Recombinant chimeric yellow fever-dengue type 2 virus is immunogenic and protective in nonhuman primates

A chimeric yellow fever (YF)-dengue type 2 (dengue-2) virus (ChimeriVax-D2) was constructed using a recombinant cDNA infectious clone of a YF vaccine strain (YF 17D) as a backbone into which we inserted the premembrane (prM) and envelope (E) genes of dengue-2 virus (strain PUO-218 from a case of den gue fever in Bangkok, Thailand). The chimeric virus was recovered from the supernatant of Vero cells transfected with RNA transcripts and amplified on ce in these cells to yield a titer of 6.3 log(10) PFU/ml, The ChimeriVax-D2 was not neurovirulent for 4-week-old outbred mice inoculated intracerebral ly, This virus was evaluated in rhesus monkeys for its safety (induction of viremia) and protective efficacy (induction of anti-dengue-2 neutralizing antibodies and protection against challenge). In one experiment, groups of non-YF-immune monkeys received graded doses of ChimeriVax-D2; a control gro up received only the vaccine diluents, All monkeys (except the control grou p) developed a brief viremia and showed no signs of illness. Sixty-two days postimmunization, animals were challenged with 5.0 log(10) focus forming u nits (FFU) of a wild-type dengue-2 virus. No viremia (<1.7 log(10) FFU/ml) was detected in any vaccinated group, whereas ail animals in the placebo co ntrol group developed viremia. All vaccinated monkeys developed neutralizin g antibodies in a dose-dependent response. In another experiment, viremia a nd production of neutralizing antibodies were determined in YF-immune monke ys that received either ChimeriVax-D2 or a wild-type dengue-2 virus, Low vi remia was detected in ChimeriVax-D2-inoculated monkeys, whereas all dengue- 2-immunized animals became viremic, All of these animals were protected aga inst challenge with a wild-type dengue-2 virus, whereas all YF-immune monke ys and nonimmune controls became viremic upon challenge. Genetic stability of ChimeriVax-D2 was assessed by continuous In vitro passage in VeroPM cell s, The titer of ChimeriVax-D2, the attenuated phenotype for 4-week-old mice , and the sequence of the inserted prME genes were unchanged after 18 passa ges in Vero cells. The high replication efficiency, attenuation phenotype i n mice and monkeys, immunogenicity and protective efficacy, and genomic sta bility of ChimeriVax-D2 justify it as a novel vaccine candidate to be evalu ated in humans.