The Venezuelan equine encephalitis virus (VEEV) complex is composed of serologically related, mosquito-borne viruses belonging to the genus Alphavirus in the family Togaviridae. While generally associated with severe and fatal encephalitis in equines, humans are susceptible to VEEV with disease symptoms including fever, malaise, myalgia and severe headache . Encephalitis occurs occasionally in adults, but more frequently in children . Similar disease manifestations in laboratory workers accidentally exposed to VEEV confirm the highly infectious nature of the virus via the aerosol route . In addition to natural or accidental exposure to the virus, the U.S. Department of Defense identified VEEV as a potential biological warfare agent since VEEV can be produced in unsophisticated culture systems, can be stored for extended periods of time and is highly infectious, requiring relatively few organisms to infect humans .
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 IAB VEEV  and a formalin-inactivated vaccine derived from TC-83, designated C84 . For several decades the TC-83 and C84 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 and thus requires multiple boosters.
The limitations of the TC-83 and C84 vaccines led to the development of an investigational live-attenuated VEEV vaccine, V3526, developed from a full-length cDNA clone of VEEV IAB Trinidad donkey strain (VEEV TrD) using site-directed mutagenesis. V3526 was attenuated by deleting a furin cleavage site from the PE2 glycoprotein and incorporating a single amino acid mutation in the E1 glycoprotein . The V3526 vaccine is effective in protecting rodents, horses and nonhuman primates (NHP) against subcutaneous or aerosol challenge with fully virulent VEEV TrD (Subtype IAB), as well as other VEEV subtypes (IC, IE and IIIA) [12–15].
Based on the success of V3526 in nonclinical studies, a Phase 1 clinical trial was conducted to evaluate the safety and immunogenicity of V3526 in human subjects. The clinical findings from the Phase 1 trial showed robust immune responses in virtually all vaccine recipients, even those receiving very low dosages (~20 plaque forming units). However, a significant number of the vaccine recipients demonstrated mild to moderate adverse events (AE) including headache, fever, malaise and sore throat. Several of the vaccine recipients experienced fevers classified as grade 3, based on the current adverse event grading scale. Viral shedding that occurred in a subset of the recipients appeared to coincide with sore throat and/or fevers. Based on these findings, clinical testing of V3526 was discontinued.
Since a high frequency of adverse reactions has been associated with live-attenuated VEEV vaccines [9, 10, 16], licensure of a live-attenuated vaccine will likely be faced with significant regulatory obstacles relating to safety. Our strategy to develop a VEEV vaccine was revised to focus on a non-infectious virus vaccine. The use of C84 was not considered for further development because the Department of Defense, in 1996, deemed this vaccine in need of improvement. C84 was last manufactured between 1980 –1981 and the limited supply of C84 vaccine has been in storage for over 29 years and the recent potency and stability of this vaccine is unknown. Manufacture of new lots of C84 is unlikely to occur because this would require re-derivation of the TC-83 stock, followed by GMP production of the TC-83 in a certifiable cell line and further development of the entire TC-83/C84 manufacturing process. In addition, a technical review of the C84 manufacturing records failed to identify a credible source document describing the actual manufacturing process and testing scheme therefore this would also need to be devised.
Having a large inventory of GMP manufactured V3526 originally reserved for the clinical testing, the decision was made to inactivate V3526 for the production of VEEV vaccine candidates that would ultimately replace C84 and be used as a primary vaccine to protect personnel at risk to accidental or intentional VEEV exposure. Studies were initiated using formalin to inactivate V3526 with the intent of producing a vaccine with a significantly reduced adverse reaction profile compared to V3526, but one that retains potential as a protective immunogen against VEEV infection and performs similarly or better than C84.
Formalin inactivation of virus has been successfully used to develop safe and efficacious human and veterinary vaccines since 1955  and most recently, an inactivated vaccine for Japanese encephalitis virus . The use of formalin inactivation for virus vaccine development is attractive from a safety perspective in that the virus cannot revert to virulence, since there is no virus replication during immunization. The use of formalin to inactivate viruses is also attractive from a manufacturing perspective as the inactivation process is relatively simple to develop. In the development of a formalin inactivated VEEV vaccine candidate, we recently developed a method to inactivate V3526 using formalin and established a system of prioritized assays to evaluate residual infectivity and preservation of immunologically essential epitopes.
In that inactivated viral vaccines are non-replicating, formulation with adjuvants may be necessary to augment a protective immune response. To address this concern, we evaluated formalin-inactivated V3526 (fV3526) formulated with each of 4 adjuvants, Viprovex®, CpG oligodeoxynucleotides (ODN) 2395, Alhydrogel™ or CpG+Alhydrogel™. Viprovex® is a synthetically manufactured peptide analogue of Substance P that stimulates antigen presenting cells to utilize both the MHC Class I and II molecules and pathways, resulting in both T-helper (Th)-1 and Th2-mediated immune responses. CpG ODN 2395, is a type C CpG ODN that strongly activates B cells and induces high IFN-α production from plasmacytoid dendritic cells [20, 21]. CpG ODN2395 has demonstrated reactivity to human and murine TOLL-like receptor 9 (TLR9) ligand. Alhydrogel™ commonly known as aluminum hydroxide, binds antigen and incorporates into an insoluble, gel-like precipitate and is believed to continually stimulate the immune system by functioning as an antigen depot . The use of CpG and Alhydrogel™ as a combination adjuvant is reported to enhance immune responses significantly greater than the use of either adjuvant alone [22–24] and was also evaluated.
The current study was designed to evaluate the immunogenicity and efficacy of fV3526 alone and in combination with adjuvants in BALB/c mice following subcutaneous (SC) or intramuscular (IM) administration. The protective efficacy of the immunological response was evaluated by challenge with VEEV TrD via the SC and aerosol routes. As the identification of a new VEEV vaccine candidate was dependent on it being as good as or better than the existing inactivated VEEV vaccine, C84 was included for comparison.