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Hepatitis C Core, truncated from C_terminal, 16kD, P. pastoris expression system, recombinant; ELISA_WB

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[#OBT0920] Hepatitis C Core, truncated from C_terminal, 16kD, P. pastoris expression system, recombinant; ELISA_WB


OBT0920 | Hepatitis C Core, truncated from C_terminal, 16kD, P. pastoris expression system, recombinant; ELISA_WB, 100 µg.
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(1) Targeting naturally occurring epitope variants of hepatitis C virus with high-affinity T-cell receptors.[TOP]

Pubmed ID :27902325
Publication Date : //
Hepatitis C virus (HCV) readily establishes chronic infection, which is characterized by failure of virus-specific CD8+ T cells. HCV uses epitope mutation and T-cell exhaustion to escape from the host immune response. Previously, we engineered high-affinity T-cell receptors (HATs) targeting human immunodeficiency virus escape mutants. In this study, the affinity of a T-cell receptor specific for the HLA-A2-restricted HCV immunodominant epitope NS3 1406-1415 (KLVALGINAV) was improved from a KD of 6.6 µM to 40 pM. These HATs could also target HCV NS3 naturally occurring variants, including an escape variant vrt1 (KLVVLGINAV), with high affinities. The HATs can be used as high-affinity targeting molecules at the centre of the immune synapse for the HLA-restricted NS3 antigen. By fusing the HAT with a T-cell activation molecule, an anti-CD3 single-chain variable fragment, we constructed a molecule called high-affinity T-cell activation core (HATac), which can redirect functional CTLs possessing any specificity to recognize and kill cells presenting HCV NS3 antigens. This capability was verified with T2 cells loaded with prototype or variant peptides and HepG2 cells expressing the truncated NS3 prototype or variant proteins. The results indicate that HATac targeting the HLA-restricted NS3 antigen may provide a useful tool for circumventing immune escape mutants and T-cell exhaustion caused by HCV infection.

Authors : Zhang Huajun, Zhang Jianbing, Chen Lei, Weng Zhiming, Tian Ye, Zhao Haifeng, Li Youjia, Chen Lin, Liang Zhaoduan, Zheng Hongjun, Zhao Wenzhuo, Zhong Shi, Li Yi,

(2) Charge neutralization as the major factor for the assembly of nucleocapsid-like particles from C-terminal truncated hepatitis C virus core protein.[TOP]

Pubmed ID :27867765
Publication Date : //
Hepatitis C virus (HCV) core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124) is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs) . The specificity and propensity of C124 to the assembly and its implications on HCV pathogenesis are not well understood.

Authors : de Souza Theo Luiz Ferraz, de Lima Sheila Maria Barbosa, Braga Vanessa L de Azevedo, Peabody David S, Ferreira Davis Fernandes, Bianconi M Lucia, Gomes Andre Marco de Oliveira, Silva Jerson Lima, de Oliveira Andréa Cheble,

(3) Comparative Immunogenicity in Rabbits of the Polypeptides Encoded by the 5' Terminus of Hepatitis C Virus RNA.[TOP]

Pubmed ID :26609538
Publication Date : //
Recent studies on the primate protection from HCV infection stressed the importance of immune response against structural viral proteins. Strong immune response against nucleocapsid (core) protein was difficult to achieve, requesting further experimentation in large animals. Here, we analyzed the immunogenicity of core aa 1-173, 1-152, and 147-191 and of its main alternative reading frame product F-protein in rabbits. Core aa 147-191 was synthesized; other polypeptides were obtained by expression in E. coli. Rabbits were immunized by polypeptide primes followed by multiple boosts and screened for specific anti-protein and anti-peptide antibodies. Antibody titers to core aa 147-191 reached 10(5); core aa 1-152, 5 × 10(5); core aa 1-173 and F-protein, 10(6). Strong immunogenicity of the last two proteins indicated that they may compete for the induction of immune response. The C-terminally truncated core was also weakly immunogenic on the T-cell level. To enhance core-specific cellular response, we immunized rabbits with the core aa 1-152 gene forbidding F-protein formation. Repeated DNA immunization induced a weak antibody and sustained proliferative response of broad specificity confirming a gain of cellular immunogenicity. Epitopes recognized in rabbits overlapped those in HCV infection. Our data promotes the use of rabbits for the immunogenicity tests of prototype HCV vaccines.

Authors : Sominskaya Irina, Jansons Juris, Dovbenko Anastasija, Petrakova Natalia, Lieknina Ilva, Mihailova Marija, Latyshev Oleg, Eliseeva Olesja, Stahovska Irina, Akopjana Inara, Petrovskis Ivars, Isaguliants Maria,

(4) HCV core protein uses multiple mechanisms to induce oxidative stress in human hepatoma Huh7 cells.[TOP]

Pubmed ID :26035647
Publication Date : //
Hepatitis C virus (HCV) infection is accompanied by the induction of oxidative stress, mediated by several virus proteins, the most prominent being the nucleocapsid protein (HCV core). Here, using the truncated forms of HCV core, we have delineated several mechanisms by which it induces the oxidative stress. The N-terminal 36 amino acids of HCV core induced TGF\(\upbeta\)1-dependent expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 1 and 4, both of which independently contributed to the production of reactive oxygen species (ROS). The same fragment also induced the expression of cyclo-oxygenase 2, which, however, made no input into ROS production. Amino acids 37-191 of HCV core up-regulated the transcription of a ROS generating enzyme cytochrome P450 2E1. Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1\(\upalpha\). The latter triggered efflux of Ca2+ from ER to mitochondria via mitochondrial Ca2+ uniporter, leading to generation of superoxide anions, and possibly also H2O2. Suppression of any of these pathways in cells expressing the full-length core protein led to a partial inhibition of ROS production. Thus, HCV core causes oxidative stress via several independent pathways, each mediated by a distinct region of the protein.

Authors : Ivanov Alexander V, Smirnova Olga A, Petrushanko Irina Y, Ivanova Olga N, Karpenko Inna L, Alekseeva Ekaterina, Sominskaya Irina, Makarov Alexander A, Bartosch Birke, Kochetkov Sergey N, Isaguliants Maria G,

(5) [Immunogenicity and heterologous protection in mice with a recombinant adenoviral-based vaccine carrying a hepatitis C virus truncated NS3 and core fusion protein].[TOP]

Pubmed ID :25997323
Publication Date : //
To develop a safe and broad-spectrum effective hepatitis C virus (HCV) T cell vaccine,we constructed the recombinant adenovirus-based vaccine that carried the hepatitis C virus truncated NS3 and core fusion proteins. The expression of the fusion antigen was confirmed by in vitro immunofluorescence and western blotting assays. Our results indicated that this vaccine not only stimulated antigen-specific antibody responses,but also activated strong NS3-specific T cell immune responses. NS3-specific IFN-γ+ and TNF-α+ CD4+ T cell subsets were also detected by a intracellular cytokine secretion assay. In a surrogate challenge assay based on a recombinant heterologous HCV (JFH1,2a) vaccinia virus,the recombinant adenovirus-based vaccine was capable of eliciting effective levels of cross-protection. These findings have im- portant implications for the study of HCV immune protection and the future development of a novel vaccine.

Authors : Guan Jie, Deng Yao, Chen Hong, Yang Yang, Wen Bo, Tan Wenjie,

(6) Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays.[TOP]

Pubmed ID :25796431
Publication Date : //
Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection.

Authors : Ali Amjad, Nisar Muhammad, Idrees Muhammad, Rafique Shazia, Iqbal Muhammad,

(7) Ligand bioactive conformation plays a critical role in the design of drugs that target the hepatitis C virus NS3 protease.[TOP]

Pubmed ID :24144444
Publication Date : //
A ligand-focused strategy employed NMR, X-ray, modeling, and medicinal chemistry to expose the critical role that bioactive conformation played in the design of a variety of drugs that target the HCV protease. The bioactive conformation (bound states) were determined for key inhibitors identified along our drug discovery pathway from the hit to clinical compounds. All adopt similar bioactive conformations for the common core derived from the hit peptide DDIVPC. A carefully designed SAR analysis, based on the advanced inhibitor 1 in which the P1 to P3 side chains and the N-terminal Boc were sequentially truncated, revealed a correlation between affinity and the relative predominance of the bioactive conformation in the free state. Interestingly, synergistic conformation effects on potency were also noted. Comparisons with clinical and recently marketed drugs from the pharmaceutical industry showed that all have the same core and similar bioactive conformations. This suggested that the variety of appendages discovered for these compounds also properly satisfy the bioactive conformation requirements and allowed for a large variety of HCV protease drug candidates to be designed.

Authors : LaPlante Steven R, Nar Herbert, Lemke Christopher T, Jakalian Araz, Aubry Norman, Kawai Stephen H,

(8) Hepatitis C genotype 6: A concise review and response-guided therapy proposal.[TOP]

Pubmed ID :24073301
Publication Date : //
Hepatitis C genotype 6 is endemic in Southeast Asia [prevalence varies between 10%-60% among all hepatitis C virus (HCV) infection], as well as also sporadically reported outside the area among immigrations. The diagnosis of HCV genotype can be inaccurate with earlier methods of genotyping due to identical 5'-UTR between genotype 6 and 1b, hence the newer genotyping methods with core sequencing are preferred. Risk factors and clinical course of HCV genotype 6 do not differ considerably from other genotypes. Treatment outcome of HCV genotype 6 with a combination of pegylated interferon and ribavirin is superior to genotype 1, and nearly comparable to genotype 3, with expected sustained virological response (SVR) rates of 60%-90%. Emerging data suggests that a shorter course 24-wk treatment is equally effective as a standard 48-wk treatment, particularly for those patients who attained undetectable HCV RNA at week 4 (RVR). In addition, baseline and on-treatment predictors of response used for other HCV genotypes appear effective with genotype 6. Although some pan-genotypic direct-acting antivirals have completed phase II/III studies (sofosbuvir and simeprevir) with clinical benefit demonstrated in small number of patients with genotype 6, broad availability of these agents in Southeast Asia may not be expected in the near future. While awaiting the newer therapy, response-guided therapy seems appropriate for patients with HCV genotype 6. Patients with RVR (representing > 70% of patients) are suitable for 24-wk treatment with expected SVR rates > 80%. Patients without RVR and/or those with poor response predictors may benefit from 48 wk of therapy, and a detectable HCV RNA at week 12 (with no early virological response) serves as a stopping rule. This treatment scheme is likely to have a major economic impact on HCV therapy, particularly in Southeast Asia, wherein treatment can be truncated securely in the majority of patients with HCV genotype 6.

Authors : Bunchorntavakul Chalermrat, Chavalitdhamrong Disaya, Tanwandee Tawesak,

(9) Significance of monoclonal antibodies against the conserved epitopes within non-structural protein 3 helicase of hepatitis C virus.[TOP]

Pubmed ID :23894620
Publication Date : //
Nonstructural protein 3 (NS3) of hepatitis C virus (HCV), codes for protease and helicase carrying NTPase enzymatic activities, plays a crucial role in viral replication and an ideal target for diagnosis, antiviral therapy and vaccine development. In this study, monoclonal antibodies (mAbs) to NS3 helicase were characterized by epitope mapping and biological function test. A total of 29 monoclonal antibodies were produced to the truncated NS3 helicase of HCV-1b (T1b-rNS3, aa1192-1459). Six mAbs recognized 8/29 16mer peptides, which contributed to identify 5 linear and 1 discontinuous putative epitope sequences. Seven mAbs reacted with HCV-2a JFH-1 infected Huh-7.5.1 cells by immunofluorescent staining, of which 2E12 and 3E5 strongly bound to the exposed linear epitope (1231)PTGSGKSTK(1239) (EP05) or core motif (1373)IPFYGKAI(1380) (EP21), respectively. Five other mAbs recognized semi-conformational or conformational epitopes of HCV helicase. MAb 2E12 binds to epitope EP05 at the ATP binding site of motif I in domain 1, while mAb 3E5 reacts with epitope EP21 close to helicase nucleotide binding region of domain 2. Epitope EP05 is totally conserved and EP21 highly conserved across HCV genotypes. These two epitope peptides reacted strongly with 59-79% chronic and weakly with 30-58% resolved HCV infected blood donors, suggesting that these epitopes were dominant in HCV infection. MAb 2E12 inhibited 50% of unwinding activity of NS3 helicase in vitro. Novel monoclonal antibodies recognize highly conserved epitopes at crucial functional sites within NS3 helicase, which may become important antibodies for diagnosis and antiviral therapy in chronic HCV infection.

Authors : Bian Yixin, Zhao Shuoxian, Zhu Shaomei, Zeng Jinfeng, Li Tingting, Fu Yongshui, Wang Yuanzhan, Zheng Xin, Zhang Ling, Wang Wenjing, Yang Baocheng, Zhou Yuanping, Allain Jean-Pierre, Li Chengyao,

(10) Construction and preparation of three recombinant adenoviruses expressing truncated NS3 and core genes of hepatitis C virus for vaccine purposes.[TOP]

Pubmed ID :23087750
Publication Date : //
In spite of dozens of clinical trials to establish effective therapeutic and/or preventive vaccine to resolve HCV infection, no real vaccine has been proved to date. Genetic vaccines based on replication-defective adenoviruses have proved to elicit strong and long lasting T-cell responses against a number of viral antigens and are even currently being used for vaccine trials in humans. According to the controversy in the immune modulatory effects of both core and NS3 full length genes, it seemed more practical to employ some parts of these HCV proteins for vaccine design.

Authors : Hosseini Seyed Younes, Sabahi Farzaneh, Moazzeni Seyed Mohammad, Modarressi Mohammad Hossein, Saberi Firoozi Mehdi, Ravanshad Mehrdad,