West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays

Abstract

Introduction of West Nile (WN) virus into the United States in 1999 created major human and animal health concerns. Currently, no human or veterinary vaccine is available to prevent WN viral infection, and mosquito control is the only practical strategy to combat the spread of disease. Starting with a previously designed eukaryotic expression vector, we constructed a recombinant plasmid (pCBWN) that expressed the WN virus prM and E proteins. A single intramuscular injection of pCBWN DNA induced protective immunity, preventing WN virus infection in mice and horses. Recombinant plasmid-transformed COS-1 cells expressed and secreted high levels of WN virus prM and E proteins into the culture medium. The medium was treated with polyethylene glycol to concentrate proteins. The resultant, containing high-titered recombinant WN virus antigen, proved to be an excellent alternative to the more traditional suckling-mouse brain WN virus antigen used in the immunoglobulin M (IgM) antibody-capture and indirect IgG enzyme-linked immunosorbent assays. This recombinant antigen has great potential to become the antigen of choice and will facilitate the standardization of reagents and implementation of WN virus surveillance in the United States and elsewhere.

FIG. 1

Map of the WN virus genomic region (top) and oligonucleotides used in RT-PCR to construct the transcription unit for the expression of WN virus prM and E coding regions (bottom). Potential transmembrane helices of viral proteins are indicated by black boxes.

FIG. 2

Map of the recombinant WN virus plasmid pCBWN. The transcription unit contains the human cytomegalovirus early gene promoter (CMV), JE virus signal sequence, WN virus prM and E gene region, and bovine growth hormone poly(A) signal (BGH).

FIG. 3

Comparison of Western blot reactivity between NRA produced by pCBWN-transformed COS-1 cells (A) and gradient-purified WN virion proteins (V). WN virus-specific mouse HIAF, flavivirus E-specific cross-reactive MAb 4G2, and eastern equine encephalitis virus monoclonal antibody (EEE MAb) were used at a 1:200 dilution in the assay. Sizes are shown in kilodaltons.

FIG. 4

WN virus-specific reactivity of pre- and postchallenge serum specimens obtained from mice and horses immunized with WN virus DNA vaccine. Pooled serum specimens from the mice and horses used in the experiments were tested at a 1:25 dilution by Western blot analysis using purified WN virion as the antigen. Western blot results obtained with pooled horse sera collected before DNA vaccination (week 0), 5 weeks postvaccination or before virus challenge (week 5), and 2 weeks postchallenge (week 7). Positive horse serum (lane +) was from control horse 16, which had PRNT titer of >1,280 on week 4 after virus challenge. Western blot results obtained with pooled mouse sera collected before DNA vaccination (week 0), 3 and 6 weeks postvaccination or before virus challenge (weeks 3 and 6), and 3 weeks postchallenge (week 9). Mouse HIAF at a 1:250 dilution was used as the positive control (lane +). Sizes are shown in kilodaltons.