Hepatitis C is a major cause of chronic liver disease, with 170 million individuals infected worldwide and no available vaccine. We analyzed the effects of an induced T-cell response in 3 chimpanzees, targeting nonstructural proteins in the absence of neutralizing antibodies. In all animals the specific T-cell response modified the outcome of infection, producing a 10-to 1,000-fold reduction in peak virus titers. The challenge of 2 immunized animals that had been previously exposed to hepatitis C virus resulted in subclinical infections. Immune responses in the third animal, naive prior to immunization, limited viral replication immediately, evidenced by a 30-fold reduction in virus titer by week 2, declining to a nonquantifiable level by week 6. After 10 weeks of immunological control, we observed a resurgence P ersistent infections caused by hepatitis C virus (HCV) occur in 70%-80% of the acutely infected population, most of whom will develop chronic hepatitis and be at risk for cirrhosis, end-stage liver disease, and/or hepatocellular carcinoma. 1 Antiviral therapy at present is successful in about 50% of patients, but treatment carries significant side effects and is very costly. 2,3 At present, about 25,000 new HCV infections occur each year in the United States, making the development of a vaccine against this virus imperative.Natural infection with HCV had previously been thought to afford no protective immunity from reinfection. 4,5 However, we and others have shown that following rechallenge, viremia and liver disease are significantly reduced in both intensity and duration 6-8 ; this appears to be mediated through faster T-cell activation in peripheral blood and liver in chimpanzees 8,9 and possibly in humans. 10 Despite developments with transgenic mouse systems using transplanted human hepatocytes, 11 the only established animal model for HCV is the chimpanzee, 12 which makes challenge vaccine experiments difficult and expensive. The development of HCV vaccines has also been hampered by the lack of an effective in vitro cell culture
Aims and Methods To facilitate antigenic characterization of the influenza A 2009 pandemic H1N1 [A(H1N1)pdm09] hemagglutinin (HA), we generated a panel of murine monoclonal antibodies (mAbs) using as the immunogen mammalian‐derived virus‐like particles containing the HA of the A/California/04/2009 virus. The antibodies were specific for the A/California/04/2009 HA, and individual mAbs suitable for use in several practical applications including ELISA, immunofluorescence, and Western blot analysis were identified. Results and Conclusions As the panel of mAbs included antibodies with hemagglutination inhibition (HI) and virus neutralizing activities, this allowed identification and characterization of potentially important antigenic and neutralizing epitopes of the A/California/04/2009 HA and comparison of those epitopes with the HAs of other influenza viruses including seasonal H1N1 viruses as well as the A/South Carolina/1918 and A/New Jersey/1976 H1N1 viruses. Three mAbs with the highest HI and neutralizing titers were able to provide passive protection against virus challenge. Two other mAbs without HI or neutralizing activities were able to provide partial protection against challenge. HA epitopes recognized by the strongest neutralizing mAbs in the panel were identified by isolation and selection of virus escape mutants in the presence of individual mAbs. Cloned viruses resistant to HI and antibody neutralization were sequenced to identify mutations, and two unique mutations (D127E and G155E) were identified, both near the antigenic site Sa. Using human post‐vaccination sera, however, there were no differences in HI titer between A/California/04/2009 and either escape mutant, suggesting that these single mutations were not sufficient to abrogate a protective antibody response to the vaccine.
Vaccinia virus encodes an enzyme with DNA modifying activity that cleaves and inefficiently cross-links cruciformic DNA. This enzyme is contained within the virion, expressed at late times postinfection, and processes DNA in an energy-independent, Mg 2؉ ion-independent manner. Viral nuclease activity was measured in extracts from cells infected with well-defined viral mutants. Since some viral extracts lacked nuclease activity, the gene encoding the activity was postulated to be one of the open reading frames absent in the viruses lacking activity. Inducible expression of each candidate open reading frame revealed that only the gene VACWR035, or K4L, was required for nuclease activity. A recombinant virus missing only the open reading frame for K4L lacked nuclease activity. Extracts from a recombinant virus expressing K4L linked to a FLAG polypeptide were able to cleave and cross-link cruciformic DNA. There were no significant differences between the virus lacking K4L and wild-type vaccinia virus WR with respect to infectivity, growth characteristics, or processing of viral replicative intermediate DNA, including both telomeric and cross-linked forms. Purification of the K4L FLAG polypeptide expressed in bacteria yielded protein containing nicking-joining activity, implying that K4L is the only vaccinia virus protein required for the nicking-joining enzymatic activity.Viruses have evolved numerous strategies for the selective replication of their genomes in the infected cell. Vaccinia virus contains a double-stranded DNA genome with covalently continuous hairpin termini (22). The replication of the poxvirus genome requires two distinct processes using different sets of viral genes (20). The initial stage, the synthetic phase, requires members of the early gene class, including a virus-encoded DNA polymerase complex, for the rapid replication of viral DNA in the cytoplasm. The mechanism for initiation of DNA synthesis remains unknown, as DNA replication occurs in the absence of a typical origin of replication (5, 21). The most likely mechanism for initiation of DNA replication takes advantage of the structure of the genomic termini by the introduction of a single-stranded nick near the terminus followed by strand displacement synthesis to copy the terminal hairpin. The resulting double-stranded copy of the hairpin can rearrange to form two self-complementary hairpins, one of which contains the necessary 3Ј OH for the elongation strand displacement reaction to copy the genome. However, the synthesis phase of DNA replication does not generate simple catenated genomes. The replicative intermediates of DNA replication are complicated multiple-branched DNA molecules, with the hairpin represented as double-stranded concatemer junctions (2,3,20,23). These structures may arise from a combination of strand elongation synthesis and strand invasion utilizing both DNA replication and recombination. The vaccinia virus DNA polymerase can promote strand annealing in vitro (30), and the gene products required for recombination i...
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