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Assessment of neurovirulence is a standard test for vaccines derived from virulent neurotropic viruses. This study evaluated the potential neurovirulence of V3526, a live attenuated vaccine derived from a full-length infectious clone of Venezuelan equine encephalitis virus (VEEV) Trinidad donkey strain (TrD), a comparator VEEV vaccine (TC-83), TrD, and process control material (PCM) in juvenile rhesus macaques. Following intrathalamic/intraspinal (IT/IS) or subcutaneous (SC) inoculations, animals were observed for periods of 18, 91 or 181 days for paresis, paralysis, neurological disorders and other signs of clinical illness. Blood was collected for measurement of viremia, VEEV neutralizing antibodies, hematologic parameters, and liver enzymes. Gross necropsies and histopathological examinations were conducted with emphasis on detecting lesions in the brain and spinal cord. Elevated temperatures (1–2 °C) were noted in several of the TrD and vaccine inoculated animals on Day 6 following inoculation and mean temperatures for the V3526 IT/IS and TC-83 groups were higher than PCM group throughout the study Day 18. No significant differences were seen for weight or clinical chemistry results between vaccine and PCM inoculated groups. Clinically significant signs (Grades 3 or 4) were noted in three of 21 V3526 IT/IS and three of 12 TC-83 inoculated animals, however, these signs resolved within 3 weeks for all V3526 IT/IS and for two of three TC-83 inoculated animals. At Day 18 extensive lesions indicative of a viral infection were seen in brain sections of all four TrD inoculated animals and one of seven V3526 IT/IS inoculated animals. Only scattered lesions, characterized by foci of gliosis and vessels with perivascular inflammation, were found in the sections from four TC-83 and six V3526 IT/IS inoculated animals. The minimal histological changes observed at Day 18 resolved to baseline levels by Day 181 comparable to the PCM group. V3526 was immunogenic and essentially nonneurovirulent when administered via the clinically relevant subcutaneous route.

A new vaccine, V3526, is a live-attenuated virus derived by site-directed mutagenesis from a virulent clone of the Venezuelan equine encephalitis virus (VEEV) IA/B Trinidad donkey (TrD) strain, intended for human use in protection against Venezuelan equine encephalitis (VEE). Two studies were conducted in horses to evaluate the safety, immunogenicity, ability to boost and protective efficacy of V3526 against challenges of TrD and VEEV IE 64A99. Horses were vaccinated subcutaneously (SC) with 10 7, 10 5, 10 3 or 10 2 plaque-forming units (pfu) of V3526. Control horses were sham immunized. In the first study, challenge viruses (TrD or 64A99) were administered SC 28 days post-vaccination (PV). No viremia and only mild fluctuation in white blood cell counts were observed PV. None of the V3526 vaccinated horses showed clinical signs of disease or pathology of VEE post-challenge (PC). In contrast, control horses challenged SC with 10 4 pfu TrD became viremic and showed classical signs of VEE beginning on Day 3 PC, including elevated body temperature, anorexia, leukopenia and malaise. Moderate to severe encephalitis was found in three of five control horses challenged with TrD. Control horses challenged with 64A99 failed to develop detectable viremia, but did exhibit a brief febrile episode at 1–3 days PC. None of the 10 immunized horses challenged with 64A99 became pyrexic. Twenty four of 25 horses immunized with V3526 in the first study developed serum neutralizing antibody to TrD and 64A99 within 14 days PV. Vaccinations with V3526, at doses as low as 10 2 pfu, were safe and efficacious in protecting horses against a virulent TrD virus challenge. The second study supported that repeat dosing resulted in an increase in serum neutralizing antibody to TrD.

A cell culture process adaptable to produce smallpox vaccine at large scale has been developed. To achieve this, Design of Experiments (DOE) was applied to identify and optimize critical cell culture process parameters for MRC-5 cell growth and recovery during cell expansion. For cell growth, a 25−1 partial factorial (two level, five factor, 16 conditions) study was designed to evaluate the effects of basal media, seeding density, culture volume, feeding frequency and serum concentration on population doubling level (PDL) after 6–7 days in adherent T-flask cultures. Results indicated that lowering the cell density to 1×104 cells/cm2, increasing the culture volume to 0.5 ml/cm2 and increasing serum concentration to 20% significantly improved cell expansion. These findings correlated with PDLs above 2.0 and cell densities above 1×105 cells/cm2 at the end of the study period. For cell recovery at passaging, a similar DOE was used to evaluate the effect of trypsin concentration, solution temperature, duration of treatment, incubation temperature and duration of standing time between quenching and reseeding. By increasing the trypsin treatment duration to 60 min and lowering the standing time between quenching and reseeding to within 1 h, the recovery of the MRC-5 cells was greatly improved. By using these newly defined conditions, a two-fold improvement in cell expansion was consistently achieved in both roller bottles and 10 layer Nunc® Cell Factories (Cell Factories). Application of these new conditions for current Good Manufacturing Practices (cGMP) production of MRC-5 cell banks and clinical material demonstrated predictably high cell expansion as well as significantly higher production of vaccinia virus, thus providing the basis for manufacturing vaccinia virus at large scale. These findings demonstrate the need for cell culture optimization and the effectiveness of DOE to rapidly define processes suitable for cGMP manufacturing of a smallpox vaccine or other viral vaccine products.