Construction, safety, and immunogenicity in nonhuman primates of a chimeric yellow fever-dengue virus tetravalent vaccine

Abstract

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 ~7.5 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.

FIG. 1

Synthesis of pYD1-5′3′ and YF/DEN1 fragment H. Overlap-extension PCR was used to fuse the prME sequence of DEN1 strain PUO359 (fragment B) to the YF capsid gene. The resulting PCR product was then subcloned as a NotI-NheI fragment. A silent BstBI site was introduced to facilitate ligation to form the full-length cDNA chimeric clone. Overlap-extension PCR was used to fuse the DEN1 envelope gene to the YF NS genes from pYFM5.2/JE-S (fragment H). In vitro ligation of the BstBI-AatII fragment with plasmid pYD1-5′3′ produced the full-length clone for transcription.

FIG. 2

RT-PCR amplification of the prME region of DEN3 parent strain PaH881/88. The prME region was amplified in two fragments by using H-87 strain-specific primers (arrows). Hydrophobic signals are shadowed. The introduced BstBI site did not result in any amino acid changes, whereas a NarI site introduced a change (Q to G) at the penultimate residue of E. 5′UTR, 5′ untranslated region.

FIG. 3

Construction of the YF/DEN3 chimera. YF- and DEN3-specific sequences are shown as shadowed and black boxes, respectively. The chimeric YF/DEN3 genome was reconstituted by in vitro ligation of three DNA fragments (see Materials and Methods). mutXhoI, mutated XhoI site.

FIG. 4

YF/DEN4 two-plasmid system. Overlap-extension PCR was used to fuse the prME sequence of DEN4 strain 1228 (fragment B) to the YF capsid gene. The resulting PCR product was then subcloned as a NotI-NheI fragment into plasmid pYF5′3′IV/JE-S. A silent BstBI site was introduced to facilitate ligation to form the full-length cDNA chimeric clone. For synthesis of pYD4-5.2, an overlap-extension PCR was used to fuse the DEN4 envelope gene to the YF NS genes in plasmid pYFM5.2/JE-S. An SfoI site replaced the NarI site during subcloning of the fragment into plasmid pYD4-5.2. In vitro ligation of the BstBI-AatII fragment with plasmid pYD4-5′3′ produced a full-length clone for transcription.