Identification of mutations in the HVR1 and PKR-BD regions in HCV-infected patients resistant to PEG-IFNα/RBV therapy.

The identification of mutations in the HVR1 region of hepatitis type C virus (HCV) is time-consuming and expensive, and there is a need for a rapid, inexpensive method of screening for these mutations to predict the ineffectiveness of pegylated interferon alpha combined with ribavirin (PEG-IFNα/RBV) therapy. The project was designed to evaluate the usefulness of the high resolution melting (HRM) technique to screen for mutation in the cDNAs encoding the HVR1 and protein kinase R-binding domain (PKR-BD) regions in a group of 36 patients infected with HCV and resistant to 12 months of combined therapy with PEG-IFNα/RBV. Viral RNA was isolated, reverse transcribed, and the fragments encoding the HVR1 and PKR-BD regions were polymerase chain reaction (PCR)-amplified, cloned, sequenced, and the melting profiles and the melting temperature (Tm) were determined by the HRM technique. After the treatment, the melting profiles of HVR1 cDNAs revealed a dominant peak corresponding to the Tm of about 85 °C (HCVs85) in almost all patients. One or more minor peaks were also observed, indicating the existence of cDNA(s) of different Tm. The HMR analysis suggested four typical forms of response to treatment. These suppositions were supported by sequencing. The HRM analysis revealed no changes in the melting profiles of PKR-BD cDNAs in the same patient before and after the therapy, suggesting that, within 12 months of treatment, new mutations were not introduced in PKR-BD. These findings were substantiated by sequencing. The HRM technique can be applied for the rapid screening for mutations in the cDNAs encoding the HVR and PKR-BD regions of HCV. We suggest that the detection of HCVs85 peak before the IFNα/RBV therapy might predict the ineffectiveness of treatment.

Application of a high-resolution melting technique for the rapid detection of partial replacement of HCV-1b by HCV-1a after PEG-IFNα/RBV therapy.

A modified method which can be used for the rapid screening of mutations in the protein kinase R-binding domain (PKR-BD) region and the hypervariable region 1 (HVR1) of hepatitis C virus (HCV) is described. This method is based on a high-resolution melting (HRM) technique used for genotyping single nucleotide polymorphisms and allows the detection of single nucleotide substitutions in the DNA sequence by measuring its Tm. The modified method, in addition to precisely measuring the Tm, allows the recording of the melting curve of the investigated cDNA fragment, which can provide provisional information about the number of different quasi-species present in the sample. The HRM analysis of the amplified cDNAs encoding the PKR-BD and HVR1 allowed the detection of partial replacement of HCV-1b by HCV-1a subspecies in one of our patients, as well as evaluation of the effectiveness of pegylated interferon α/ribavirin (PEG-IFNα/RBV) therapy. The HRM technique has never been used for the rapid screening of sequence variations in these regions and may be used for a similar purpose in any viral genome.

A correlation between the heterogeneity of hypervariable region 1 of E2 glycoprotein of Hepatitis C virus (HCV) and HCV antibody profile: a case study.

A correlation between the heterogeneity of hypervariable region 1 (HVR1) of E2 glycoprotein (gp) and Hepatitis C virus (HCV) antibody profile was investigated. Of 6 patients studied two were in acute phase, two in chronic phase and two showed signs of long-time HCV infection, i.e. liver cirrhosis. All the patients exhibited a vigorous antibody response to viral proteins C, NS3, NS4 and NS5. An antibody response to HVR1 of E2 was found in one patient in acute phase and in one or two patients in chronic phase. Such a response was not found in the two patients with liver cirrhosis. Single-stranded conformation polymorphism (SSCP) and sequence analyses of HVR1 of E2 showed the lowest HVR1 heterogeneity in patients in acute phase and the highest one in those in chronic phase, while the long-time carriers of the virus showed an intermediate heterogeneity. This may reflect a specific interplay between the virus and immune system. The HVR1 heterogeneity may rise in the course of infection as a means of evading the immune pressure. Then, when an organism is unable to clear the virus, because the responses to HVR1 epitopes are weakened or exhausted, a population of less heterogeneous HVR1 variants may be established.

Enhancement of cellular and humoral immune responses to human immunodeficiency virus type 1 Gag and Pol by a G/P-92 fusion protein expressing highly immunogenic Gag p17/p24 and Pol p51 antigens.

OBJECTIVES: Immunity to the human immunodeficiency virus type-1 (HIV-1) G/P-92 fusion protein consisting of highly immunogenic regions of Gag (p17 and p24) and Pol (p51) expressed in recombinant vaccinia virus (vG/P-92) was compared with responses to the entire viral Gag-Pol precursor protein (vVK1). STUDY DESIGN/METHODS: We analyzed the level of Gag and Pol protein expression in vG/P-92-infected cells as well as the ability of the G/P-92 fusion protein to form virus-like particles (VLP) in infected cultures. The efficacy of vG/P-92 and vVK1 vaccines was evaluated in a murine model by measuring T helper (Th), cytotoxic T lymphocyte (CTL), and antibody responses to Gag and Pol antigens. RESULTS: The deletion of a frameshift site resulted in an increased level of Pol in cells expressing the G/P-92 fusion protein. Particles budding from the plasma membrane were detected in both vG/P-92- and vVK1-infected cells, but the release of VLP was less efficient from cells expressing the G/P-92 fusion protein than the entire gag-pol gene product. Immunization with vG/P-92 vector elicited a higher level of cellular and humoral responses to both Gag and Pol antigens than the vVK1 vaccine. CONCLUSIONS: The enhanced immunogenicity of the G/P-92 fusion protein compared with the entire viral gag-pol gene product might be related to a higher intracellular level of Pol and Gag expression due to the deletion of a frameshift site and less efficient transport of VLP from vG/P-92-infected cells, respectively.

The effect of deletion of the V3 loop of gp120 on cytotoxic T cell responses and HIV gp120-mediated pathogenesis.

New strategies for improving the efficacy of HIV vaccines are of significant importance. In this study, we analyzed the effect of deletion of the hypervariable V3 loop of gp120 on envelope (env)-specific CTL responses in PBMC of HIV-infected individuals. We showed increased CTL activities against conserved epitopes of the env glycoprotein in cultures induced with the AV3 mutant compared with those stimulated with the full-length env gene products. In contrast to the wild-type env, the AV3 mutant-expressing cells were resistant to Ab-dependent cell-mediated cytotoxicity, formed no syncytia, and neither underwent nor induced apoptosis in CD4+ cells. Thus, the AV3 mutant may redirect immune responses toward conserved epitopes of gp160, has longer expression time due to increased resistance to Ab-dependent cell-mediated cytotoxicity, and does not trigger cytopathic effects associated with apoptosis and syncytium formation. This approach may apply to other Ags of HIV, where deletions of highly variable or immunosuppressive epitopes may improve the efficacy of HIV vaccines.

The effect of epitope variation on the profile of cytotoxic T lymphocyte responses to the HIV envelope glycoprotein.

To address the relationship between viral and host factors during HIV infection, we analyzed the effect of viral mutations on T cell responses in seropositive, asymptomatic HLA-A2+ individuals using four envelope (env)-specific peptides with the HLA-A*0201 binding motif. We showed that the natural sequence variation was frequent within epitopes located in the C-terminal region of the env glycoprotein and was largely responsible for a lower env-specific cytotoxic T lymphocyte (CTL) activity in the peptide-stimulated cultures. The highest CTL responses in vitro were induced with conserved epitopes D1 and 4.3 that mapped to the N-terminal region of the env glycoprotein. These peptides exhibited high binding affinity for HLA-A*0201 molecules and stimulated CD8+ T cells of relatively limited TCR Vbeta chain repertoire. Decreased CTL activities to the D1 epitope were observed in the absence of any detectable viral mutation, and were associated with lower proliferative responses and expression of the CD28 antigen. Results of this study demonstrate that the degree of sequence variation within a stimulatory epitope of the viral quasispecies, as well as proliferative potential of the effector cells, are among the factors underlying decreased CTL activity in HIV-infected patients. These experiments also provide evidence that the D1 peptide might be useful for the development of vaccines and immune-based therapy.

Role of the V2, V3, and CD4-binding domains of GP120 in curdlan sulfate neutralization sensitivity of HIV-1 during infection of T lymphocytes.

A sulfated polysaccharide, curdlan sulfate (CRDS) with 1,3-beta-D-glucan as a main chain, inhibits HIV-1 infection of human peripheral blood lymphocytes (PBLs) by binding to the V3 region of gp 120. We previously showed that T cell (T)-tropic HIV-1 isolates are over 10-fold more sensitive to neutralization by CRDS than macrophage (MT)-tropic viruses, which possesses a relatively less charged amino acid composition in the V3 sequence. To analyze the interaction of CRDS with V3 and its association with neutralization sensitivity of HIV-1 isolates, we examined the effect of CRDS on the binding of neutralizing antibodies to monomeric and oligomeric gp 120 mutants of T- and MT-tropic HIV-1 clones in which the V3 loop was either deleted or substituted by V3 of another isolate. Our results showed that the presence and the amino acid composition of the V3 loop appears to determine the extent of interaction of CRDS with the V2 and CD4-binding regions on native gp 120 monomers; however, the positive charge of V3 has less effect on this interaction on oligomeric gp 120. Furthermore, our results established that only the CRDS-induced masking of V3 on oligomeric gp120 appears to be associated with the anti-HIV-1 activity of CRDS in vitro. Our findings underline the usefulness of CRDS for understanding the structural constraints on gp 120 that drive the transition from MT- to T-tropic isolates in vivo and enable the virus to use multiple fusion cofactors.