaccine efficacy. This study investigated the circulating lineages of VP4 and VP7 proteins of human RVA isolates and their phylogeny in ≤24-month-old symptomatic, rotavirus-positive children with transudative diarrhea within 48 h of admission to Mofid Children's Hospital between December 2020 and March 2022 in Tehran, Iran. Antigen detection was performed by ELISA, RNA extraction, and semi-nested multiplex PCR for G/P genotypes, followed by sequencing and bioinformatic analysis using multiple sequence alignments in MEGA and phylogenetic analysis by Geneious Prime. The similarity of VP7 and VP4 amino acids with the RotaTeq and Rotarix vaccine strains for cytotoxic T cell and antigenic epitopes was evaluated using the UCSF Chimera Molecular Modeling System. Overall, 27.3 % of the samples were RVA positive, showing untypeable (2.5 %), single (76.9 %), and mixed (20.5 %) genotypic characteristics. The strains clustered in the G1/II, G2/IV, G3/I, G4/I, G9/III, P[8]/III, P[4]/V, and P[6]/I lineages. Comparative analysis of VP7 antigenic epitopes showed that the G1/II strains were completely conserved, while the G2/IV, G3/I, G4/I, G6, G9/III strains contained 2, 3–5, 2, 4 and 9 amino acid substitutions, respectively. The P[8]/III genotypes differed by 3 amino acids, while the P[6]/I genotype had the most substitutions. CTL epitopes were completely conserved in G3/I strains, but other genotypes differed by 1–4 amino acids compared to the vaccine strains. Given the diversity of circulating RVA genotypes and the observed mutations in neutralizing and CTL epitopes, immune escape by some of the strains is likely in Iran. This finding underscores the importance of evaluating the effect of rotavirus vaccines on local genotypes and related lineages before implementing a vaccination program.

Identification of Two Linear Epitopes on MGF_110-13L Protein of African Swine Fever Virus with Monoclonal Antibodies.

African swine fever caused by African swine fever virus (ASFV) is an acute, highly contagious swine disease with high mortality. To facilitate effective vaccine development and find more serodiagnostic targets, fully exploring the ASFV antigenic proteins is urgently needed. In this study, the MGF_110-13L was identified as an immunodominant antigen among the seven transmembrane proteins. The main outer-membrane domain of MGF_110-13L was expressed and purified. Two monoclonal antibodies (mAbs; 8C3, and 10E4) against MGF_110-13L were generated. The epitopes of two mAbs were preliminary mapped with the peptide fusion proteins after probing with mAbs by enzyme-linked immunosorbent assay (ELISA) and Western blot. And the two target epitopes were fine-mapped using further truncated peptide fusion protein strategy. Finally, the core sequences of mAbs 8C3 and 10E4 were identified as 48WDCQDGICKNKITESRFIDS67, and 122GDHQQLSIKQ131, respectively. The peptides of epitopes were synthesized and probed with ASFV antibody positive pig sera by a dot blot assay, and the results showed that epitope 10E4 was an antigenic epitope. The epitope 10E4 peptide was further evaluated as a potential antigen for detecting ASFV antibodies. To our knowledge, this is the first report of antigenic epitope information on the antigenic MGF_110-13L protein of ASFV.

Liposome-based in situ antigen-modification strategy for "universal" T-cell-receptor engineered T cell in cancer immunotherapy.

T-cell receptor (TCR) engineered T-cell therapy, unlike chimeric antigen receptor T-cell therapy, relies on the inherent ability of TCRs to detect a wider variety of antigenic epitopes, such as protein fragments found internally or externally on cells. Hence, TCR-T-cell therapy offers broader possibilities for treating solid tumors. However, because of the complicated process of identifying specific antigenic peptides, their clinical application still encounters significant challenges. Thus, we aimed to establish a novel "universal" TCR-T "artificial antigen expression" technique that involves the delivery of the antigen to tumor cells using DSPE-PEG-NY-ESO-1157-165 liposomes (NY-ESO-1 Lips) to express TCR-T-cell-specific recognition targets. In vitro as well as in vivo studies revealed that they could accumulate efficiently in the tumor area and deliver target antigens to activate the tumor-specific cytotoxic T-cell immune response. NY-ESO-1 TCR-T therapy, when used in combination, dramatically curbed tumor progression and extended the longevity of mice. Additionally, PD-1 blockage enhanced the therapeutic effect of the aforementioned therapy. In conclusion, NY-ESO-1 Lips "cursed" tumor cells by enabling antigenic target expression on their surface. This innovative technique presents a groundbreaking approach for the widespread utilization of TCR-T in solid tumor treatment.

Identification of broad-spectrum B-cell and T-cell epitopes of H9 subtype avian influenza virus HA protein using polypeptide scanning1

The H9N2 subtype avian influenza virus (AIV) hemagglutinin (HA) protein is a major immunogen in which HA1 is a genetic variant and HA2 is relatively conserved. Identifying broad-spectrum antigen epitopes targeting HA1 is crucial for vaccine design and detection. Based on the phylogenetic and serological analyses, we identified 2 antigenic groups and 3 representative viruses: A/chicken/Jiangsu/JY040218C/2019, A/pigeon/Jiangsu/JY020616/2019, and A/chicken/Jiangsu/WX090312/2018. An overlapping peptide library was synthesized using HA1 amino acid sequences of the viruses as templates. Through peptide scanning of the sera against different strains of H9N2 subtype AIV, we identified peptides from 4 regions (H9-2/3, H9-20/21, H9-26, and H9-29/30/31) that demonstrated broad-spectrum reactivity. Immunological assay results demonstrated that H9-21 (219RIFKPLIGPRPLVNGLMGRI239), H9-26 (269SGESHGRILKTDLKMGSCTV289), and H9-30 (309YAFGNCPKYI GVKSLKLAVG329) effectively induced antibody generation and conferred partial protective efficacy against the parent virus JY040218C. The results of lymphocyte proliferation and ELISpot assays indicated that peptides H9-15 (159MRWLTQKNNAYPTQDAQYTN179), H9-22 (229PLVNGLMGRINYYWSVLKP G249), and H9-23 (239NYYWSVLKPGQTLRIKSDGN259) could effectively stimulate the expression of interferon-gamma in peripheral blood lymphocytes of chickens immunized against different strains of H9N2 AIV. Collectively, 5 novel cell epitopes H9-15, H9-22, H9-23, H9-26, and H9-30, including the best B cell epitope H9-26 and the best T cells epitope H9-22, were identified that could be targeted for vaccine design or detection approaches against H9N2 AIVs.

Development of a novel multi-epitope vaccine for brucellosis prevention

Brucellosis, a zoonotic disease caused by Brucella, presents a significant threat to both animal and human health. In animals, the disease can lead to infertility, miscarriage, and high fever, while in humans, symptoms may include recurrent fever, fatigue, sweating, hepatosplenomegaly, and joint and muscle pain following infection. Treatment often involves long-term antibiotic therapy, placing a substantial psychological and financial burden on patients. While vaccination is crucial for prevention, current animal vaccines have drawbacks such as residual virulence, and a safe and effective human vaccine is lacking. Hence, the development of a vaccine for brucellosis is imperative. In this study, we utilized bioinformatics methods to design a multi-epitope vaccine targeting Brucella. Targeting Heme transporter BhuA and polysaccharide export protein, we identified antigenic epitopes, including six cytotoxic T lymphocyte (CTL) dominant epitopes, six helper T lymphocyte (HTL) dominant epitopes, one conformation B cell dominant epitope, and three linear B cell dominant epitopes. By linking these epitopes with appropriate linkers and incorporating a Toll-like receptor (TLR) agonist (human beta-defensin-2) and an auxiliary peptide (Pan HLA-DR epitopes), we constructed the multi-epitope vaccine (MEV). The MEV demonstrated high antigenicity, non-toxicity, non-allergenicity, non-human homology, stability, and solubility. Molecular docking analysis and molecular dynamics simulations confirmed the interaction and stability of the MEV with receptors (MHCI, MHCII, TLR4). Codon optimization and in silico cloning validated the translation efficiency and successful expression of MEV in Escherichia coli. Immunological simulations further demonstrated the efficacy of MEV in inducing robust immune responses. In conclusion, our findings suggest that the engineered MEVs have the potential to stimulate both humoral and cellular immune responses, offering valuable insights for the future development of safe and efficient Brucella vaccines.

Chemical Synthesis and Antigenicity Evaluation of an Aminoglycoside Trisaccharide Repeating Unit of Pseudomonas aeruginosa Serotype O5 O-Antigen Containing a Rare Dimeric-ManpN3NA.

Pseudomonas aeruginosa bacteria are becoming increasingly resistant against multiple antibiotics. Therefore, the development of vaccines to prevent infections with these bacteria is an urgent medical need. While the immunological activity of lipopolysaccharide O-antigens in P. aeruginosa is well-known, the specific protective epitopes remain unidentified. Herein, we present the first chemical synthesis of highly functionalized aminoglycoside trisaccharide 1 and its acetamido derivative 2 found in the P. aeruginosa serotype O5 O-antigen. The synthesis of the trisaccharide targets is based on balancing the reactivity of disaccharide acceptors and monosaccharide donors. Glycosylations were analyzed by quantifying the reactivity of the hydroxyl group of the disaccharide acceptor using the orbital-weighted Fukui function and dual descriptor. The stereoselective formation of 1,2-cis-α-fucosylamine linkages was achieved through a combination of remote acyl participation and reagent modulation. The simultaneous SN2 substitution of azide groups at C2' and C2″ enabled the efficient synthesis of 1,2-cis-β-linkages for both 2,3-diamino-D-mannuronic acids. Through a strategic orthogonal modification, the five amino groups on target trisaccharide 1 were equipped with a rare acetamidino (Am) and four acetyl (Ac) groups. Glycan microarray analyses of sera from patients infected with P. aeruginosa indicated that trisaccharides 1 and 2 are key antigenic epitopes of the serotype O5 O-antigen. The acetamidino group is not an essential determinant of antibody binding. The β-D-ManpNAc3NAcA residue is a key motif for the antigenicity of serotype O5 O-antigen. These findings serve as a foundation for the development of glycoconjugate vaccines targeting P. aeruginosa serotype O5.

A Novel Multi-epitope Vaccine Based on Virulence Stimulating Two-component Protein EsrA Against Fish Pathogenic Edwardsiella tarda – An Immunoinformatic Approach

Edwardsiellosis, caused by the gram-negative pathogen Edwardsiella tarda, is a major global aquaculture threat. Despite vaccination being a potential strategy, development of effective vaccines remains a challenge. The study aimed to develop a multi-epitope vaccine (EsrAmev) against virulent EsrA antigen in fish pathogenic E. tarda using an immunoinformatic approach. The study identified antigenic, non-allergenic, and nontoxic epitopes for cytotoxic T lymphocytes, helper T-lymphocytes, and linear Blymphocytes. They were combined to create the EsrAmev construct, with TLR4 agonist added for the immunogenicity. The physiochemical analysis of EsrAmev by ProtParam server showed a molecular weight of 29 kDa, an antigenicity score of 0.5966, and a solubility of 0.823563 with immunogenic, non-allergenic and non-toxic properties. Robetta, Mod, and Galaxy refiner servers refined the EsrAmev 3D structure, achieving 97.4% RAMA-favoured region, stable interactions, binding stability, and structural stiffness with TLR4 receptor. The EsrAmev construct, optimized for high immune responses in Escherichia coli K12 model, was successfully cloned into the pET28a (+) vector in silico for future wet lab applications. These findings suggests that the EsrAmev multi-epitope vaccine might have potential for the prophylaxis of E.tardacausing Fish Edwardsiellosis.

CO-DELIVERY of glutamic acid-extended peptide antigen and imidazoquinoline TLR7/8 agonist via ionizable lipid nanoparticles induces protective anti-tumor immunity

Cancer vaccines aim at generating cytotoxic CD8+ T cells that kill cancer cells and confer durable tumor regression. Hereto, CD8+ peptide epitopes should be presented by antigen presenting cells to CD8+ T cells in lymphoid tissue. Unfortunately, in unformulated soluble form, peptide antigens are poorly taken up by antigen presenting cells and do not efficiently reach lymph nodes. Hence, the lack of efficient delivery remains a major limitation for successful clinical translation of cancer vaccination using peptide antigens. Here we propose a generic peptide nanoformulation strategy by extending the amino acid sequence of the peptide antigen epitope with 10 glutamic acid residues. The resulting overall anionic charge of the peptide allows encapsulation into lipid nanoparticles (peptide-LNP) by electrostatic interaction with an ionizable cationic lipid. We demonstrate that intravenous injection of peptide-LNP efficiently delivers the peptide to immune cells in the spleen. Peptide-LNP that co-encapsulate an imidazoquinoline TLR7/8 agonist (IMDQ) induce robust innate immune activation in a broad range of immune cell subsets in the spleen. Peptide-LNP containing the minimal CD8+ T cell epitope of the HPV type 16 E7 oncoprotein and IMDQ induces high levels of antigen-specific CD8+ T cells in the blood, and can confer protective immunity against E7-expressing tumors in both prophylactic and therapeutic settings.

The N545S and K717N substitution at the N-glycosylation sites of the S2 subunit of avian infectious bronchitis virus can significantly enhance viral pathogenicity

The S2 subunit of infectious bronchitis virus (IBV) is a heavily glycosylated protein that can impact various characteristics of the virus. It is currently known that N-glycosylation modifications are predominantly located on the S2 subunit. However, the exact role of their N-glycosylation modification remains undisclosed. To elucidate the function of these N-glycosylation sites, we identified 14 common sites distributed on the S2 subunit of the 5 genotypes of IBV in present study. Subsequently, we selected 7 sites to generate mutants and assessed their impact on viral virulence, replication ability, and antigenicity. Our finding revealed that only 2 substitutions, N545S and K717N, increased the viral replication titer and antigenicity, and ultimately the pathogenicity in chicks. To delve into the mechanisms underlying this increased pathogenicity, we discovered that K717N can change the structure of antigenic epitopes. The N545S substitution not only influenced antigenic epitope structure, but also enhanced the ability of the virus to enter CEKs during the early stages of viral replication. These results suggest that the enhanced viral pathogenicity associated with N545S and K717N substitutions is multifaceted, with acceleration of the viral membrane fusion process and alterations in epitope structure representing crucial factors in the capability of N-glycosylation modifications to boost viral virulence. These insights provide valuable guidance for the efficient development of live attenuated vaccines.

Nucleotide sequence characterization, amino acid variations and 3D structural analysis of HN protein of the NDV VIId genotype.

Haemagglutinin-neuraminidase (HN) is one of the membrane proteins of Newcastle disease virus (NDV) that plays a significant role during host viral infection. Therefore, antibodies against HN are vital for the host's ability to protect itself against NDV infection due to their critical functions in viral infection. As a result, HN has been a candidate protein in vaccine development against the Newcastle disease virus. This report used the full-length sequence of the HN protein of NDV isolated in Iran (VIId subgenotype). We characterize and identify amino acid substitutions in comparison to other more prevalent NDV genotypes, VII subgenotypes and vaccine strains. Furthermore, bioinformatics tools were applied to determine the three-dimensional structure, molecular dynamics simulation and prediction of B-cell antigenic epitopes. The results showed that the antigenic regions of our isolate are quite comparable to the other VII subgenotypes of NDV isolated from different geographical places. Moreover, by employing the final 3D structure of our HN protein, the amino acid residues are proposed as a B-cell epitope by epitope prediction servers, which leads to the introduction of linear and conformational antigenic sites. Immunoinformatic vaccine design principles currently exhibit tremendous potential for developing a new generation of candidate vaccines quickly and economically to eradicate infectious viruses, including the NDV. In order to accomplish this, focus is directed on residues that might be considered antigenic.

Universal characterization of epitope immunodominance from a multiscale model of clonal competition in germinal centers.

We introduce a multiscale model for affinity maturation, which aims to capture the intraclonal, interclonal, and epitope-specific organization of the B-cell population in a germinal center. We describe the evolution of the B-cell population via a quasispecies dynamics, with species corresponding to unique B-cell receptors (BCRs), where the desired multiscale structure is reflected on the mutational connectivity of the accessible BCR space, and on the statistical properties of its fitness landscape. Within this mathematical framework, we study the competition among classes of BCRs targeting different antigen epitopes, and we construct an effective immunogenic space where epitope immunodominance relations can be universally characterized. We finally study how varying the relative composition of a mixture of antigens with variable and conserved domains allows for a parametric exploration of this space, and we identify general principles for the rational design of two-antigen cocktails.

Dual chemical bonding construction of electrochemical peptide sensor based on GDY/MOFs(Fe) composite for ultra-low determination of prostate-specific antigen

A novel “double chemical bonding” electrochemical peptide biosensor 2FcP-GA-GDY(Fe)@NMIL-B was developed for highly selective, ultrasensitive, and ultrastable identification of prostate-specific antigen (PSA). The C–Fe–O chemical bond linking Fe-Graphdiyne (Fe-GDY) with NH2-MIL88B(Fe) (NMIL88B) as the first chemical bonding of electrode carrier Fe-GDY@NH2-MIL88B(Fe) (GDY(Fe)@NMIL) significantly accelerates electron transport. With glutaraldehyde (GA) as a crosslinking agent, the Schiff-base –NC- formed by GDY(Fe)@NMIL nanocomposites links the two Fc molecules labeled peptides (2FcP) as the second chemical bonding, facilitating high-density attachment of peptides to the electrode carrier in a firm manner. When the PSA analyte is introduced to identify and cleave the specific peptide, the release of ferrocene from its head leads to a decrease in the electrical signal, enabling sensitive detection. The prepared sensing platform exhibits exceptional analytical performance for PSA with an extended linear response range from 10 fg mL−1 to 50 ng mL−1. Additionally, the detection limit has been significantly reduced to an ultra-low level of only 0.94 fg mL−1, surpassing those reported in most literature by several orders of magnitude. Moreover, the 2FcP-GA-GDY(Fe)@NMIL-B sensor has excellent selectivity and stability while also showcasing great potential for practical application of PSA detection in human serum using the standard addition method.

Monoclonal antibody development for early detection of ASFV I73R protein: Identification of a linear antigenic epitope

African swine fever virus (ASFV), which was first identified in northern China in 2018, causes high mortality in pigs. Since the I73R protein in ASFV is abundantly expressed during the early phase of virus replication, it can be used as a target protein for early diagnosis. In this study, the I73R protein of ASFV was expressed, and we successfully prepared a novel monoclonal antibody (mAb), 8G11D7, that recognizes this protein. Through both indirect immunofluorescence and Western blotting assays, we demonstrated that 8G11D7 can detect ASFV strains. By evaluating the binding of the antibody to a series of I73R-truncated peptides, the definitive epitope recognized by the monoclonal antibody 8G11D7 was determined to be 58 DKTNTIYPP 66. Bioinformatic analysis revealed that the antigenic epitope had a high antigenic index and conservatism. This study contributes to a deeper understanding of ASFV protein structure and function, helping establish ASFV-specific detection method.

Silica Nanochannels as Nanoreactors for the Confined Synthesis of Ag NPs to Boost Electrochemical Stripping Chemiluminescence of the Luminol-O2 System for the Sensitive Aptasensor.

Herein, we, for the first time, synthesize silver nanoparticles (Ag NPs) within the nanochannels of amino group-functionalized vertically ordered mesoporous silica films (NH2-VMSF) and investigate their coreaction accelerator role in the luminol-dissolved oxygen (O2) electrochemical stripping chemiluminescence (ESCL) system. The synthesized Ag NPs are capable of electrocatalytic reduction of O2 to superoxide radicals, and meanwhile, sliver ions (Ag+) electrochemically stripped from Ag NPs can promote the amount of luminol anion radicals, generating the boosted ECL intensity of the luminol-dissolved O2 system. This proposed Ag NPs@NH2-VMSF on the indium tin oxide electrode was applied to construct the ESCL aptasensor for quantitative determination of prostate-specific antigen (PSA), yielding a low detection limit [0.19 pg/mL (S/N = 3)] and a broad linear dynamic range (1 pg/mL to 100 ng/mL). Furthermore, good analytical performance of PSA in serum with satisfactory recoveries and low relative standard deviation values is achieved by our developed ESCL aptasensor, rendering it a convenient and sensitive method for PSA determination in clinical applications and further broadening the strategy of ESCL techniques.

Virtual Discovery of Immune-Stimulating Epitopes in Chikungunya Virus for Vaccine Design

Epitope identification is a key step in vaccine development, and this can be achieved much faster and less expensively with in silico methods, compared to traditional methods for vaccine production. In silico methods applied in this research utilised both bioinformatics and immunoinformatics approaches for chikungunya virus vaccine design, which involved the retrieval of sequences from databases, and identification of conserved regions within the sequences by multiple sequence alignment on the MEGA X software (Pennsylvania State University, State College, USA). The epitopes in the conserved regions were selected, and various immunological predictions and screenings were carried out by employing immunological databases and tools. This process identifies epitopes such as conservation of cytotoxic T lymphocyte, helper T lymphocytes, and B cell epitopes. The primary, secondary, and tertiary structure of the vaccine was also predicted using structure predicting servers, and finally, the vaccine candidate was docked to toll-like receptor 4 to study its binding affinity and configuration. A total of 125 conserved antigenic epitopes were selected from capsid, 6K, and E1 proteins, which were found to be non-allergens and conform to acceptable physicochemical standards, as reported by other authors with similar work. The epitopes were predicted to be capable of inducing cytotoxic T lymphocytes, helper T lymphocytes, and B cell production. Construction of secondary structure was done using the Self-Optimized Prediction Method with Alignment (SOPMA), which predicted 17.96% α-helices, and 4.69% β-turns, among others. Predicting the tertiary structure provided five models, of which Model 1 was selected on the bases of its confidential score of 0.59, estimated TM-score of 0.79±0.09, and root mean square deviation of 8.0±4.4Å. Validity analysis revealed a Ramachandran plot where 97.2% of the vaccine residue was within the favoured region, and the peptide showed a Z-score of -1.52. The predicted peptide effectively docked with toll-like receptor 4 with a binding energy of -1,072.8. From the data obtained, it was revealed that the selected epitopes are highly immunogenic, non-allergenic, conform to native protein, and form a peptide capable of vaccine application. The authors can conclude this is a promising candidate for vaccine design and development.

Comparative functional characterization and in vitro immunological cross-reactivity studies on Daboia russelii and Craspedocephalus malabaricus venom.

Snake venom is a complex mixture of organic and inorganic constituents, including proteins and peptides. Several studies showed that antivenom efficacy differs due to intra- and inter-species venom variation. In the current study, comparative functional characterization of major enzymatic proteins present in Craspedocephalus malabaricus and Daboia russelii venom was investigated through various in vitro and immunological cross-reactivity assays. The enzymatic assays revealed that hyaluronidase and phospholipase A2 activities were markedly higher in D. russelii. By contrast, fibrinogenolytic, fibrin clotting and L-amino acid oxidase activities were higher in C. malabaricus venom. ELISA results suggested that all the antivenoms had lower binding potential towards C. malabaricus venom. For D. russelii venom, the endpoint titration value was observed at 1:72 900 for all the antivenoms. In the case of C. malabaricus venom, the endpoint titration value was 1:2700, except for Biological E (1:8100). All these results, along with the avidity assays, indicate the strength of venom-antivenom interactions. Similarly, the western blot results suggest that all the antivenoms showed varied efficacies in binding and detecting the venom antigenic epitopes in both species. The results highlight the need for species-specific antivenom to better manage snakebite victims.

Functional and structural basis of human parainfluenza virus type 3 neutralization with human monoclonal antibodies.

Human parainfluenza virus type 3 (hPIV3) is a respiratory pathogen that can cause severe disease in older people and infants. Currently, vaccines against hPIV3 are in clinical trials but none have been approved yet. The haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins of hPIV3 are major antigenic determinants. Here we describe naturally occurring potently neutralizing human antibodies directed against both surface glycoproteins of hPIV3. We isolated seven neutralizing HN-reactive antibodies and a pre-fusion conformation F-reactive antibody from human memory B cells. One HN-binding monoclonal antibody (mAb), designated PIV3-23, exhibited functional attributes including haemagglutination and neuraminidase inhibition. We also delineated the structural basis of neutralization for two HN and one F mAbs. MAbs that neutralized hPIV3 in vitro protected against infection and disease in vivo in a cotton rat model of hPIV3 infection, suggesting correlates of protection for hPIV3 and the potential clinical utility of these mAbs.