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.

Shared epitope classification methods according to Gregersen and de Vries allow adequate characterization of patients with rheumatoid arthritis in a Colombian population

Introduction: The most important genetic association in rheumatoid arthritis (RA) is presented with some alleles from the HLA-DRB1 gene that encode the shared epitope (SE). Objectives: To apply the SE classification methods of Gregersen, de Vries, Raychaudhuri, Mattey, and Tezenas du Montcel in a group of Colombian patients with RA and determine the most common HLA-DRB1 alleles in the population. Methods: RA diagnosis, genetic study of the HLA-DRB1 region using Luminex technology in 50 RA and 50 healthy subjects. For the classification analysis, Fisher's exact test and chi-squared test were applied. Tables were created to count the RA-related alleles. We used odds ratio to determine the risk between the presence of the shared epitope (SE) and anti-cyclic citrullinated peptides (Anti-CCP). Results: Gregersen and de Vries methods were suitable for the characterization of RA in this population (p = .006). The most prevalent HLA-DRB1 alleles in the RA group were 14:02,04:04, 08:02,04:05, and 10:01. High frequencies of the 07:01, 03:01,13:02,01:02, and 12:01 HLA-DRB1 alleles were found in the healthy population. HLA-DRB1 alleles with similar distribution in both populations were 04:07, 15:01, 11:01, 16:02, and 01:01. A high frequency of SE + was observed in Anti-CCP + individuals (63.15%); however, this was not statistically significant [OR2.4 (.63-9.01); p = .19]. Conclusion: The SE classification methods of Gregersen and de Vries were adequate in characterizing RA in a Colombian population group. An equivalence of 100% was verified between the susceptibility alleles defined by de Vries and the alleles assigned as SE according to Gregersen.

Introducción: La asociación genética más importante en artritis reumatoide (AR) se presenta con algunos alelos del gen HLA DRB1 que codifican el epítope compartido (EC). Objetivos: Aplicar los métodos de clasificación de EC de Gregersen et al., de Vries et al., Raychaudhuri et al., Mattey et al., y Tezenas du Montcel et al., en un grupo de pacientes colombianos con AR, y determinar los alelos HLA DRB1 más frecuentes en esta población. Métodos: Diagnóstico para AR, estudio genético de la región HLA DRB1 por tecnología Luminex® de 50 sujetos AR y 50 sanos. Para análisis comparativos de clasificaciones EC, se aplicaron las pruebas test exacto de Fisher y Chi-cuadrado y se realizaron tablas de conteos para los alelos relacionados con AR. Se estimó la razón de odds para determinar el riesgo entre la presencia de EC y los anticuerpos antipéptidos cíclicos citrulinados (anti-PCC). Resultados: Los métodos de Gregersen et al. y de Vries et al. fueron adecuados para la caracterización de AR en esta población (p = 0,006). Los alelos HLA DRB1 más prevalentes en el grupo AR fueron 14:02, 04:04, 08:02, 04:05 y 10:01. Se encontraron altas frecuencias de los alelos HLA DRB1 07:01, 03:01,13:02, 01:02 y 12:01 en población sana. Alelos HLA DRB1 con distribución similar en ambas poblaciones fueron: 04:07, 15:01, 11:01, 16:02 y 01:01. Se observó alta frecuencia de individuos EC+ en el grupo AR anti-PCC+ (63,15%); no obstante, sin asociación estadística (OR: 2,4 [0,63-9,01]; p = 0,19). Conclusión: Los métodos de clasificación para EC de Gregersen et al. y de Vries et al. fueron adecuados caracterizando AR en un grupo de población colombiana. Se corroboró equivalencia del 100% entre los alelos de susceptibilidad definidos por de Vries y los alelos asignados como EC según Gregersen et al.

Design of a multi-epitope subunit vaccine candidate for chikungunya virus using computational methodology

@#Chikungunya virus (CHIKV) is a neglected tropical pathogen that causes fever and long-lasting severe arthralgia. Despite its high morbidity, there is still no licensed specific therapeutic option for it. This study proposes a multi-epitope subunit vaccine candidate for CHIKV, designed using computational methods. It was based on the E2 spike glycoprotein in CHIKV, from which T- and B-cell epitopes were predicted and then refined. The pan HLA DR-binding epitope (PADRE) was added to this refined construct, then simulated compared with the native protein, where it was predicted to elicit more than twice the number of antibody titers. Thus, this construct is potentially effective against CHIKV, which further experimentation using live models would be able to verify. This study also demonstrates the feasibility of using rational tools in the future to further optimize vaccine design.

Epitope identification of calcitonin gene-related peptide antibodies based on ELISA and NMR methods / 中国生物制品学杂志

@#Objective To develop an indirect ELISA-based peptide scanning method combined with nuclear magnetic resonance(NMR)technique for the epitope identification of calcitonin gene-related peptide(CGRP)antibodies.Methods The antigen binding activities of two antibodies(new CGRP antibody and control antibody)were determined by indirect ELISA using each truncated CGRP fragment as coating antigen,and the linear epitope was analyzed according to the EC50value of four-parameter curve. Two-dimensional hydrogen-nitrogen correlation(2D1H-15N HSQC)spectrum of CGRP were acquired by NMR technique,and the binding of antibodies to the arginine of CGRP were analyzed through the disturbance of the antibodies to CGRP signals. Specific arginine modifications were detected by liquid chromatography-mass spectrum(LCMS) and NMR technique,and two arginine resonances were assigned on CGRP by correlating the rank order of the modification rate.ResultsThe antigen binding activities of two antibodies with CGRP(1-37),CGRP(19-37)and CGRP(25-37)showed dose-response relationships,and were fitted with four-parameter equation. However,there were no significant antigen binding with CGRP(1-18),CGRP(19-24)and CGRP(25-37)without C-terminal amide. The linear epitopes of both antibodies were located at the C-terminal of CGRP. The resonances of arginine ε-NH in 2D1H-15N HSQC spectrum disappeared in the presence of the control antibody;and the resonances appeared in the presence of the new antibody. The arginine R11 and R18 of CGRP could bind to the control antibody,but not to the new antibody. The NMR assignment for the arginine resonances were made by correlating the relative ranking of the modification rate where signals A and B arose from R11 ε-NH and R18 ε-NH respectively.ConclusionIn this study,the linear and conformational epitopes of new CGRP antibody and control antibody were identified based on the methods of ELISA and NMR,which may provide a theoretical basis for the design of the candidate therapeutic CGRP antibodies.

Matching strategy for patients with platelet transfusion refractoriness caused by compound antibodies against HLA and CD36 / 中国输血杂志

【Objective】 To search compatible and suitable platelets for platelet transfusion refractoriness (PTR) patient caused by compound antibodies against HLA and CD36. 【Methods】 ELISA method was used to detect the antibody against platelet antigens and the specificity of HLA-I antibody in PTR patients. The specificity of HLA-I antibody and corresponding epitopes of patients were analyzed using MATCH IT! and HLA Matchmaker software. The HLA genotype of both donor and patient was obtained by HLA-SSO method. Compatible or suitable donor platelets for PTR patients were searched through cross-reactive group (CREG) of HLA-I and HLA epitope-matched approach (Eplet). The matching degree was identified using monoclonal antibody-specific immobilization of platelet antigens (MAIPA) and the platelet suspension immunofluores-cence test (PIFT). Finally, the transfusion effect was evaluated according to the corrected count increment (CCI). 【Results】 Compound antibodies against both CD36 and HLA-I antigens were detected in two PTR patients, and their phenotype of CD36 was conformed to be type I deficient. Through LSA testing, high-frequency of HLA -I antibodies was found in these two patients, and the panel reactive antibody in patients 1 and 2 was 56% (54/96) and 53% (51/96), respectively. According to HLA-CREG and Eplet matching strategies, one donor of grade C-matching with patient 1 and one donor of grade D-matching with patient 2 were screened from the CD36 deficiency donor bank, respectively. And the selected donors avoided the antigen of HLA-I antibody epitope. These results of MAIPA and PIFT also confirmed that no immune response was detected between the patient and the donor. And a CCI of >4.5 within 24 hour of transfusion of compatible platelets was obtained in patient 2. 【Conclusion】 For PTR patients caused by HLA and CD36 compound antibodies, a combination strategy including serological cross-matching, HLA-CREG and Eplet approach should be used to select the CD36 deficient donor platelets which evaded the antigen corresponding to HLA-I antibodies and had the compatible HLA epitopes.

Viral selection pressure and CD4+T cell epitope in HLA-DRB1 * 11:01 of HCV infected patients / 中国输血杂志

【Objective】 HLA-DRB1 * 11:01, as a class HLA-Ⅱ gene, was reported to be associated with spontaneous clearance of HCV in Han and Li population. Our study was to investigate the effects of viral selection pressure and CD4+T cell epitope on the natural outcome of HCV infection in HLA-DRB1 * 11:01 positive infected patients. 【Methods】 The positive selection sites and population growth of E1E2 and NS3 genes of common HCV 6a in HLA-DRB1 * 11:01 positive and negative groups in Guangdong were respectively analyzed. The peptide library covering the conserved regions of common HCV genotypes was used to stimulate HCV spontaneous clearance group and chronic infection group using ELISPOT method. Reactive peptides were obtained according to the number of spot-forming cells per well and the frequency of occurrence in different groups. 【Results】 The positive selection sites (PSSs) of E1E2 and NS3 of common HCV 6a in HLA-DRB1 * 11:01 negative group were greater than those in HLA-DRB1 * 11:01 positive group. Furthermore, the number of PPSs in CD4+T cell peptide in HLA-DRB1 * 11:01 negative group were also greater than those in HLA-DRB1 * 11:01 positive group; Both groups of HCV 6a had a population growth in Guangdong, and the expansion trend of HLA-DRB1 * 11:01 negative group was significantly higher than that of HLA-DRB1 * 11 :01 positive group. Compared to HCV chronic infection group, the response rate of HCV spontaneous clearance group to five peptides (C-52 E2691-707, C-119 NS31545-1560, C-134 NS4A1669-1684, C-154 NS4B1912-1927, C-159 NS4B1929-1944) was higher. However, the HCV chronic infection group showed a higher response rate to two of the peptides(C-111 NS31497-1512, C-130 NS31650-1665). When HLA-DRB1 * 11:01 typing was considered, there was no significant difference in HCV-specific immune response generated by PBMCs between HLA-DRB1 * 11:01 positive and HLA-DRB1 * 11:01 negative groups. 【Conclusion】 This study revealed the relationship between viral selection pressure of HLA-DRB1 * 11:01 HCV infected persons and CD4+T cell antigen epitopes. At the same time, CD4+ T cell antigen epitopes of HCV pan-genotype were obtained, providing basic data for the development of T cell vaccine suitable for HCV pan-genotype.

Bioinformatics analysis of VP1 of Coxsackievirus A10 / 公共卫生与预防医学

Objective To predict and analyze the physicochemical properties, structural characteristics, and antigenic epitopes of viral protein (VP) VP1 of Coxsackievirus A10 (CV-A10) by bioinformatics methods. Methods The physicochemical properties and structural characteristics of CV-A10 VP1 were predicted by ProtParam, SOPMA, SWISS-MODEL, PDBsum, and ProSA-web. The antigenic epitopes of CV-A10 VP1 were predicted and analyzed by DNAstar, ABCpred, Bepipred 2.0, ElliPro, DiscoTope-2.0, NetMHCpan-4.1, NetMHCIIpan-4.0, Consurf, VaxiJen v.2.0, AllerTOP v.2.0, ToxinPred2, and IEDB immunogenicity. Results Bioinformatics analysis showed that CV-A10 VP1 was a basic, unstable, and hydrophilic protein, of which the secondary structure mainly consisted of random coil. The analysis revealed that CV-A10 VP1 had multiple potential B and T cell antigenic epitopes as well as a dominant antigenic epitope based on the potential epitope. Conclusion CV-A10 VP1 has multiple potential sites that induce specific humoral and cellular immunity, providing important support for its experimental identification, molecular epidemiological studies, and vaccine development.

Targets of chimeric antigen receptor T-cell in breast cancer and the application progress / 肿瘤研究与临床

Breast cancer is the most common malignant tumor in women. It is particularly important to seek targeted therapy other than surgery, chemoradiotherapy, endocrine therapy. With the continuous exploration of tumor immunotherapy, more and more therapeutic targets have been discovered. This paper reviews therapeutic targets of chimeric antigen receptor T-cell (CAR-T) and the application in breast cancer.

Preliminary immunological evaluation of Mycobacterium tuberculosis multicomponent protein vaccine candidates EPDPA015f and EPDPA015m / 中华微生物学和免疫学杂志

Objective:To preliminarily evaluate the immunogenicity and efficacy of two novel tuberculosis vaccine candidates (a fusion multicomponent protein EPDPA015f and a mixed multicomponent protein EPDPA015m) and to provide a new antigen combination for the development of tuberculosis vaccines.Methods:Recombinant plasmids for the expression of EPDPA015f and EPDPA015m proteins were constructed. Six-week-old BALB/c mice were immunized with EPDPA015f or EPDPA015m in combination with aluminium adjuvant (50 μg/mouse) for three times with an interval of 10 d. The mice were sacrificed 10 d after the last immunization to collect blood and spleen samples. Serum antibody titers and cytokine levels were measured by ELISA, Luminex technique and enzyme-linked immunospot assay (ELISPOT). Mycobacterial growth inhibition assay (MGIA) was used to detect the ability of mouse splenocytes to inhibit the growth of Mtb in vitro. One-way analysis of variance and t-test were used for statistical analysis. Results:Both EPDPA015f and EPDPA015m could induce the production of various cytokines and IgG antibodies at a high level. The levels of cytokines related to Th1 (IL-2, TNF-α, IFN-γ), Th2 (IL-4, IL-6, IL-10) and Th17 (IL-17) as well as other proinflammatory cytokines (GM-CSF, IL-12) were higher in the EPDPA015f group than in the adjuvant group ( P<0.05). The titer of IgG antibody induced by EPDPA015f was as high as 1∶4×10 6. The results of MGIA showed that the numbers of Mtb (lgCFU) in the PBS, adjuvant, EPDPA015f and EPDPA015m groups were 3.46±0.11, 3.51±0.06, 2.98±0.09 and 3.19±0.08, respectively. The number of colonies in the EPDPA015f group was the least as compared with that in the other three groups ( P<0.001, P<0.001, P<0.01). Conclusions:The vaccine candidate EPDPA015f could elicit more comprehensive and high-level cellular and humoral immune responses, and exhibited superior in vitro inhibitory activity against the growth of Mtb. EPDPA015f had the potential to be used as a preventive vaccine or a booster vaccine

Epitope spreading in autoimmune bullous diseases / 中华皮肤科杂志

Epitope spreading often occurs in patients with autoimmune bullous diseases (ABDs), resulting in exposure of more antigenic epitopes, aggravation or transformation of pre-existing diseases, or concurrence of other diseases. With the increase in the immunological evidence for epitope spreading, more and more scholars have realized that epitope spreading plays an important role in the development of ABDs. This review introduces the phenomenon of epitope spreading in ABDs from 4 aspects, including the concurrence of or transformation between different types of pemphigoid, different types of pemphigus, pemphigus and pemphigoid, as well as between ABDs and other skin diseases.

Sequence characteristics of Rhipicephalus microplus Enolase gene and prediction of structure and antigenic epitopes of its encoding protein / 中国血吸虫病防治杂志

Objective To analyze the sequence characteristics of Rhipicephalus microplus Enolase gene, and to predict the secondary and tertiary structure and antigenic epitopes of the Enolase protein. Methods Sixty-two engorged female R. microplus were sampled from a yellow cattle breeding farm in Zhijiang County, Huaihua City, Hunan Province in June 25, 2022. Genomic DNA was isolated from R. microplus, and the Enolase gene was amplified using PCR assay, followed by cloning, sequencing and expression of the amplification product. The sequence characteristics of the Enolase gene were analyzed using the software Clustal X, and the gene sequence was translated into amino acid sequences. The secondary and tertiary structures of the Enolase protein were deduced using the software PRABI, and the physicochemical properties of the Enolase protein were analyzed using the software PRABI. In addition, the B- and T-cell epitopes of the Enolase protein were predicted using the software ABCpred Prediction, Scratch, IEDB and NetCTL. Results The R. microplus Enolase gene sequence was 1 323 bp in size, and the contents of A, T, G and C bases were 24.5%, 22.5%, 27.0% and 26.0%,with 47.0% of A + T content and 53.0% of G + C content. The R. microplus Enolase gene encoded 434 amino acids, and the Enolase protein had a molecular weight of 47.12 kDa. The secondary structure of the Enolase protein contained 186 α-helixes (42.86%), 32 β-turns (7.37%), 144 random coils (33.18%) and 72 extended strands (16.59%). The Enolase protein was most probably present in cytoplasm (76.7%), followed by in mitochondrion (39.1%) and nucleus (21.7%), and the Enolase protein had no signal peptide or transmembrane domain. In addition, the Enolase protein had 14 B-cell dominant epitopes and 8 T-cell dominant epitopes. Conclusions The R. microplus Enolase gene sequence exhibits a GC preference, and its encoding Enolase protein is an acidic and hydrophilic protein, with α-helixes and random coils as its primary structure, and presenting B- and T-cell dominant epitopes, which is a potential target for development of vaccines against R. microplus.

Design and antitumor activity of programmed cell death ligand 1 epitope peptide vaccine / 中国药科大学学报

@#Several programmed cell death protein 1 (PD-1) or its ligand (PD-L1) immune checkpoint blocking antibodies are available for clinical treatment, but only some patients show clinical response, so an alternative strategy for tumor immunotherapy is needed.A therapeutic tumor vaccine targeting PD-L1 is a meaningful attempt.In this study, we designed an epitope peptide vaccine targeting PD-L1, and then screened the immunogenic PD-L1 epitope peptide based on the humanized immune system (HIS) mouse model and further investigated its anti-tumor activity.The results show that the designed and screened PD-L1-B1 epitope peptide vaccine not only successfully induced PD-L1-specific humoral and cellular immunity, but also exhibit anti-tumor activity.In addition, immunotherapy increased T-lymphocyte infiltration of tumors and reshaped the tumor immunosuppressive microenvironment.In conclusion, PD-L1-B1 epitope peptide vaccine exhibits potent anti-tumor activity and may be an effective alternative immunotherapeutic strategy for patients insensitive to PD-1/PD-L1 blockade.

Preliminary identification of CD4 T cell epitopes in genotype 1 and 6 of hepatitis C virus / 中国输血杂志

【Objective】 To study the CD4 T cell epitopes in Core and NS3 protein of genotype 1(GT1) and 6(GT6) of hepatitis C virus(HCV). 【Methods】 A total of 298 overlapping peptides(16-mer) spanning Core and NS3 protein of GT1 and GT6 HCV were synthesized. Peripheral blood mononuclear cells(PBMCs) from 17 HCV+ and 7 healthy blood donors were stimulated by peptide pools, followed by evaluating T cell response by IFN-γ ELISPOT, by which 21 peptides with positive results were found. These peptides were further applied to individually stimulate 20 HCV+ and 18 healthy PBMCs. The differences of responsive frequencies to the 21 positive peptides between the two study groups were compared. 【Results】 Pooled and individual peptide stimulation tests showed that HCV+ PBMCs were responsive to the stimulation of 5 peptides(GT1 NS31273-1288 and NS31315-1330; GT6 NS31033-1048, NS31087-1102 and NS31351-1366), with a responsive frequency ranging 18.9%-27.0%. In contrast, healthy PBMCs were not or low responsive(0%-4.0%) to these five peptides. The responsive frequencies were statistically different between the two groups(P<0.05). No reported epitopes in IEDB were found identical with these 5 peptides via sequence alignment. 【Conclusion】 Our study identified novel CD4 T cell epitopes in NS3 protein of GT1 and GT6 HCV, which has potential application value for the research and development of HCV vaccine.

Establishment and clinical evaluation of dry fluorescent luminescencemethod for detection of antibodies to hepatitis C virus / 中国输血杂志

【Objective】 To establish a dry fluorescent luminescence method for the detection of antibodies to hepatitis C virus (HCV) and evaluate its clinical application. 【Methods】 Anti-HCV antibody was detected by double-antigen sandwich dry fluorescent luminescence method established using multi-epitope chimeric antigen. The established method was used to detect national reference samples(positive 20, negative 20), and a total of 349 clinical samples, including 108 HCV patients, 36 patients with other diseases and 205 healthy individuals, which were tested in parallel with enzyme-linked immunoassay (ELISA) to evaluate the performance of the established method. 【Results】 The concordance rate of positive and negative(each 20) reference samples were both 100% (20/20), and the CV of precision reference sample was 9.16%, which met the requirements of national reference samples. In clinical performance evaluation, the AUC value was 0.984, and the sensitivity and specificity of the dry fluorescent luminescence method were 96.30% (104/108) and 96.27% (233/241). The overall concordance rate between dry fluorescent luminescence method and ELISA was 97.71% (341/349) (Kappa=0.952). 【Conclusion】 The dry fluorescence luminescence method of HCV antibody is simple and rapid, with high sensitivity and high specificity, and can be used in clinical application.

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.

Immunogenicity of chimeric peptide gene of Xinjiang hemorrhagic fever virus glycoprotein / 国际生物医学工程杂志

Objective:To investigate the effects of different chimerism strategies and different immune ways on the two antigen-dominant regions of Xinjiang hemorrhagic fever virus (XHFV) glycoprotein.Methods:The 5' end was added or not added with interleukin-2 (IL-2) signal peptide and the general-purpose auxiliary T cell epitopes as different design strategies. GcⅠ and GcⅡ and the epitopes previously identified on GcⅠ (Gc 233-248, Gc 241-256 and Gc 281-296) were fused and constructed into the eukaryotic expression vector pVAX1 and the prokaryotic expression vector pET-28a. The recombinant prokaryotic plasmid transformed into E.coli BL21 was induced and purified, and the recombinant eukaryotes were extracted by indirect immunofluorescent assay. BALB/c mice were immunized by protein immunity, gene immunity, and DNA prime-protein boost immunity. The IgG antibody level was measured by ELISA. The immune effect was evaluated by the proliferation of T-lymphocytes and the content of cytokines in the spleen. Results:The results of double enzyme digestion and sequencing showed that eight recombinant plasmids were successfully constructed, and the recombinant eukaryotes were successfully expressed in vitro by fluorescence microscopy. After three times of immunization, the IgG level and the proliferation of T-lymphocytes in the spleen of mice in the experimental group were significantly higher than those in the control group ( P<0.01). The mass concentration test results of Th2 cytokines IL-4 and Th1 cytokines interferon-gamma (IFN-γ) revealed that the response of the DNA prime-protein boost immunity was biased to Th1. Conclusions:The multi-epitope chimeric vaccine of XHFV glycoprotein was successfully constructed, and the target antigen could be expressed effectively in vivo. The immune groups stimulated stronger humoral and cellular immune responses compared with the control group. Among them, the immune effect of pVAX1-ST(GcⅠe+GcⅡ) combined with recombinant protein r(GcⅠe+GcⅡ) was the best, and it is expected to be a new candidate vaccine for XHFV.

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.

Systematic mutational analysis of epitope-grafted ED3 immunogenicity reveals a DENV3-DENV4 bi-serospecific ED3 mutant

Objective: To identify the residue determinants of the serospecificity and sero-cross-reactivity of dengue virus (DENV) envelope protein domain 3 (ED3), which contains two major putative epitopes of DENV. Methods: We constructed ED3 from DENV3 (3ED3) and DENV4 (4ED3), and six epitope-grafted variants, where we transferred epitope 1 (L304I, K305D, V309M, and S310A) and/or epitope 2 (D383N, K384S, K387T, and N389H) of 4ED3 onto 3ED3 and vice-versa. Swiss albino mice aged 3-4 weeks were immunized against wildtype and epitope-grafted ED3 variants and anti-ED3 IgG antibody responses were determined using ELISA. Results: Mouse immunization using 3ED3 and 4ED3 generated serotype-specific antisera, as expected. Similarly, most epitopegrafted ED3s produced antisera serospecific to the template ED3 with little or no cross-recognition of ED3 of the serotype from which the epitopes were taken. These indicated that a mere grafting of the epitope was not sufficient to transfer serospecificity, contrary to our expectations. However, one epitope-grafted ED3 mutant, where epitope 1 of 3ED3 was grafted onto 4ED3 (4ED3epi1), generated antisera that was serospecific to both 4ED3 and 3ED3. Conclusions: The 4ED3epi1 is a chimeric ED3 that produces antisera possessing serospecificity to both 3ED3 and 4ED3 onto a common 4ED3 scaffold. The 4ED3epi1, therefore, provides a unique tool for analyzing serospecificity and sero-cross-reactivity in dengue. We believe that chimeric ED3 may provide a template for future recombinant ED3 possessing serospecificity of multiple DENVs onto a single scaffold and may pave a way developing tri-and/or tetravalent anti-DENV antisera.

Preparation and characterization of a recombinant poly-epitopic vaccine EgG1Y162-2 (4) against cystic echinococcosis based on the linker GSGGSG / 中国血吸虫病防治杂志

Objective To perform prokaryotic expression and preliminary characterization of the recombinant poly-epitope vaccine EgG1Y162-2 (4) against cystic echinococcosis. Methods The recombinant poly-epitope vaccine EgG1Y162-2 (4) against Echinococcus granulosus based on the linker GSGGSG was subjected to structural three-dimensional (3D) modeling using immunoinformatics to analyze the structural changes and evaluate the antigenicity of the vaccine. The pET30a-EgG1Y162-2 (4) recombinant plasmid was generated using double digestion with EcoR I and Sal I, and then transformed into competent cells. Following protein induction with isopropyl-β-D-thiogalactoside (IPTG), the prokaryotic expression proteins were characterized using Western blotting, and the antigenicity of the recombinant protein was analyzed using sera from cystic echinococcosis patients and health volunteers. Results The four EgG1Y162-2 proteins coupled by the 3D structure of the recombinant vaccine EgG1Y162-2 (4) presented independent and effective expression and good antigenicity. The highest protein expression was detected in the supernatant following induction of the recombinant plasmid pET30a-EgG1Y162-2 (4) by 0.2 mmol/L IPTG at 37 °C for 4 h, and a pure protein component was seen following elution with 60 mmol/L imidazole. Western blotting analysis of the recombinant multiepitope protein HIS-EgG1Y162-2 (4) showed a band at approximately 39 kDa, and this band was recognized by sera from cystic echinococcosis patients. Conclusion A recombinant poly-epitope vaccine EgG1Y162-2 (4) against cystic echinococcosis has been successfully constructed, which provides a preliminary basis for researches on recombinant multi-epitope vaccine against cystic echinococcosis.

Characterization of a monoclonal antibody against the hemagglutinin stem of H7N9 subtype avian influenza virus / 生物工程学报

The conserved hemagglutinin (HA) stem region of avian influenza virus (AIV) is an important target for designing broad-spectrum vaccines, therapeutic antibodies and diagnostic reagents. Previously, we obtained a monoclonal antibody (mAb) (5D3-1B5) which was reactive with the HA stem epitope (aa 428-452) of H7N9 subtype AIV. To systematically characterize the mAb, we determined the antibody titers, including the HA-binding IgG, hemagglutination-inhibition (HI) and virus neutralizing (VN) titers. In addition, the antigenic epitope recognized by the antibody as well as the sequence and structure of the antibody variable region (VR) were also determined. Moreover, we evaluated the cross-reactivity of the antibody with influenza virus strains of different subtypes. The results showed that the 5D3-1B5 antibody had undetectable HI and VN activities against H7N9 virus, whereas it exhibited strong reactivity with the HA protein. Using the peptide-based enzyme-linked immunosorbent assay and biopanning with a phage-displayed random peptide library, a motif with the core sequence (431W-433Y-437L) in the C-helix domain in the HA stem was identified as the epitope recognized by 5D3-1B5. Moreover, the mAb failed to react with the mutant H7N9 virus which contains mutations in the epitope. The VR of the antibody was sequenced and the complementarity determining regions in the VR of the light and heavy chains were determined. Structural modeling and molecular docking analysis of the VR verified specific binding between the antibody and the C-helix domain of the HA stem. Notably, 5D3-1B5 showed a broad cross-reactivity with influenza virus strains of different subtypes belonging to groups 1 and 2. In conclusion, 5D3-1B5 antibody is a promising candidate in terms of the development of broad-spectrum virus diagnostic reagents and therapeutic antibodies. Our findings also provided new information for understanding the epitope characteristics of the HA protein of H7N9 subtype AIV.