In vitro neutralization of HCV by goat antibodies against peptides encompassing regions downstream of HVR-1 of E2 glycoprotein.

This article aims at testing several in vitro systems with various viral sources and cell lines for propagation of HCV to evaluate goat antibodies raised against three E2 epitopes in viral neutralization experiments. Four human cell lines (Huh-7, Huh-7.5, HepG2, and CaCo2) were tested using two different HCV viral sources; Genotype 4 infected sera and J6/JFH HCV cc particles. Neutralization capacity of goat Abs against conserved E2 epitopes; p412 (a.a 412-419), p517 (a.a 517-531), and p430 (a.a 430-447) were examined in the above mentioned in vitro systems. Although infection with patients' sera seems to mimic the in vitro situation, it has limited replication rates as compared with HCV cc particularly in Huh7.5 cells. Non-HCV adapted Huh-7 cells were also found susceptible for transfection with J6/JFH virus but at much slower kinetics. The results of the neutralization assay showed that anti p412 and anti p517 were highly neutralizing to HCVcc. Our data demonstrate that antibodies directed against the viral surface glycoprotein E2 reduced the infectivity of the J6/JFH virus and are promising agents for immunotherapy and HCV vaccine development.

A Study of CC-Chemokine Receptor 5 (CCR5) Polymorphism on the Outcome of HCV Therapy in Egyptian Patients.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is a globally serious public health issue. OBJECTIVES: In this study, we investigated CC chemokine receptor 5 (CCR5-59029) polymorphism which is considered an important component of the immune system in determining the outcome of HCV infection. Its critical role as a marker in response to interferon therapy of HCV infection is also investigated besides its effect on other clinical patient factors. PATIENTS AND METHODS: This study was conducted on 82 Egyptian patients with chronic Hepatitis C Virus (HCV) infection who received PEG-INF + Ribavirin treatment for 48 weeks. The study was also conducted on 50 healthy controls (with negative results for HCV antibody and RNA PCR). Full history of patients in this study was recorded. Clinical and histological examinations, qualitative HCV nested RT-PCR, quantitative real -time PCR, and genotyping of HCV RNA genome were performed. CCR5-59029 polymorphism with nucleotide substitution from G to A was amplified. The amplicons were digested with restriction endonuclease Bsp 1286I, and produced RFLPs of the CCR5 genotypes were determined. RESULTS: The present study showed a significant association between the functional SNP of CCR5 gene and the viral response to interferon in chronic HCV Egyptian patients. It was shown that the higher fibrosis stages (F2-F4) had significant association with nonresponse to treatment compared to the lower fibrosis stages (F0-F1) (95% confidence: 5.497 - 55.074, P = 0.0001). In addition, worse liver activity grade (A2-A3) had a very highly significant association with non-responder HCV patients compared to those with better liver activity grade (A1) (95% confidence: 2.242 - 20.974, P = 0.0007). Most importantly HCV patients with G allele had a high significant association with nonresponse to treatment, higher fibrosis stages and worse liver activity grades, while the A allele had a high significant association with sustained response, low fibrosis stages and relatively better liver activity grade (95% confidence: 3.347 - 15.036, P = 0.0001). CONCLUSIONS: SNPs within the CCR5 gene should be considered as an important factor used in combination with other host gene SNPs when developing a mathematical model for anticipating response to HCV therapy.

Neutralizing activities of caprine antibodies towards conserved regions of the HCV envelope glycoprotein E2.

Anti HCV vaccine is not currently available and the present antiviral therapies fail to cure approximately half of the treated HCV patients. This study was designed to assess the immunogenic properties of genetically conserved peptides derived from the C-terminal region of HVR-1 and test their neutralizing activities in a step towards developing therapeutic and/or prophylactic immunogens against HCV infection. Antibodies were generated by vaccination of goats with synthetic peptides derived from HCV E2. Viral neutralizing capacity of the generated anti E2 antibodies was tested using in vitro assays. Goats immunized with E2 synthetic peptides termed p412 [a.a 412-419], p430 [a.a 430-447] and p517 [a.a 517-531] generated high titers of antibody responses 2 to 4.5 fold higher than comparable titers of antibodies to the same epitopes in chronic HCV patients. In post infection experiments of native HCV into cultured Huh7.5 cells anti p412 and anti p 517 were proven to be neutralizing to HCV genotype 4a from patients' sera (87.5% and 75% respectively). On the contrary anti p430 exhibited weak viral neutralization capacity on the same samples (31.25%). Furthermore Ab mixes containing anti p430 exhibited reduced viral neutralization properties. From these experiments one could predict that neutralization by Abs towards different E2-epitopes varies considerably and success in the enrichment of neutralization epitope-specific antibodies may be accompanied by favorable results in combating HCV infection. Also, E2 conserved peptides p517 and p412 represent potential components of a candidate peptide vaccine against HCV infection.

Circulating viral core and E1 antigen levels as supplemental markers for HCV chronic hepatitis.

The performance of polyclonal monospecific rabbit anti-sera raised against synthetic peptides derived from conserved HCV sequences of genotype 4 was evaluated for efficient detection of viral core and E1 antigens in circulating immune complexes (ICs) precipitated from 65 serum samples of HCV patients. The infection was established in those patients by the presence of HCV RNA in their sera. A novel enzyme-linked immunosorbent assay (ELISA) was developed for the detection of HCV core and E1 antigen in serum samples. Western blot analyses were used to demonstrate the presence of the core and E1 target antigen in serum samples. The mean OD readings of both core and E1 antigens were significantly higher (P < 0.05) among the viremic patients when compared to controls. Also a significant positive correlation (P < 0.05, r = 0.98) between the values of both core and E1 was recorded. Western blot analysis based on monospecific antibodies against core and E1 recognized the 38-kDa and 88 -kDa bands respectively in the sera of all infected patients. No specific reaction was observed with the sera from uninfected individuals. Interestingly the results of core and E1 antigen levels displayed no positive correlation with the HCV copy number as measured by bDNA. Liver enzymes (ALT and AST) showed a moderate positive correlation (r = 0.44 and 0.47 respectively) with the viral core antigens level. The same trend holds true for E1 (r = 0.43 and 0.64 for ALT and AST respectively). HCV load in infected patients revealed extremely poor correlation with serum ALT and AST levels (r = 0.022 and 0.002 respectively). In conclusion we present a new combination of serological tools correlating with liver enzyme levels that could be utilized as supplemental tests to viral load testing. Also, a sensitive and specific immunoassay was developed for the detection of HCV core and E1 in human serum. This test can be applied for laboratory diagnosis of HCV infection.

Antibody to E1 peptide of hepatitis C virus genotype 4 inhibits virus binding and entry to HepG2 cells in vitro.

AIM: To analyze the neutralizing activity of antibodies against E1 region of hepatitis C virus (HCV). Specific polyclonal antibody was raised via immunization of New Zealand rabbits with a synthetic peptide that had been derived from the E1 region of HCV and was shown to be highly conserved among HCV published genotypes. METHODS: Hyper-immune HCV E1 antibodies were incubated over night at 4 degree Celsius with serum samples positive for HCV RNA, with viral loads ranging from 615 to 3.2 million IU/ mL. Treated sera were incubated with HepG2 cells for 90 min. Blocking of viral binding and entry into cells by anti E1 antibody were tested by means of RT-PCR and flow cytometry. RESULTS: Direct immunostaining using FITC conjugated E1 antibody followed by Flow cytometric analysis showed reduced mean fluorescence intensity in samples pre-incubated with E1 antibody compared with untreated samples. Furthermore, 13 out of 18 positive sera (72%) showed complete inhibition of infectivity as detected by RT-PCR. CONCLUSION: In house produced E1 antibody, blocks binding and entry of HCV virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. Isolation of these antibodies that block virus attachment to human cells are useful as therapeutic reagents.

Molecular study on Y chromosome microdeletions in Egyptian males with idiopathic infertility.

AIM: To determine the frequency of genetic deletions within the azoospermia factors in Egyptian infertile males. METHODS: The Yq microdeletions in 33 infertile males with undetectable chromosomal anomalies were examined by mutiplex polymerase chain reaction (PCR). Deletions were confirmed using single PCR amplifications. RESULTS: Four out of the total 33 (12 %) men had Yq(11) microdeletions, thus supporting the average reported figures in other populations. Three of those 4 cases had single short tandem sequence deletions with discrete histological findings of their testes. Single sY272 deletion within AZFc was associated with Sertoli cell only syndrome, whereas a patient with isolated sY84 deletion within AZFa had immature testicular structure. The remaining case had a large deletion in AZFa-c and short stature. CONCLUSION: The present study supports the hypothesis that the Yq(11) encompasses genetic determinants of stature besides genes controlling spermatogenesis.