A high incidence of community-acquired hepatitis C virus infection that can lead to the progressive development of chronic active hepatitis, liver cirrhosis, and primary hepatoceflular carcinoma occurs throughout the world. A vaccine to control the spread of this agent that represents a major cause of chronic liver disease is therefore needed. Seven chimpanzees (Pan troglodytes) have been immunized with both putative envelope glycoproteins [El (gp33) (4) and leads to the development of chronic hepatitis and liver cirrhosis in "50%o and 10% of cases, respectively (5). A significant proportion of patients with liver cirrhosis will also develop primary hepatocellular carcinoma (6). The prevalence of HCV infection around the world is generally between 0.4 and 2% (7-10), although a much higher level has been reported in Egypt (14%; see ref. 11). Therefore, HCV constitutes a major cause of chronic liver disease throughout the world. With the recent development of recombinant-based diagnostic assays for the detection of circulating HCV antibodies (2, 3), the risk of being infected with HCV after transfusion of blood or cellular components has been substantially reduced (12,13). However, community-acquired infection is much more common and occurs at various frequencies in high-risk groups such as i.v. drug users, health-care workers, and sexual and household contacts ofhepatitis patients, although -40%o of cases in the United States appear to have no known risk factor for acquisition of infection (14). Thus, the development of an HCV vaccine to prevent transmission within the community is highly desirable.HCV is distantly related genetically to both the pestiviruses and flaviviruses and, like these relatives, appears to process virion structural proteins from the N-terminal region of the polyprotein precursor encoded by the positivestranded RNA genome (15). The host signal peptidase mediates the cleavage of a basic, presumed nucleocapsid protein (C; -20 kDa) from the N terminus of the polyprotein precursor followed by two glycoproteins (El, glycoproteins (gp33 and gp72) under nondenaturing conditions from the endoplasmic reticulum. A fraction of the purified material was shown to exist in the form of a large E1/E2 oligomeric complex (22). We now report on the efficacy of this purified preparation in vaccinating chimpanzees against experimental infection with HCV-1. MATERIALS AND METHODSVaccine Preparation. A Stu I-Bgl II cDNA restriction fragment ofthe HCV-1 genome (nt 63 to +2901; aa 1-967; ref. 15) encoding the complete C (20 kDa), El (gp33 kDa), and E2 (gp72 kDa) proteins along with a C-terminally truncated NS2 product was cloned into the Sma I site of plasmid SC59 downstream of a hybrid early/late vaccinia promoter (S. Chakrabarti and B.M., unpublished work). BSC40 cells preinfected with wild-type WR vaccinia were transfected with the SC59 recombinant and thymidine kinase-negative recombinants, selected, and purified through three rounds ofplaque purification (23). Spinner cultures of HeLa cells (109 c...
Boceprevir is a hepatitis C virus (HCV) nonstructural protein (NS) 3/4A protease inhibitor that is currently being evaluated in combination with peginterferon alfa-2b and ribavirin in phase 3 studies. The clinical resistance profile of boceprevir is not characterized in detail so far. The NS3 protease domain of viral RNA was cloned from HCV genotype 1-infected patients (n ؍ 22). A mean number of 47 clones were sequenced before, at the end, and after treatment with 400 mg boceprevir twice or three times daily for 14 days for genotypic, phenotypic, and viral fitness analysis. At the end of treatment, a wild-type NS3 protease sequence was observed with a mean frequency of 85.9%. In the remaining isolates, five previously observed resistance mutations (V36M/A, T54A/S, R155K/T, A156S, V170A) and one mutation (V55A) with unknown resistance to boceprevir were detected either alone or in combination. Phenotypic analysis in the HCV replicon assay showed low (V36G, T54S, R155L; 3.8-to 5.5-fold 50% inhibitory concentration [IC 50 ]), medium (V55A, R155K, V170A, T54A, A156S; 6.8-to 17.7-fold IC 50 ) and high level (A156T; >120-fold IC 50 ) resistance to boceprevir. The overall frequency of resistant mutations and the level of resistance increased with greater declines in mean maximum HCV RNA levels. Two weeks after the end of treatment, the frequency of resistant variants declined and the number of wild-type isolates increased to 95.5%. With the exception of V36 and V170 variants all resistant mutations declined by more than 50%. Mathematical modeling revealed impaired replicative fitness for all single mutations, whereas for combined mutations a relative increase of replication efficiency was suggested. Conclusion: During boceprevir monotherapy, resistance mutations at six positions within the NS3 protease were detected by way of clonal sequence analysis. All mutations are associated with reduced replicative fitness estimated by mathematical modeling and show cross-resistance to telaprevir.
Rodent tumor cells engineered to secrete cytokines such as interleukin 2 (IL-2) or IL-4 are rejected by syngeneic recipients due to an enhanced antitumor host immune response. An adenovirus vector (AdCAIL-2) containing the human IL-2 gene has been constructed and shown-to direct secretion of high levels of human IL-2 in infected tumor cells. AdCAIL-2 induces regression of tumors in a transgenic mouse model of mammary adenocarcinoma following intratumoral injection. Elimination of existing tumors in this way results in immunity against a second challenge with tumor cells. These findings suggest that adenovirus vectors expressing cytokines may form the basis for highly effective immunotherapies of human cancers.
Several small molecule drugs that bind to the host CCR5 co-receptor and prevent viral entry have been developed for the treatment of HIV-1 infection. The innate variability found in HIV-1 envelope and the complex viral/cellular interactions during entry makes defining resistance to these inhibitors challenging. Here we found that mapping determinants in the gp160 gene from a primary isolate RU570-VCV(res), selected in culture for resistance to the CCR5 entry inhibitor vicriviroc, was complicated by inactivity of the cloned envelope gene in pseudovirus assays. We therefore recombined the envelope from RU570-VCV(res) into a highly active and susceptible ADA gp160 backbone. The chimeric envelopes generated robust signals in the pseudovirus assay and a 200 amino acid fragment, encompassing a C2-V5 region of the RU570-VCV(res) envelope, was required to confer resistance in both the single-cycle assay and in replicating virus. In contrast, a chimeric envelope that contained only the V3-loop region from this resistant virus was completely susceptible suggesting that the V3-loop changes acquired are context dependent.
Antibodies against hexon, the major coat protein of adenovirus (Ad), are an important component of the neutralizing activity in serum from naturally infected humans and experimentally infected animals. The mechanisms by which antihexon antibodies neutralize the virus have not been defined. As a model system, murine monoclonal antibodies raised against Ad type 5 (Ad5) were screened for antihexon binding and neutralization activity; one monoclonal antibody, designated 9C12, was selected for further characterization. The minimum ratio of 9C12 to Ad5 required for neutralization was 240 antibody molecules per virus particle, or 1 antibody per hexon trimer. Analysis of antibody-virus complexes by dynamic light scattering and negativestain electron microscopy (EM) showed that the virus particles were coated with electron-dense material but not aggregated at neutralizing ratios. Cryo-EM image reconstruction of the antibody-virus complex showed that the surface of the virus particle was covered by a meshwork of 9C12 antibody density, consistent with bivalent binding at multiple sites. Confocal analysis revealed that viral attachment, cell entry, and intracellular transport to the nuclear periphery still occur in the presence of neutralizing levels of 9C12. A model is presented for neutralization of Ad by an antihexon antibody in which the hexon capsid is cross-linked by antibodies, thus preventing virus uncoating and nuclear entry of viral DNA.
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