Prediction of promiscuous multiepitope-based peptide vaccine against RdRp of rotavirus using immunoinformatics studies

ABSTRACT Rotavirus, a dsRNA virus in the Reoviridae family, shows a segmented genome. The VP1 gene encodes the RNA-dependent RNA polymerase (RdRp). This study aims to develop a multiepitope-based vaccine targeting RdRp using immunoinformatic approaches. In this study, 100 available nucleotide sequences of VP1-Rotavirus belonging to different strains across the world were retrieved from NCBI database. The selected sequences were aligned, and a global consensus sequence was developed by using CLC work bench. The study involved immunoinformatic approaches and molecular docking studies to reveal the promiscuous epitopes that can be eventually used as active vaccine candidates for Rotavirus. In total, 27 highly immunogenic, antigenic, and non-allergenic T-cell and B-cell epitopes were predicted for the Multiepitope vaccine (MEV) against rotavirus. It was also observed that MEV can prove to be effective worldwide due to its high population coverage, demonstrating the consistency of this vaccine. Moreover, there is a high docking interaction and immunological response with a binding score of −50.2 kcal/mol, suggesting the vaccine's efficacy. Toll-like receptors (TLRs) also suggest that the vaccine is physiologically and immunologically effective. Collectively, our data point to an effective MEV against rotavirus that can effectively reduce viral infections and improve the health status worldwide.

Computer-Aided vaccine design for selected positive-sense single stranded RNA viruses

Spontaneous mutations and lack of replication fidelity in positive-sense single stranded RNA viruses (+ssRNA virus) result in emergence of genetic variants with diverse viral morphogenesis and surface proteins that affect its antigenicity. This high mutability in +ssRNA viruses has induced antiviral drug resistance and ability to overcome vaccines that subsequently resulted in rapid viral evolution and high mortality rate in human and livestock. Computer aided vaccine design and immunoinformatics play a crucial role in expediting the vaccine production protocols, antibody production and identifying suitable immunogenic regions or epitopes from the genome sequences of the pathogens. T cell and B cell epitopes can be identified in pathogens by immunoinformatics algorithms and methods that enhance the analysis of protective immunity, vaccine safety, immunity modelling and vaccine efficacy. This rapid and cost-effective computational vaccine design promotes development of potential vaccine that could induce immune response in host against rapidly mutating pathogens like +ssRNA viruses. Epitope-based vaccine is a striking concept that has been widely employed in recent years to construct vaccines targeting rapidly mutating +ssRNA viruses. Therefore, the present review provides an overview about the current progress and methodology in computeraided vaccine design for the most notable +ssRNA viruses namely Hepatitis C virus, Dengue virus, Chikungunya virus and Coronaviruses. This review also highlights the applications of various immunoinformatics tools for vaccine design and for modelling immune response against +ssRNA viruses.

Analysis of lactate dehydrogenase gene polymorphisms and prediction of B cell epitopes in four human Plasmodium species / 中国血吸虫病防治杂志

Objective To analyze the polymorphism of Plasmodium lactate dehydrogenase (pLDH) gene and predict B-cell epitopes in pLDH peptides in four species of human malaria parasites. Methods The blood samples and epidemiological characteristics were collected from malaria cases in Yunnan Province registered in the National Notifiable Disease Report System. The pLDH genes of four human Plasmodium species were amplified using nested PCR assay and sequenced. The polymorphisms of pLDH genes was analyzed using the software MEGA version 7.0.26 and DnaSP version 5.10, and the B-cell epitopes were predicted in pLDH peptides using the Immune Epitope Database (IEDB). Results The sequences of P. vivax LDH (PvLDH), P. falciparum LDH (PfLDH), P. ovale LDH (PoLDH) and P. malariae LDH (PmLDH) genes were obtained from 153, 29, 17 and 11 blood samples from patients with P. vivax, P. falciparum, P. ovale and P. malariae malaria, respectively, which included 15, 2, 4 and 2 haplotypes and had a nucleotide diversity (π) of 0.104. A high level of intra-species differentiation was seen in the PoLDH gene (π = 0.012), and the π values were all < 0.001 for PvLDH, PfLDH and PmLDH genes. Active regions of B-cell antigen were predicted in the pLDH peptide chain of four human malaria parasites, of 4 to 5 in each chain, and the activity score was approximately 0.430. Among these peptide chains, the “86-PGKSDKEWNRD-96” short-peptide was a B-cell epitope shared by all four species of human malaria parasites, and the “266-GQYGHS (T)-271” short-peptide was present in PvLDH and PoLDH peptide chains, while “212-EEVEGIFDR-220” was only found in the PvLDH peptide chain, and “208-LISDAE-213” was only seen in the PfLDH peptide chain. Conclusions The PoLDH gene polymorphism may be derived from the weak negative purification selection, while PvLDH, PfLDH and PmLDH genes may maintain a relatively conservative state. There may be two B-cell epitopes “212-EEVEGIFDR-220” and “208-LISDAE-213” in the proximal region of the C terminal in the pLDH peptide chain, which is feasible to differentiate between P. vivax and P. falciparum infections.

An immunoglobulin y that specifically binds to an in silico-predicted unique epitope of Zika virus non-structural 1 antigen

Objective: To identify unique immunogenic epitopes of Zika virus non-structural 1 (NS1) antigen and produce immunoglobulin Y (IgY) for potential use in he diagnosis of of Zika virus infection. Methods: Immunogenic epitopes were identified using in silico B-cell epitope prediction. A synthetic peptide analog of the predicted epitope was used to induce antipeptide IgY production in hens which was purified using affinity chromatography. Presence of purified IgY and its binding specificity were performed by gel electrophoresis and ELISA, respectively. Results: Out of the nine continuous epitopes identified, the sequence at position 193-208 (LKVREDYSLECDPAVI) was selected and used to produce anti-peptide IgY. The produced IgY was found to bind to the synthetic analog of the Zika virus NS1 immunogenic epitope but not to other flaviviruses and random peptides from other pathogens. Conclusions: In this study, we identified an immunogenic epitope unique to Zika virus that can be used to develop a serodiagnostic tool that specifically detect Zika virus infection.

Prediction and analysis of T-cell and B-cell epitopes of Mycobacterium tuberculosis Rv2657c protein / 实用医学杂志

Objective To predict the epitopes of Mycobacterium tuberculosis Rv2657c protein, to understand its immunogenicity Methods The T?cell and B?cell epitopes of Mycobacterium tuberculosis Rv2657c protein were predicted by DNAStar software package. The homology of Rv2657c amino acid sequence with the human protein sequences was prepared using Blast method, then the CTL epitopes were predicted using SYFPEITHI supermotif method, BIMAS quantitative motif method and NetCTL prediction method, and the Th epitopes were predicted by RANKPEP and SYFPEITHI supermotif prediction method. Results The prediction using DNAStar software package showed that Rv2657c protein had 5 B?cell epitopes and 6 T?cell epitopes. The protein had 6 CTL epitopes and there were 38 Th epitopes. Conclusion Rv2657c protein has both B?cell epitopes and T?cell epitopes. It may be a candidate target antigen for the studies of vaccine and diagnosis of tuberculosis.

Identification of novel Mycobacterium tuberculosis peptides as diagnostic reagents for tuberculosis among human immunodeficiency virus infected population / 中华传染病杂志

Objective To screen and validate the major histocompatibility complex class-Ⅰ(MHC-Ⅰ) restricted tuberculosis peptides as potential diagnostic reagents in tuberculosis interferon-gamma release assay (IGRA) used among human immunodeficiency (HIV)-infected population.Methods Candidate peptides were encoded by Mycobacterium tuberculosis (TB) RD (Region of difference).Computer software was used to predict and select CD8+ T cell epitopes restricted by MHC-Ⅰ molecules with high frequency and high affinity among HIV-infected people.Then peptides containing CD8+ T cell epitope were synthesized and screened in vitro.The sensitivity and specificity of IGRA using the above mixed peptides as stimulants were compared with those of IGRA using early secretory antigen target-6 (ESAT-6,molecular weight of 6 000) and culture filtrate protein-10 (CFP-10,molecular weight of 10 000) as stimulants among HIV-infected population.Results Eight overlapping peptides,including Rv0222176-191,Rv1980c122-138,Rv1985c105-120,Rv3425141-165,Rv3873133-151,Rv3873158-166,Rv387878-86,Rv3879c673-690,were obtained finally,which were able to stimulate the production of interferon-gamma from peripheral CD8+ T cells of tuberculosis patients,but not from peripheral blood mononuclear cells (PBMC) of healthy controls.Among the 25 patients with HIV/TB co-infection,the sensitivities of IGRA using the combination peptides (CP) and that using rESAT-6/CFP-10 (CE) were low (68％ vs 48％,x2 =2.052,P=0.152).However,the sensitivity increased to 92％ by using the combination of CP and CE,which was significantly higher than that using rESAT-6/CFP-10 alone (x2 =11.523,P ＜ 0.01),and the specificity was not affected.Conclusion These RD peptides with CD8+ T cell epitopes can increase the sensitivity of IGRA in detecting HIV/TB co-infection,which may improve the detection rate of tuberculosis in HIV infected population.

Preparation of monoclonal antibodies against gamma-type phospholipase A2 inhibitors and immunodetection of these proteins in snake blood

Background: Paracoccidioidomycosis (PCM) is a neglected systemic mycosis caused by a dimorphic fungus of the Paracoccidioides genus. The standard diagnosis is based on isolation of the fungi in culture, and by microscopic visualization of characteristic multiple budding yeast cells in biological samples. However, in some situations, access to the site of injury prevents the collection of biological material. A variety of immuno-serological techniques has proven useful for allowing inferring diagnosis with a certain degree of certainty, thus optimizing time. The aim of this study was to standardize and validate the Dot-ELISA (DE) assay, comparing it with the serological standard, double immunodiffusion (DI). Methods: In order to standardize the DE assay, 143 serum samples were used. Out of those, 23 were from apparently healthy patients, 77 were from patients with confirmed PCM and 43 were from patients with other lung infections (tuberculosis, aspergillosis and histoplasmosis). To validate the DE technique, 300 serum samples from patients with PCM clinical suspicion (probable and possible cases) were employed, and these results were compared with those of DI. Results: The DE assay showed sensitivity of 91%, specificity of 95.4%, positive predictive value of 96%, negative predictive value of 98.2%, accuracy of 93%, and great precision (k = 0.93). In addition, the nitrocellulose membranes have proved to be viable for using at least 90 days after P. brasiliensis B-339 antigen sensitization. Conclusion: Dot-ELISA method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity. Furthermore, Dot-ELISA shows the prospect of being transferred to laboratories of mycoserology including those with fewer resources or even to be used directly in the field. It has an excellent shelf life ­ membranes coated with antigen can be used for testing without changes in the pattern of reactivity among laboratories ­ and presents reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological standard methodology. Based on the present findings, it possible to state that this technique constitutes a remarkable option to be used in routine diagnosis for public health centers.(AU)

Preparation of monoclonal antibodies against gamma-type phospholipase A2 inhibitors and immunodetection of these proteins in snake blood

Abstract Background: The gamma-type phospholipase A2 inhibitor (LI) is a natural protein commonly found in snake serum, which can neutralize pathophysiological effects of snake venom phospholipases A2. Therefore, this protein is a potential candidate to the development of a novel antivenom. To the best of our knowledge, there is no antibody currently available for PLI identification and characterization. Methods: Bioinformatics prediction of epitope using DNAStar software was performed based on the sequence of Sinonatrix annularis PLI (SaPLI). The best epitope 151CPVLRLSNRTHEANRNDLIKVA172 was chosen and synthesized, and then conjugated to keyhole limpet hemocyanin and bovine serum albumin for use as an immunogen and plate-coating antigen, respectively. Results: Eighteen IgG anti-PLI mAb hybridoma cell strains were obtained, and all the mAbs had positive interaction with recombinant His6-PLI and natural SaPLI. Moreover, the mAb from 10E9 strain was also successfully used for the immunodetection of other snake serum PLIs. cDNA sequence alignment of those PLIs from different snake species showed that their epitope segments were highly homologous. Conclusions: The successful preparation of anti-PLImAb is significant for further investigation on the relationship between the structure and function of PLIs, as well as the interaction between PLIs and PLA2s.

In silico analysis of Brucella abortus Omp2b and in vitro expression of SOmp2b

PURPOSE: At present, there is no vaccine available for the prevention of human brucellosis. Brucella outer membrane protein 2b (Omp2b) is a 36 kD porin existed in common Brucella pathogens and it is considered as priority antigen for designing a new subunit vaccine. MATERIALS AND METHODS: In the current study, we aimed to predict and analyze the secondary and tertiary structures of the Brucella abortus Omp2b protein, and to predict T-cell and B-cell epitopes with the help of bioinformatics tools. Subsequently, cloning and expression of the short form of Omp2b (SOmp2b) was performed using pET28a expression vector and Escherichia coli BL21 host, respectively. The recombinant SOmp2b (rSOmp2b) was purified with Ni-NTA column. RESULTS: The recombinant protein was successfully expressed in E. coli host and purified under denaturation conditions. The yield of the purified rSOmp2b was estimated by Bradford method and found to be 220 microg/mL of the culture. CONCLUSION: Our results indicate that Omp2b protein has a potential to induce both B-cell- and T-cell-mediated immune responses and it can be evaluated as a new subunit vaccine candidate against brucellosis.

Sequence analysis of the 5′ third of glycoprotein C gene of South American bovine herpesviruses 1 and 5

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) share high genetic and antigenic similarities, but exhibit marked differences in tissue tropism and neurovirulence. The amino-terminal region of glycoprotein C (gC), which is markedly different in each of the viruses, is involved in virus binding to cellular receptors and in interactions with the immune system. This study investigated the genetic and antigenic differences of the 5′ region of the gC (5′ gC) gene (amino-terminal) of South American BoHV-1 (n=19) and BoHV-5 (n=25) isolates. Sequence alignments of 374 nucleotides (104 amino acids) revealed mean similarity levels of 97.3 and 94.2% among BoHV-1 gC (gC1), respectively, 96.8 and 95.6% among BoHV-5 gC (gC5), and 62 and 53.3% between gC1 and gC5. Differences included the absence of 40 amino acid residues (27 encompassing predicted linear epitopes) scattered throughout 5′ gC1 compared to 5′ gC5. Virus neutralizing assays testing BoHV-1 and BoHV-5 antisera against each isolate revealed a high degree of cross-neutralization between the viruses, yet some isolates were neutralized at very low titers by heterologous sera, and a few BoHV-5 isolates reacted weakly with either sera. The virus neutralization differences observed within the same viral species, and more pronounced between BoHV-1 and BoHV-5, likely reflect sequence differences in neutralizing epitopes. These results demonstrate that the 5′ gC region is well conserved within each viral species but is divergent between BoHV-1 and BoHV-5, likely contributing to their biological and antigenic differences.

Prediction and identification of T-cell epitopes in major group 3 allergen de-rived from Dermatophagoides farina / 中国血吸虫病防治杂志

Objective To predict and identify T cell epitopes of major group 3 allergen derived from Dermatophagoides fari-na(Der f 3). Methods The T cell epitopes of Der f 3 were analyzed through the sequence analysis by using the bioinformatics online tools. The five predicted peptides of T-cell epitopes were artificially synthesized. The spleen lymphocytes were co-cultured with the five T cell epitopes by using the modified MTT method and the levels of IL-2,IFN-γ,IL-4 and IL-5 in the supernatant of the cultures were detected by ELISA. Results Five T cell epitopes of Der f 3 were predicted and three of which could pro-mote the proliferation of the mouse spleen lymphocytes. The secretions of IL-2 and IFN-γwere significantly induced and the se-cretions of IL-4 and IL-5 were significantly decreased by three of five prediction epitopes of Der f 3:37GDCPYQISLQSSSHFC-GG54,98IYQHENYDSMTIDNDVALIKLKTPMT123 and 164SELQRVDIDVVSREQCDQLYS184. Conclusion Three T cell epitopes of Der f 3 have been initially identified,which lays the foundation of the diagnosis and treatment of asthma.

Prediction and identification of B-cell epitopes on monkey B virus envelope protein gD / 中国比较医学杂志

Objective To Identify B-cell epitopes on monkey B virus envelope protein gD.Methods Base on bioinformatics software, secondary structure, hydrophilicity, surface accessibility, antigenic index and flexibility of monkey B virus envelope protein gD was analysed and some potential peptide epitopes were forecasted.Then, the interactions between synthetic peptides and BV positive sera were detected by Enzyme-linked immunosorbent assay ( ELISA) .At last, The sensitivity and specificity of synthetic peptides was evaluated used 20 samples of Standard sera by ELISA.Result Seven epitopes were forecasted by Bioinformatics analysis. Four synthetic peptides, sequence as 46 LPPLEQKTD54 , 106 RGAPEATRSDA116 , 291 PELAPEERGTSRTPGD306 and 361 AVYLVRRRGR370 could be reacted with positive sera pool.The sensitivity of 4 synthetic peptides changed form 40% to 70% and the specificity were 100% for 4 synthetic peptides. Conclusion There are at least four linear epitope on B virus gD protein.

Prediction and identification of linear B-cell epitopes in major group 3 aller-gen derived from Dermatophagoides farina / 中国血吸虫病防治杂志

Objective To predict and identify the linear B-cell epitopes in the major group 3 allergen derived from Derma-tophagoides farina(Der f 3). Methods The linear B-cell epitopes of Der f 3 allergen were analyzed based on the physicochemi-cal properties of amino acids including antigenicity,surface accessibility,flexibility,hydrophilicity,beta-turn by online bioinfor-matics softwares. The eight predicted peptides of linear B-cell epitopes were artificially synthesized and incubated with three aller-gic serum pools(4 serum samples in each),which were consisted of total 12 serum samples from the allergic individuals,and the strong positive epitopes were selected. Results Eight B-cell epitopes from Der f 3 were predicted successfully. Five of eight B-cell epitopes were identified with strong IgE-binding abilities followed by specific IgE assay. The amino acid sequences of them were following:33KAKAGDCP40, 86HASGGEKIQVAEIYQHENYDSMTID110, 118LKTPMTLDQTNAKPVPLPPQGSDVKVG144, 156QEGSYSLP163 and 199DVANGGVDSCQGDSGGPVVD218. Conclusions Five linear B-cell epitopes of Der f 3 allergen have been identified successfully. This result might provide a basis of the diagnosis and treatment for asthma.

Advances of Bioinformatics Tools Applied in Virus Epitopes Prediction / 中国病毒学·英文版

In recent years, the in silico epitopes prediction tools have facilitated the progress of vaccines development significantly and many have been applied to predict epitopes in viruses successfully. Herein, a general overview of different tools currently available, including T cell and B cell epitopes prediction tools, is presented. And the principles of different prediction algorithms are reviewed briefly. Finally, several examples are present to illustrate the application of the prediction tools.

Prediction of the B cell epitopes of human heparanase protein and determination of their immunogenicity / 中华微生物学和免疫学杂志

Objective To predict the secondary structure and the B cell epitopes of human heparanase protein, and to identify its immunogenicity. Methods The flexible regions of secondary structure and the B cell epitopes of human heparanase amino acid sequence were predicted by DNAStar and Bcepred software. The multiple antigenic peptides (MAP) of the epitopes were synthesized in 8-branch form. Rabbits were immunized with the 8-branch MAPs mixed with a universal T-helper epitope human IL-1β peptide (VQGEESNDK, amino acid 163-171 ). The immunogenicity of the synthesized peptides was evaluated by ELISA, Western blot and immunohistochemistry. Results Amino acid 1 -15 ( MAP1), 279-293 (MAP2) and 175-189(MAP3) of large-subunit of human heparanase protein was predicted as the most potential epitopes of human heparanase protein. All the three synthesized MAPs induced high titer of antibodies. ELISA, Western blot and immunohistochemistry analysis showed all the three MAPs could produce high titer serum antibodies, antibodies induced by MAP1 and MAP2 had high specific binding activity , and MAP2 antibody showed the strongest binding activity with liver cancer tissues. Conclusion The large-subunit No. 1-15, 279-293 amino acid of human heparanase protein may be the B cell preponderant epitopes and the strongest immunogenicity may be No. 279-293 peptide, which provided a theoretic basis for the antibody and vaccine development of heparanase subunit peptide.

Prediction of HLA-A2.1 restricted CTL epitope of SSX gene family and affinity assay of these epitopes with MHC-Ⅰ molecule / 第三军医大学学报

Objective To explore the immunotherapeutic strategy based on SSX tumor gene family by the CTL epitope prediction of the members of SSX tumor gene family and affinity assay of the epitope combining with MHCⅠ molecule.Methods The CTL epitope in the members of SSX gene family were predicted with the predictive software of HLA-A2.1 restricted CTL epitope.The 4 predicted epitope peptides were synthesized: P1(AMTKLGFKA),P4 (AMTKLGFNV),P5(AMTKLGFKV) and P6(VMTKLGFKV).After the affinity between these peptides and MHC-Ⅰ molecule were examined respectively,the combining stability between these peptides and the MHC-Ⅰ molecule were investigated respectively.Results As compared with positive control peptide HBcAg18-27,all of the synthesized peptides except P1 showed high affinity with MHC-Ⅰ molecule.The combining stability assay indicated that dissociation complex 50(DC_(50)) for P1,P4 and the positive control HBcAg 18-27 was all above 8.0 h,but DC_(50) for P5 and P6 was between 2 h and 4 h.Conclusion Two ideal peptides were screened out from the predicted CTL epitope peptides by in vitro MHC-Ⅰ combining affinity and stability assay,which provided the experimental data for the further identification of these epitope peptides in vivo.

Prediction of the secondary structure and B cell epitope of human decay-accelerating factor / 中国免疫学杂志

Objective:To predict the secondary structure and B cell epitope of human decay accelerating factor Methods:The accuracy of two secondary structure prediction methods,Chou & Fasman and PHDsec,was compared,and then human DAF secondary structure was analyzed by PHDsec Hydrophilicity scores and solvent accessibility of DAF SCR1 4 were obtained by methods of Hopp & Woods and PHDacc respectively Combined with the secondary structure of DAF,the B cell epitopes in DAF were predicted Results:PHDsec which use multiple sequence alignment,achieve a better prediction accuracy There is no alpha helix in SCR1 4 of human DAF but only beta sheet and loop segments; The computer predicted most possible epitope in DAF localize within amino acid residues Pro73 Val79?Arg130 Leu139 and Glu156 Cys163 Conclusion:These results will be useful in estimate of the epitope and activity sites localized within human DAF

Prediction of MHC class Ⅰ binding peptides using neural network ensembles / 中国病理生理杂志

AIM: To predict MHC class Ⅰ binding peptides by using neural network ensembles. METHODS: As a combination of neural networks, neural network ensemble (NNE) was here used to improve the predictive performance. Based on a database of 628 nonamers and their classified binding capacities, the generalized NNEs were used to classify peptides respectively with non, low, moderate and high binding capacities to MHC class I molecule encoded by gene HLA-A*0201. The predictive power of NNE was further evaluated by running generalized NNE on a set of actual T-cell epitopes. RESULTS: The generalized NNEs achieved an average predictive hit rate of 0.8 for the above classifications. In addition, NNE was also efficient in the prediction of the potential T-cell epitopes, and about 84% of the actual T-cell epitopes were among the potentially antigenic peptides with high and moderate affinities. CONCLUSION: The NNEs can be applied in the prediction of MHC class Ⅰ binding peptides, and moreover, after proper modifications, they can be conveniently extended to cover peptides with any length and thus suitable for the prediction of peptides binding to other MHC class Ⅰ or even class Ⅱ molecules.

Prediction for Helper T Cell Epitopes and its Application in Vaccine Development against Parasite Infection / 中国寄生虫学与寄生虫病杂志

Cellular immunity plays an important role in defense against diseases, such as pathogenic infection,autoimmunity and tumor. With the progress of molecular immunology, mechanisms of T cellular immunity, and the T cell epitopes and functional genomics, studies on the prediction based on data-drived for T cell epitopes has been highlighted, and could be one of the useful tools for application in vaccine development. This review summarizes theory and methodology of prediction for helper T cell epitopes, and their application in vaccine development against parasites, and new research directions are also discussed.