Human-like antibodies neutralizing Western equine encephalitis virus

Abstract

This study describes the development of the first neutralizing antibodies against Western equine encephalitis virus (WEEV), a member of the genus Alphavirus. WEEV is transmitted by mosquitoes and can spread to the human central nervous system, causing symptoms ranging from mild febrile reactions to life-threatening encephalitis. WEEV has been classified as a biological warfare agent by the US Centers for Disease Control and Prevention. No anti-WEEV drugs are currently commercially available. Neutralizing antibodies are useful for the pre- and post-exposure treatment of WEEV infections. In this study, two immune antibody gene libraries were constructed from two macaques immunized with inactivated WEEV. Four antibodies were selected from these libraries and recloned as scFv-Fc, with a human Fc part. These antibodies bound WEEV specifically in ELISA with little or no cross-reaction with other alphaviruses. They were further analyzed by immunohistochemistry. All binders were suitable for the intracellular detection of WEEV particles. Neutralizing activity was determined in vitro. Three of the four antibodies were found to be neutralizing; about 1 ng/mL of the best antibody (ToR69-3A2) neutralized 50% of 5x10(4) TCID 50/mL. Due to its human-like nature with a germinality index of 89% (VH) and 91% (VL), the ToR69-3A2 antibody is a promising candidate for future passive vaccine development.

Keywords: Alphavirus; NHP antibodies; Western equine encephalitis virus (WEEV); immune antibody library; passive vaccine; phage display; scFv; scFv-Fc.

Figure 1. Immunization scheme for macaque 1 and 2. *timepoint for WEEV library construction. +, timepoint for VEEV library construction.

Figure 2. Anti-WEEV immune responses. Sera were tested by antigen ELISA using supernatants from VRS-purified WEEV-infected Vero cells or non-infected cells (negative control). Bound antibodies were detected by incubation with rabbit anti-monkey IgG conjugated to horseradish peroxidase (HRP).

Figure 3. Anti-WEEV scFv-Fc antibody sandwich ELISA. Plates coated with a dilution series of WEEV suspensions captured by anti-WEEV mAb SFV12/2 (3 µg/mL). Three titration ELISAs were made in parallel. Staining was performed by incubation with 1 µg/mL biotinylated anti-WEEV antibodies, followed by streptavidin-HRP conjugate (1:4,000). As a negative control, we assessed antibody binding to Vero cell culture material.

Figure 4. Analysis of cross-reactivity with other alphaviruses by a qualitative sandwich ELISA. The monoclonal SFV12/2, mAb Pix c/t 6/2 or VEE-WIS1 (3 µg/mL) were coated for capturing and different alphaviruses were applied (TCID50/mL WEEV: 4x10⁵; EEEV: 6x10⁵; VEEV: 8x10⁶; SINV: 8x10⁵; SFV: 2.5x10⁶; CHIKV: 7x10⁵; PIXV: 8x10⁵). Staining was performed with 2 µg/mL biotinylated anti-WEEV scFv-Fc antibodies (200 ng/mL for ToR68–3G2), followed by streptavidin-HRP conjugate (1:4,000). Three ELISA experiments were performed in parallel, with the exception of CKIKV and PIXV for ToR68–3G2. Here, only two ELISA experiments were performed. As negative control, antibody binding to Vero cell culture material was tested.

Figure 5. IHC analysis of anti-WEEV scFv-Fc antibody binding to VEEV infected Vero cells. Vero cells were infected with WEEV and fixed in formalin, for staining with a 1:5000 dilution of the antibodies ( = 200 ng/mL for the recombinant antibodies), followed by incubation with streptavidin-HRP conjugate (1:6,000). As positive controls, the anti-WEEV antibody MAB8742 and the anti-alphavirus antibody SFV12/2 were used. As negative control, the streptavidin-HRP conjugate was used without an anti-WEEV antibody.

Figure 6. Inhibition of WEEV infection by anti-WEEV scFv-Fc antibodies. Neutralization was analyzed in NPLA assays. Antibody dilution series were incubated with 5x10⁴ TCID₅₀/mL before 2x10⁵ Vero cells were infected. As controls, the non-neutralizing mAb SFV12/2 was used or the cells were not infected. The infection of cells was demonstrated by the specific immunostaining of viral antigens by incubation with the biotinylated mAb SFV 12/2 (1:5,000) followed by streptavidin-HRP conjugate (1:4,000).