Abstract
The three encephalitic alphaviruses, namely, the Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV), are classified by the Centers for Disease Control and Prevention (CDC) as biothreat agents. Currently, no licensed medical countermeasures (MCMs) against these viruses are available for humans. Neutralizing antibodies (NAbs) are fast-acting and highly effective MCMs for use in both pre- and post-exposure settings against biothreat agents. While significant work has been done to identify anti-VEEV NAbs, less has been done to identify NAbs against EEEV and WEEV. In order to develop anti-EEEV or -WEEV NAbs, mice were immunized using complementary strategies with a variety of different EEEV or WEEV immunogens to maximize the generation of NAbs to each of these viruses. Of the hybridomas generated, three anti-EEEV and seven anti-WEEV monoclonal antibodies were identified with in vitro neutralization activity. The most potent neutralizers (two anti-EEEV NAbs and three anti-WEEV NAbs) were further evaluated for neutralization activity against additional strains of EEEV, a single strain of Madariaga virus (formerly South American EEEV), or WEEV. Of these, G1-2-H4 and G1-4-C3 neutralized all three EEEV strains and the Madariaga virus strain, whereas G8-2-H9 and 12 WA neutralized six out of eight WEEV strains. To determine the protective efficacy of these NAbs, the five most potent neutralizers were evaluated in respective mouse aerosol challenge models. All five NAbs demonstrated various levels of protection when administered at doses of 2.5 mg/kg or 10 mg/kg 24 h before the respective virus exposure via the aerosol route. Of these, anti-EEEV NAb G1-4-C3 and anti-WEEV NAb 8C2 provided 100% protection at both doses and all surviving mice were free of clinical signs throughout the study. Additionally, no virus was detected in the brain 14 days post virus exposure. Taken together, efficacious NAbs were developed that demonstrate the potential for the development of cross-strain antibody-based MCMs against EEEV and WEEV infections.
Highlights
The three encephalitic alphaviruses, namely, the Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV), belong to the Alphavirus genus of the family Togaviridae [1]
The recombinant adenoviral construct pAd-EEEV PE6 was linearized with Pac I and transfected into HEK 293 cells cultured in Dulbecco’s minimal essential medium (DMEM) with 5% fetal bovine serum (FBS) for amplification and the amplified adenovirus was purified via chromatography
The Neutralizing antibodies (NAbs) titer was identified as the highest dilution that resulted in 50% inhibition of cytopathic effects (CPE)
Summary
The three encephalitic alphaviruses, namely, the Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV), belong to the Alphavirus genus of the family Togaviridae [1]. A subgenomic positive-stranded RNA (the 26S RNA) is identical to the 3 one-third of the genome and serves as the translational template for the structural proteins, capsid (C), E3, E2, 6K, and E1 [3] Three of these proteins, C, E1, and E2 are found on all mature encephalitic alphavirus virions, while the E3 protein has only been positively identified in VEEV capsids to date [4]. The South American strains are classified as a separate species, namely, the Madariaga virus, which has an approximately 23% difference in nucleotide sequence from EEEV [7] In nature, these viruses primarily circulate through animal populations and infect humans via bites from mosquito carriers that have fed on infected animals. In an in vivo pre-exposure prophylaxis setting, all five NAbs that were tested demonstrated a level of protection against EEEV or WEEV aerosol infection
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