Genetically delivered antibody protects against West Nile virus

Abstract

Gene-based delivery of recombinant antibody genes is a promising therapeutic strategy offering numerous advantages including sustained antibody levels, better safety profile and lower production cost. Here we describe generation of a recombinant antibody Fc-9E2 comprising a fusion protein between human Fc of IgG1 and a single-chain Fv derived from a hybridoma 9E2 secreting a mAb neutralizing West Nile virus (WNV). Fc-9E2 was shown to retain parental mAb's specificity and WNV-neutralizing capacity. Adenovirus-mediated in vivo delivery of the antibody gene resulted in sustained Fc-9E2 serum levels leading to abrogation of lethal WNV infection in an animal model.

Fig. 1

Schematic outline of the study. Genetic material form a hybridoma secreting the WNV-neutralizing mAb 9E2 (a) was used to generate scFv 9E2 (b) and then Fc-9E2 (c) coding sequences. Expression cassette comprising Fc-9E2 ORF, preceded by mouse kappa chain leader peptide sequence and followed by a polyadenylation signal, placed under the control of the CMV promoter was introduced into an Ad genome, which was further used to generate recombinant Ad/Fc-9E2 (d). The vector was used to inoculate mice (e) achieving Fc-9E2 in vivo production resulting in WNV neutralization and animal protection.

Fig. 2

Characterization of Fc-9E2. (a) Western blot of purified Fc-9E2. A549 cells were transduced with the Ad/Fc-9E2. The recAb was protein A-purified from culture supernatant. Aliquots of the purified recAb were separated by SDS-PAGE followed by Western blot with HRP-labeled goat anti-human IgG Abs and DAB staining. (R) Sample in reducing (2-ME, boiling) conditions; (UR) sample in unreducing (no 2-ME, no boiling) conditions. (b) Indirect ELISA with WNV-infected cell lysate. Anti WNV mAb 5H6 (squares) or an irrelevant mAb (diamonds) were used as capturing Abs. The wells were incubated with WNV-infected (Eg-101) Vero cell lysate followed by Fc-9E2, anti-human IgG/HRP and DAB staining. (c) Radioimmunoprecipitation with the lysate of BHK cells transfected with WN956 infectious clone. Cell lysate was incubated with Fc-9E2 (lane 1) and WNV E protein-specific mAb E31 (lane 2) followed by precipitation with protein A agarose. Proteins were separated by 12% SDS-PAGE and visualized on X-ray film. (d) Fc-9E2 ELISA with WNVEC. Fc-9E2 was incubated with either WNVEC (squares) or casein (diamonds) followed by HRP-labeled goat anti-human IgG Abs and DAB staining.

Fig. 3

Ad delivered recAb blood circulation in mice. Female BALB/c mice were inoculated i.p. with either 10⁸ (diamonds) or 10⁹ (squares) pfu of Ad/Fc9E2. Sera samples were collected at days 1, 2, 3, 4, 5, 7, 14 and 21 post-inoculation. Fc9E2 concentrations in individual sera were determined using Human IgG ELISA Quantitation Kit. Bars represent mean ± S.D.

Fig. 4

Ad/Fc-9E2 mediated protection against WNV. Ad/Fc-9E2 (a), or Ad/Fc-G19 encoding an irrelevant recAb (b), were inoculated i.p. at 10⁹ pfu/mouse: 24 h before (squares); simultaneously with (triangles); and 24 h after (circles) i.p. challenge with WNV NY99. Swiss Webster mice were used, 6 mice per group. One group (diamonds) was used as control for viral infection. The mice were monitored for survival for 14 days.

Fig. 5

Anti WNV antibodies in WNV challenged mice treated with recAb-encoding Ad vectors. Sera samples were collected from the mice described in Fig. 4 at indicated time points and tested by ELISA for Fc-9E2 circulation (a–c) and anti-WNV mouse IgM (d–f). (squares) Animals treated with Ad/Fc-9E2; (diamonds) with Ad/Fc-G19. (a and d) Ads given 24 h before; (b and e) Ads given simultaneously with; (c and f) Ad given 24 h after WNV challenge. Bars represent mean ± S.D.