We previously reported construction of a chimeric yellow fever-dengue type 2 virus (YF/DEN2) and determined its safety and protective efficacy in rhesus monkeys (F. Guirakhoo et al., J. Virol. 74:5477-5485, 2000). In this paper, we describe construction of three additional YF/DEN chimeras using premembrane (prM) and envelope (E) genes of wild-type (WT) clinical isolates: DEN1 (strain PUO359, isolated in 1980 in Thailand), DEN3 (strain PaH881/88, isolated in 1988 in Thailand), and DEN4 (strain 1228, isolated in 1978 in Indonesia). These chimeric viruses (YF/DEN1, YF/DEN3, and YF/DEN4) replicated to ϳ7.5 log 10 PFU/ml in Vero cells, were not neurovirulent in 3-to 4-week-old ICR mice inoculated by the intracerebral route, and were immunogenic in monkeys. All rhesus monkeys inoculated subcutaneously with one dose of these chimeric viruses (as monovalent or tetravalent formulation) developed viremia with magnitudes similar to that of the YF 17D vaccine strain (YF-VAX) but significantly lower than those of their parent WT viruses. Eight of nine monkeys inoculated with monovalent YF/DEN1 -3, or -4 vaccine and six of six monkeys inoculated with tetravalent YF/DEN1-4 vaccine seroconverted after a single dose. When monkeys were boosted with a tetravalent YF/ DEN1-4 dose 6 months later, four of nine monkeys in the monovalent YF/DEN groups developed low levels of viremia, whereas no viremia was detected in any animals previously inoculated with either YF/DEN1-4 vaccine or WT DEN virus. An anamnestic response was observed in all monkeys after the second dose. No statistically significant difference in levels of neutralizing antibodies was observed between YF virus-immune and nonimmune monkeys which received the tetravalent YF/DEN1-4 vaccine or between tetravalent YF/DEN1-4-immune and nonimmune monkeys which received the YF-VAX. However, preimmune monkeys developed either no detectable viremia or a level of viremia lower than that in nonimmune controls. This is the first recombinant tetravalent dengue vaccine successfully evaluated in nonhuman primates.
Although the smallpox virus was eradicated over 20 years ago, its potential release through bioterrorism has generated renewed interest in vaccination. To develop a modern smallpox vaccine, we have adapted vaccinia virus that was derived from the existing Dryvax vaccine for growth in a human diploid cell line. We characterized six cloned and one uncloned vaccine candidates. One clone, designated ACAM1000, was chosen for development based on its comparability to Dryvax when tested in mice, rabbits and monkeys for virulence and immunogenicity. By most measures, ACAM1000 was less virulent than Dryvax. We compared ACAM1000 and Dryvax in a randomized, double-blind human clinical study. The vaccines were equivalent in their ability to produce major cutaneous reactions ('takes') and to induce neutralizing antibody and cell-mediated immunity against vaccinia virus.
Chimeric yellow fever (YF)-dengue (DEN) viruses (ChimeriVax-DEN) were reconstructed to correct amino acid substitutions within the envelope genes of original constructs described by Guirakhoo et al. (2001, J. Virol. 75, 7290-7304). Viruses were analyzed and compared to the previous constructs containing mutations in terms of their growth kinetics in Vero cells, neurovirulence in mice, and immunogenicity in monkeys as monovalent or tetravalent formulations. All chimeras grew to high titers [ approximately 7 to 8 log(10), plaque-forming units (PFU)/ml] in Vero cells and were less neurovirulent than YF 17D vaccine in mice. For monkey experiments, the dose of DEN2 chimera was lowered to 3 log(10) PFU in the tetravalent mixture in an effort to reduce its dominant immunogenicity. The magnitude of viremia in ChimeriVax-DEN immunized monkeys was similar to that of YF-VAX, but significantly lower than those induced by wild-type DEN viruses. All monkeys developed high levels of neutralizing antibodies against homologous (chimeras) or heterologous (wild-type DEN viruses isolated from different geographical regions) viruses after a single dose of monovalent or tetravalent vaccine. Administration of a second dose of tetravalent vaccine 2 months later increased titers to both homologous and heterologous viruses. A dose adjustment for dengue 2 chimera resulted in a more balanced response against dengue 1, 2, and 3 viruses, but a somewhat higher response against chimeric dengue 4 virus. This indicates that further formulations for dose adjustments need to be tested in monkeys to identify an optimal formulation for humans.
Three consecutive plaque purifications of four chimeric yellow fever virus-dengue virus (ChimeriVax-DEN) vaccine candidates against dengue virus types 1 to 4 were performed. The genome of each candidate was sequenced by the consensus approach after plaque purification and additional passages in cell culture. Our data suggest that the nucleotide sequence error rate for SP6 RNA polymerase used in the in vitro transcription step to initiate virus replication was as high as 1.34 ؋ 10 ؊4 per copied nucleotide and that the error rate of the yellow fever virus RNA polymerase employed by the chimeras for genome replication in infected cells was as low as 1.9 ؋ 10 ؊7 to 2.3 ؋ 10 ؊7 . Clustering of beneficial mutations that accumulated after multiple virus passages suggests that the N-terminal part of the prM protein, a specific site in the middle of the E protein, and the NS4B protein may be essential for nucleocapsid-envelope interaction during flavivirus assembly.A fundamental feature of RNA viruses is the error-prone nature of replication of their genomes, resulting in adaptation and rapid evolution. Various in vitro, in vivo, and ex vivo methods have been used to estimate mutation rates of viral RNA-dependent RNA polymerases. The generally accepted average value of 10 Ϫ4 to 10 Ϫ5 mutations per nucleotide (nt) per round of RNA replication (reviewed in references 6, 7, 23, and 28) results in substitution of 0.1 to 1 nt per each synthesized genomic RNA molecule of ϳ10 kb. Mutation rates vary between 10 Ϫ3 and 10 Ϫ6 in individual studies, depending on the virus and method used, although none of these methods provide an exact measure of the error rate. In vitro methods that utilize purified viral RNA polymerases may overestimate the error rate due to suboptimal in vitro enzymatic conditions and experimental complexity. In ex vivo methods, such as sequencing of phenotypic mutants generated in cell culture and analysis of monoclonal antibody escape mutants or of rates of reversion of phenotypic markers, the number of rounds of RNA replication in infected cells is not precisely known. Methods that involve a reverse transcription-PCR step with cloning of cDNA in bacteria prior to sequencing can be misleading because of the accumulation of additional mutations introduced by reverse transcription-PCR.The Flavivirus genus of the Flaviviridae family consists of about 70 viruses of which 40 have been associated with human illness (reviewed in reference 2). Flaviviruses are small enveloped plus-strand RNA viruses (reviewed in reference 18). The viral particle contains a nucleocapsid composed of viral RNA and capsid protein C, surrounded by a lipid bilayer in which the envelope protein E and membrane protein M are embedded. The genomic RNA is about 11,000 nt long and contains a single long open reading frame (ORF). Translation of the ORF produces a single polyprotein precursor containing viral proteins in the order: C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-2K-NS4B-NS5, where C through E are the structural components of the virion (prM is a pr...
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