Evaluation of Truncated HCV-NS3 Protein for Potential Applications in Immunization and Diagnosis.

BACKGROUND: Hepatitis C virus infection is one of global health concern. No vaccine is available so far and nonstructural protein 3 (NS3) is one of the main target antigens for developing studies on therapeutic vaccine and diagnostic application. In the current study, we expressed a truncated recombinant HCV-NS3 protein under native condition and evaluated its potential applications in immunization and diagnosis. METHODS: The recombinant pET-NS3 containing a truncated form of HCV NS3 region was constructed, confirmed by sequencing reactions, and expressed into E.coli BL21-DE3 strain. The expressed protein was purified by affinity chromatography under native condition. Characterization of diagnostic value and immunogenicity of this recombinant protein were analyzed by using an indirect ELISA method. RESULTS: Our data showed that a truncated NS3 which contains the immunodominant region of this protein was successfully expressed and purified with a high yield of 3 mg/L. Our data showed that the immunogenicity of this truncated protein can induce a specific humoral response, as well as the usage for screening of HCV positive blood samples. CONCLUSIONS: Altogether, the present study provides a simple and efficient system for protein expression and purification of an immunodominant region of NS3-HCV in native conformation and its potential application for diagnosis and vaccine development in future.

Impact of TGF-ß1 Gene Polymorphism (rs1800469) on Treatment Response to Pegylated Interferon/Ribavirin in Iranian Patients with Hepatitis C.

BACKGROUND: Hepatitis C virus as a major cause of chronic liver disease affects more than 170 million people worldwide. Recent studies have claimed that single nucleotide polymorphisms (SNPs) for the transforming growth factor-ß1 (TGF-ß1) gene were strongly associated with the antiviral treatment response. Thus, the present study aimed at the determination of distribution of the rs1800469 (C/T) polymorphism among Iranian with chronic hepatitis C. METHODS: A total of 165 blood samples including 68 SVR positive and 21 non-responder samples from individuals suffering chronic hepatitis C and also 76 healthy individual controls were analyzed in this cross-sectional study. DNA was isolated from the samples using a DNA extraction standard kit. Then the frequency of the polymorphism was analyzed using PCR-RFLP method. Eventually, the products of interest were detected on 2.5% agarose gel electrophoresis. RESULTS: The distribution of the C/T polymorphism between healthy individuals and patients were obtained as TT: 22.4%, TC: 46%, CC: 31.6%, and TT: 19.1%, TC: 48.3%, CC: 32.6%, respectively. Furthermore, the CC genotype was identified in 20 patients of whom 68 achieved SVR, while the CT heterozygous was found in 43 patients and SVR was achieved in 38. Finally, the TT was detected in 17 patients, and 7 patients did not achieve SVR. CONCLUSIONS: We observed a significant difference of C allele frequency with SVR as compared to the T allele among patients (p = 0.064). On the other hand, there is no correlation between the polymorphism and susceptibility to HCV infection. However, further studies with more samples seem to be necessary.

Correlation Study Between IL-28B Gene Polymorphism (rs8099917SNP) and Sustained Virological Response in Iranian Patients with Chronic Hepatitis C.

BACKGROUND: The current standard treatment for hepatitis C is a combination of pegylated interferon alpha and ribavirin (peg-IFNα/RBV). Recent studies have shown that single nucleotide polymorphisms (SNPs) near the interleukin 28B (IL28B) gene coding for IFN-λ3 were associated with the antiviral treatment response. Therefore, in this study, we determined the distribution of the rs8099917 (T/G) polymorphism with sustained virological response (SVR) to chronic hepatitis C virus infection among Iranian patients. METHODS: This cross-sectional study was performed on 150 blood samples based on 93 patients with chronic HCV genotypes 1 and 3 including 71 SVR positive, 22 negative, and 57 healthy individual controls. DNA was extracted from the samples and the frequency of the polymorphism was analyzed the using PCR-RFLP method. Finally, the products were detected on 3.5% agarose gel electrophoresis. RESULTS: The analysis of the data for G/T polymorphism showed that the GG genotype was identified in 6 patients of 71 who achieved SVR, while the GT heterozygous was found in 33 patients and SVR was achieved in 19. Finally, the TT was detected in 53 patients and 7 patients were resistant to treatment. CONCLUSIONS: The results showed significant effects of G allele carriers on susceptibility to HCV infection com-pared to the other allele (T) in our studied population (p = 0.013, OR = 2.23, 95% CI = 1.18-4.21), but we did not find a significant correlation for SVR to therapy in patients with genotype TT (p = 0.055, OR = 0.48, 95% CI = 0.23-1.01). However, further studies with more samples are necessary.

Impact of null genotypes of GSTT1 and GSTM1 with uterine leiomyoma risk in Iranian population.

AIM: Few studies have investigated the role of the GSTM1 and GSTT1 genes in uterine leiomyoma. Therefore, in the current study the distribution of these genotypes in Iranian women and susceptibility to uterine leiomyoma was investigated. METHODS: Blood samples of 50 patients with uterine leiomyoma and 50 healthy individual controls were collected in this cross-sectional study. Genomic DNA was extracted, and subsequently GSTM1 and GSTT1 null genotypes were detected by the Gap-polymerase chain reaction method. RESULTS: A total of 42% of patients appeared to lack GSTM1 enzyme activity due to the presence of an extended deletion (GSTM1 0/0 genotype), compared with 18% in a control group (odds ratio [OR], 3.56; 95% confidence interval [CI], 1.35-9.37; P < 0.010). In addition, the prevalence of the GSTT1 null genotype in patients was higher than that in the control group (42% to 14%, P < 0.009). Also, it was shown that individuals with both null genotypes (-/-) had a 19.23-fold higher risk of developing the disease in comparison to people who showed both present genotypes (+/+). (P = 0.007; 95%CI, 2.20-167.41). Besides, it was observed that at least one null genotype increases the risk of myoma to 2.6 compared to the both present genotype (P-value < 0.03, 95%CI, 1.05-6.82). CONCLUSION: To our knowledge, this is first significant correlation between risk of uterine leiomyoma and null GSTM1 and GSTT1 genotypes among Iranian patients. Our data support the involvement of GSTM1 and GSTT1 in uterine leiomyoma liability, and especially its role as a genetic factor in the occurrence of this disease.

G2 Dendrimer as a Carrier Can Enhance Immune Responses Against HCV-NS3 Protein in BALB/c Mice.

BACKGROUND: Hepatitis C virus (HCV) infection is a major issue of public health. It seems of paramount importance to find an effective vaccine against HCV infection. The best vaccine candidate should induce robust cellular responses. The aim of the current study was to evaluate immunogenicity effects of novel conjugated dendrimer G2 with the recombinant NS3 antigen as a vaccine candidate for eliciting Th1-oriented cellular responses. METHODS: Female BALB/c mice were immunized with different regimes especially with NS3 conjugated with G2 dendrimer. The humoral responses (Total IgG and IgG iso-typing) and cellular responses (Ex vivo IFN-γ and IL-4 ELISpot assays, in vitro CTL assay and proliferation) were evaluated and compared in immunized mice. RESULTS: The results indicated that induced specific total IgG in all mice groups immunized with rNS3 formulated with different adjuvants and IgG2a subclass was the predominant isotype in rNS3-G2 (p≤0.05). For preliminary evaluation of cellular response, ex vivo ELISpot assay has shown that the higher frequency of IFN-γ producing cells was in groups immunized with rNS3+M720 and rNS3-G2 (p= 0.0012) than control groups. Finally, the rNS3-specific CTLs activity showed the highest percentage of specific lysis (LDH release) of the target cells in rNS3-G2 and rNS3+M720 groups. CONCLUSION: In the present study, as our knowledge, this is first time that the immunogenicity of nanodendrimer G2 as a biocompatible adjuvant with the HCV-NS3 antigen was evaluated. The results showed high capability of the regimen to induce strong Th1-orinted cellular response in mice model, indicating the dendrimer G2 as a novel adjuvant candidate for HCV vaccine studies.

Role of IL28-B Polymorphism (rs12979860) on Sustained Virological Response to Pegylated Interferon/Ribavirin in Iranian Patients With Chronic Hepatitis C.

BACKGROUND: The current medical treatment for hepatitis C virus (HCV) infection is pegylated interferon plus ribavirin, but just 50% of genotype 1 HCV patients and about 80% of HCV genotype 3 patients are treated completely. Recently, the rs12979860 C/T polymorphism, which is located 3 kb upstream of the IL28b gene that codes IFNλ3, shows a powerful association in response to the treatment in HCV patients. OBJECTIVES: The aim of this study was to evaluate the relationship between IL28b single nucleotide polymorphism (SNP) and treatment outcomes among chronic HCV patients in Iran. PATIENTS AND METHODS: In this cross-sectional study, 108 blood samples were collected from chronic patients in Iran; 50 unrelated healthy subject samples were also collected. Genomic DNA was extracted, and rs12979860 SNP was done by PCR-RFLP. Finally, products were detected on 12% polyacrylamide gel electrophoresis. RESULTS: The analysis of data for C/T SNP showed that the CC genotype is more common in the control group than in the group of patients. In contrast, the frequency of TT as a mutant genotype is more frequent in patients than in uninfected people. In addition, results showed a statistically significant relationship between CC, CT, and TT genotypes in sensitive and resistant groups (P value: < 0.001, Or: 0.003, CI: 0-0.047). This relationship was also examined in terms of allele frequency, to determine whether the possibility of resistance to treatment in patients with T allele is more than in patients who carry C allele (P value: < 0.001). CONCLUSIONS: These results showed a significant effect between rs12979860 SNP and sustained virological response (SVR) rate in Iranian patients with chronic HCV. To decrease the cost of long treatments and to prevent severe side effects, determining this polymorphism at the beginning of treatment can be very helpful for patients and physicians.

Application of outer membrane vesicle of Neisseria meningitidis serogroup B as a new adjuvant to induce strongly Th1-oriented responses against HIV-1.

Despite the worldwide efforts made in the field of HIV vaccine development, an efficient AIDS vaccine strategy is still vague. Virus-like particles (VLPs) are one of the introduced aspects for HIV vaccine development since the non-replicative nature of HIV VLPs, resulting from the lack of viral genomic RNA, makes them suitable for broad applications. We have previously designed and introduced non-infectious VLPs (mzNL4-3) by introduction of a deletion mutation in the reverse transcriptase and integrase coding regions of HV-1. There are evidences suggesting that an effective cellular immune response against HIV-1 is able to control and suppress viremia during primary and chronic HIV infections. In the present study we have evaluated the potency of mzNL4-3 VLPs mixed with Neisseria meningitidis serogroup B outer-membrane vesicle (OMV), which is among the microbial components with proved adjuvant properties, to induce humoral and cellular responses against HIV-1. Analysis of anti-HIV-1 responses elicited in immunized BALB/c mice following different immunization regimens indicated OMV+VLP as an immunopotent combination which significantly induced anti-HIV-1 IgG with IgG2a dominancy. Results of cytokine and ELISpot assays also showed the capability of VLP+OMV immunogen for effective induction of IFN-gamma; and IL4 secreting cells and further suggested the promotion of Th1-oriented response that was evidenced with the increased IFN-γ/IL4 secretion ratio. According to our study, HIV-1 VLPs combined with N. meningitidis B OMVs seem to be a promising approach in vaccine development against HIV-1.