Preparation and immunogenic properties of a recombinant West Nile subunit vaccine

Abstract

While several West Nile vaccines are being developed, none are yet available for humans. In this study aimed at developing a vaccine for humans, West Nile virus (WNV) envelope protein (E) and non-structural protein 1 (NS1) were produced in the Drosophila S2 cell expression system. The C-terminal 20% of the E protein, which contains the membrane anchor portion, was deleted, thus allowing for efficient secretion of the truncated protein (80E) into the cell culture medium. The proteins were purified by immunoaffinity chromatography (IAC) using monoclonal antibodies that were flavivirus envelope protein group specific (for the 80E) or flavivirus NS1 group specific (for NS1). The purified proteins were produced in high yield and used in conjunction with adjuvant formulations to vaccinate mice. The mice were tested for both humoral and cellular immune responses by a plaque reduction neutralization test and ELISA, and by lymphocyte proliferation and cytokine production assays, respectively. The results revealed that the 80E and the NS1 proteins induced both high-titered ELISA and neutralizing antibodies in mice. Splenocytes from immunized mice, cultured in vitro with the vaccine antigens as stimulants, showed excellent proliferation and production of cytokines (IFN-gamma, IL-4, IL-5, and IL-10). The level of antigen-stimulated lymphocyte proliferation and cytokine production was comparable to the level obtained from mitogen (phytohemagglutinin or pokeweed) stimulation, indicating a robust cellular response as well. These findings are encouraging and warrant further in vivo studies to determine the protective efficacy of the WNV vaccine candidate.

Fig. 1

(A) Coomassie blue stained SDS-PAGE of WNV 80E protein expressed by Drosophila S2 cells under non-reducing conditions. Lane 1, Spinner Culture #1 of cell line WN-80E-1 harvested 2/19/03; Lane 2, Spinner Culture #2 of cell line WN-80E-1 harvested 2/10/03; Lane 3, culture of a dengue transformant cell line. The migration of the WNV 80E is faster than the dengue 80E due to differences in glycosylation and tertiary structure (samples are non-reduced). (B) Western blot of duplicate SDS-PAGE gel seen in A. The blot was probed with a commericially available WNV rabbit polyclonal from BioReliance. This antibody cross-reacts slightly with the Dengue 80E.

Fig. 2

(A) Coomassie blue stained SDS-PAGE of WNV NS1 protein expressed by Drosophila S2 cells under reducing (Lanes 1 and 2) and non-reducing conditions (Lanes 3 and 4). Lanes 1 and 3, Spinner Culture #1 of cell line WN-NS1-5 harvested 7/6/03; Lanes 2 and 4, Spinner Culture #2 of cell line WN-NS1-5 harvested 7/6/03. (B) Western blot of duplicate SDS-PAGE gel seen in A. The blot was probed with the mouse monoclonal 7E11. The two approximately 40 kDa bands of WN-NS1 are two different glycoforms of the NS1 protein. The higher MW reactive band at about 80 kDa in Lanes 3 and 4 is a dimer. The 7E11 antibody reacts more strongly with reduced than non-reduced NS1.

Fig. 3

Coomassie stained SDS-PAGE gel (A) and Western blot (B) of purified WNV 80E. Both samples were run under non-reducing conditions on 10% gels. The Western blot was developed using a rabbit polyclonal antisera developed against formalin inactivated dengue virus. The sizes of the molecular weight markers (in kDa) are indicated to the left of the gel and blot. The sample loadings (in μg) are presented at the top of each.

Fig. 4

Coomassie stained SDS-PAGE gel (A) and Western blot (B) of purified WNV NS1. Both samples were run under non-reducing conditions on 10% gels. The Western blot was developed using a rabbit polyclonal antisera developed against purified dengue NS1. The sizes of the molecular weight markers (in kDa) are indicated to the left of the gel and blot. The sample loadings (in μg) are presented at the top of each.

Fig. 5

Ratio of IgG2a to IgG1 antibody titers. Panel A: antibody titers to WNV 80E. Panel B: antibody titers to WNV NS1.

Fig. 6

Antigen-stimulated lymphocyte proliferation. Splenectomies performed 4 days post booster vaccination. The total cpm recovered from tritiated thymidine incorporation per 4 × 10⁵ cells (total number of cells cultured per well) is plotted. Values derived from quadruplicate wells per stimulant condition were averaged. Histograms represent the mean of individual mice (n = 3). Error bars represent the standard deviations. Tests of significance (p < 0.05) were performed between groups with the same stimulant and between stimulants within a group using an unpaired t-test with the aid of a commercially available statistical program (GraphPad Prizm).

Fig. 7

Antigen-stimulated cytokine production in vitro. Splenectomies performed 7 days post booster vaccination. Panels A, B, and C: IFN-γ, IL-4, and IL-5, respectively. Histograms represent the mean of individual mice (n = 5). Error bars represent the standard deviations. Tests of significance (p < 0.05) were performed between groups with the same stimulant and between stimulants within a group using an unpaired t-test with the aid of a commercially available statistical program (GraphPad Prizm).