Venezuelan equine encephalitis virus (VEEV) is a highly infectious agent that can cause severe and frequently fatal encephalitis in equines and is naturally transmitted to humans by mosquitoes . VEEV is considered a potential biological weapon amenable to use in warfare and terrorism due to its high infectivity via aerosol exposure [2, 3]. To address the aerosol threat of VEEV on public health, two vaccines were developed by the U.S. government during the 1960s and 1970s: TC-83, a cell-culture attenuated vaccine developed from the Trinidad donkey (VEEV TrD) strain of subtype IA/B VEEV  and a formalin-inactivated vaccine derived from TC-83, designated C-84 . For several decades the TC-83 and C-84 vaccines have been administered by the U.S. Army Special Immunizations Program to laboratory workers and animal health field workers at risk for exposure to VEEV. While TC-83 induces long-lasting immunity against closely related VEEV subtypes , major limitations of the vaccine exist including: only an approximately 80% response rate as assessed by plaque reduction neutralization test (PRNT) ; a 25% incidence of adverse reactions ; and reversion to virulence after mouse brain passages . In addition, as a live virus vaccine, TC-83 cannot be used as a booster for subjects with waning antibody titers . C-84 is currently used to boost antibody titers following vaccination with TC-83 and to immunize TC-83 non-responders. C-84 also has limitations in that protection is of short duration thus requires multiple boosters.
As a result of the limitations of TC-83 and C-84, a new live-attenuated VEEV vaccine candidate was developed, V3526, that contains a deletion of the PE2 cleavage signal (furin cleavage site) combined with a second-site suppressor mutation in the E1 glycoprotein . These attenuating mutations were shown to eliminate the characteristic VEEV disease in animal models inoculated with the parent clone, V3000 [11–14]. In spite of the attenuation, V3526 maintains the ability to elicit a protective immune responses in animals [11, 12, 14]. Due to the success of V3526 in animal models, a Phase 1 clinical trial of V3526 was conducted to evaluate the safety and immunogenicity of this new vaccine candidate. V3526 induced a robust immune response in all vaccines but high frequency of fever and a flu-like syndrome were reported  which led to the cessation of further development of V3526 as a live-attenuated vaccine. Next generation vaccine development efforts for VEEV were then redirected toward the development of an inactivated V3526 vaccine. There are several features of V3526 that make it a good candidate for inactivation. First, the molecular basis of attenuation of V3526 is known. Second, extensive nonclinical studies combined with the recent Phase 1 clinical trial demonstrate a higher responder rate and more robust immune response following vaccination with V3526 than observed with TC-83. The superior immunogenicity of V3526 compared to TC-83 suggests a higher rate of success may be achieved using inactivated V3526 compared to C-84, the inactivated version of TC-83. In fact, C-84 has undergone extensive testing in animal models and fails to protect hamsters against aerosol exposure to virulent VEEV suggesting C-84 does not induce strong mucosal immunity . Although studies could have been conducted to optimize C-84 as a protective immunogen by modifying dosage, schedule, route and use of adjuvant, this was not pursued as C-84 is no longer being manufactured and optimization of C-84 as a vaccine would not further our development of a next generation VEE vaccine.
In addition to the well-documented safety and immunological profile of live V3526 in animal and humans, V3526 has advantages over TC-83, C-84 and other attenuated VEE viruses with respect to the manufacturing process. The manufacturing process for V3526 has been developed to meet Good Manufacturing Practice standards and meets the requirements established by the US Food and Drug Administration for biologicals seeking Investigational New Drug status. In contrast, the production of TC-83 and C-84 are based on older technology not compatible with current FDA standards and would require re-derivation of the TC-83 stock, followed by development of a GMP production process for TC-83 in a certifiable cell line and further development of the entire TC-83/C-84 manufacturing process.
Several methods have been used to inactivate infectious agents, including gamma irradiation . In the early 1970s, gamma irradiation was used to inactivate wild-type VEEV [18, 19] with the intent of developing an inactivated vaccine. In these studies, gamma-irradiated VEEV preparations were highly immunogenic and afforded protection against lethal challenge; however, further evaluation was not pursued. To further evaluate gamma-irradiation as an inactivation method for a VEEV vaccine, we optimized the gamma-irradiation process for V3526 with the intent of completely inactivating the virus while preserving immunologically important epitopes .
The objective of this study was to evaluate gamma-irradiated V3526 for immunogenicity and efficacy when administered alone and when formulated with adjuvants in BALB/c mice following SC or IM administration. The protective efficacy of the immunological responses was evaluated by challenge with VEEV TrD via the SC and aerosol routes.