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 Compound Library (Th)-1and 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] and [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 [22]. 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], [23] and [24] and was also evaluated. The current study was designed to evaluate the immunogenicity

and efficacy of fV3526 alone and in Adriamycin order 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. Live V3526 bulk drug substance (BDS) was produced by Sigma Aldrich Fine Chemicals (SAFC Pharma), Carlsbad, CA. The titer of this material was 2.9 × 107 pfu/mL. The challenge virus, VEEV TrD, was produced by Commonwealth

Biotechnologies Incorporated, Richmond, VA. For the negative control, process control material (PCM) was used, which consists of supernatant from mock infected cultures. C84 was used as a comparator and was manufactured at The Salk Institute, Government Service Division, Swiftwater, PA. Virus inactivation studies were carried out at SAFC Pharma. V3526 virus was treated with 0.1% v/v formalin (USP grade, EMD Chemicals) most in a calibrated shaking water bath set at 37 °C for 24 h. Residual formalin was reduced to less than 1 × 10−8% using a tangential flow filtration system (GE Healthcare) with a 500 kDa molecular weight cutoff membrane. The multi-system approach for evaluation of virus inactivation was developed to meet the expected regulatory requirements for documentation supporting the safety of new vaccines [25]. Inactivated virus preparations were tested for residual infectivity using a standard plaque assay previously described [12] and serial passage on baby hamster kidney (BHK)-21 cells [26].