Advanced oxidation technology for the development of a next-generation inactivated West Nile virus vaccine

Abstract

West Nile virus (WNV) is the most frequent mosquito-borne disease reported in the continental United States and although an effective veterinary vaccine exists for horses, there is still no commercial vaccine approved for human use. We have previously tested a 3% hydrogen peroxide (H₂O₂)-based WNV inactivation approach termed, HydroVax, in Phase I clinical trials and the vaccine was found to be safe and modestly immunogenic. Here, we describe an advanced, next-generation oxidation approach (HydroVax-II) for the development of inactivated vaccines that utilizes reduced concentrations of H₂O₂ in combination with copper (cupric ions, Cu²⁺) complexed with the antiviral compound, methisazone (MZ). Further enhancement of this oxidative approach included the addition of a low percentage of formaldehyde, a cross-linking reagent with a different mechanism of action that, together with H₂O₂/Cu/MZ, provides a robust two-pronged approach to virus inactivation. Together, this new approach results in rapid virus inactivation while greatly improving the maintenance of WNV-specific neutralizing epitopes mapped across the three structural domains of the WNV envelope protein. In combination with more refined manufacturing techniques, this inactivation technology resulted in vaccine-mediated WNV-specific neutralizing antibody responses that were 130-fold higher than that observed using the first generation, H₂O₂-only vaccine approach and provided 100% protection against lethal WNV infection. This new approach to vaccine development represents an important area for future investigation with the potential not only for improving vaccines against WNV, but other clinically relevant viruses as well.

Keywords: Advanced oxidation; Antibody; Hydrogen peroxide; Vaccination; Vaccine; West Nile virus.

Figure 1.. The HydroVax-II inactivation approach provides robust inactivation of WNV.

Inactivation kinetics were performed with purified WNV-KV using the indicated individual chemicals and combinations of inactivation reagents. HydroVax consisted of 3% H₂O₂, whereas HydroVax-II consisted of an optimized combination of 0.005% H₂O₂, 0.125 μM CuCl₂, 20 μM methisazone (MZ) and 0.019% formaldehyde. The list of individual reagents and reagent combinations were tested at the same concentrations used in the final optimized HydroVax-II inactivation approach. The dashed line indicates the limit of detection (50 PFU/mL) and open symbols represent data points in which no infectious virus was identified.

Figure 2.. Virus inactivation with HydroVax-II technology improves the retention of neutralizing WNV-specific epitopes during inactivation.

(A) Purified WNV-KV was treated with 3% H₂O₂ for 7 hours (HydroVax), or treated with HydroVax-II for 20 hours at room temperature. Inactivated virus was diluted, coated onto ELISA plates and assayed with a panel of WNV Env-specific MAbs to determine the structural integrity of neutralizing epitopes among different regions of the WNV Env protein. The positive control (Live) consisted of untreated live WNV incubated under the same conditions for 20 hours in the absence of inactivation reagents. (B) The maximum ELISA titer was established for each MAb based on the live virus controls and the percentage of the live virus signal was calculated for each inactivation condition. DI-lr, Domain I lateral ridge; DII-ci, Domain II central interface; DII-di, Domain II dimer interface; DII-hi, Domain II hinge interface; DII-fl, Domain II fusion loop. MGAWN1 is a humanized version of the E16 antibody and retains the same epitope specificity [45].

Figure 3.. HydroVax-II technology substantially improves WNV-specific neutralizing antibody responses.

WNV vaccine antigens were prepared using the HydroVax or HydroVax-II manufacturing and inactivation technologies and formulated with alum prior to immunizing groups of BALB/c mice (n = 5 per group) at 1 μg per dose on day 0 and day 28. Serum samples were collected at day 56 (28 days post-boost) and tested for neutralizing activity against WNV-KV. Statistical comparisons between the indicated groups were determined using one-way ANOVA.

Figure 4.. HydroVax-II vaccination provides complete protection against morbidity and mortality following WNV-NY99 challenge.

Groups of BALB/c mice (n = 5/group) were vaccinated with a limited dose of 1 μg of WNV antigen prepared using the HydroVax or HydroVax-II manufacturing and inactivation approaches and compared to unvaccinated naïve controls after challenge with WNV-NY99. At 3-months post-primary vaccination (2-months post-booster vaccination), the weights of age-matched naïve mice (A) and mice immunized with the HydroVax (B) or HydroVax-II (C) vaccine were monitored daily to measure morbidity after intraperitoneal challenge with 200 PFU (20 LD₅₀) of WNV-NY99. Animals that reached pre-determined humane euthanasia endpoints are indicated by the † symbol. (D) Comparisons of survival were made relative to naive animals using the Mantel-Cox log rank test, with significant differences relative to the naïve group indicated by an asterisk (P <0.05).