An inactivated West Nile Virus vaccine derived from a chemically synthesized cDNA system

Abstract

A cDNA comprising the complete genome of West Nile Virus (WNV) was generated by chemical synthesis using published sequence data, independent of any preformed viral components. The synthetic WNV, produced by transfection of in vitro transcribed RNA into cell culture, exhibited undistinguishable biological properties compared to the corresponding animal-derived wild-type virus. No differences were found concerning viral growth in mammalian and insect cell lines and concerning expression of viral proteins in cells. There were also no significant differences in virulence in mice following intranasal challenge. After immunizations of mice with experimental vaccines derived from the synthetic and wild-type viruses, protection from lethal challenge was achieved with similar amounts of antigen. Both vaccine preparations also induced comparable levels of neutralizing antibodies in mice. In addition, the synthetic approach turned out to be very accurate, since the rescued WNV genome contained no undesired mutations. Thus, the first flavivirus based on chemical gene synthesis was indistinguishable from the parent virus. This demonstrates that virus isolates from animal sources are dispensable to derive seed viruses for vaccine production or research.

Fig. 1

Construction of the two partial cDNA clones. A schematic drawing of the WNV genome is indicated on the top. Dark gray boxes represent the viral structural and light gray boxes the nonstructural part of the coding sequence. The 5′ and 3′ noncoding regions (NCR) are indicated as thin lines. (a) Six WNV sequence segments were generated by gene synthesis (vector backbones are not shown). The 5′TL AB segment is corresponding to WNV genomic sequence nt 1–1792, 5′TL CD to nt 1789–3632, segment 3′TL AB to nt 3622–5801, 3′TL CD to nt 5792–8028, 3′TL EF to nt 8022–10,025 and 3′TL GH to nt 10,022–11,029. (b) Fragments 5′TL AB and 5′TL CD were ligated yielding the final 5′ partial clone insert (5′TL) and two 3′ part fragments were generated by fusing fragments 3′TL AB with 3′TL CD and 3′TL EF with 3′TL GH. (c) Final partial clones, integrated in pBR322, are indicated. The 3′ partial clone was generated by ligation of the 3′TL ABCD with the 3′TL EFGH insert, taking advantage of the unique SpeI restriction site.

Fig. 2

Detection of WNV antigen in Vero cells by indirect immunofluorescence. Vero cells, uninfected (a), transfected with WNVsyn-transcribed RNA (b), infected with WNV wild-type virus (c), or infected with a WNVsyn virus stock (d) were tested for the expression of WNV proteins by indirect immunofluorescence two days after infections or transfection.

Fig. 3

Plaque morphology of wild-type WNV (a) and WNVsyn (b) at day 4 postinfection of Vero cells seeded in six-well plates with a titer of 100 TCID₅₀. Staining was carried out with neutral red for 4 h. Growth of synthetic (solid diamonds) and wild-type WNV (open squares) was analyzed in Vero (c) and C6/36 cells (d) infected with a MOI of 0.0001. The growth curve experiment was repeated once. The titers are means of two determinations ± SD indicated by the error bars.

Fig. 4

Neutralizing antibody titers in Balb/c mice after immunization using WNVwt and WNVsyn derived vaccine preparations (a) and characterization of the inactivated vaccine preparations used to immunize mice by Western blotting (b). Gray bars represent neutralizing antibody titers of mice vaccinated with WNVwt antigen and white bars neutralizing antibody titers of mice vaccinated with WNVsyn preparations (upper panel). Western blotting was carried out to analyze the antigen content used in individual formulations (lower panel); w, WNVwt; s, WNVsyn; E, WNV envelope protein; prM, pre-membrane protein; M*, dimeric membrane protein.