Semicontinuous Bioreactor Production of Recombinant Butyrylcholinesterase in Transgenic Rice Cell Suspension Cultures.

An active and tetrameric form of recombinant butyrylcholinesterase (BChE), a large and complex human enzyme, was produced via semicontinuous operation in a transgenic rice cell suspension culture. After transformation of rice callus and screening of transformants, the cultures were scaled up from culture flask to a lab scale bioreactor. The bioreactor was operated through two phases each of growth and expression. The cells were able to produce BChE during both expression phases, with a maximum yield of 1.6 mg BChE/L of culture during the second expression phase. Cells successfully regrew during a 5-day growth phase. A combination of activity assays and Western blot analysis indicated production of an active and fully assembled tetramer of BChE.

Neurovirulence evaluation of Venezuelan equine encephalitis (VEE) vaccine candidate V3526 in nonhuman primates.

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 (i.t./i.s. ) or subcutaneous (s.c.) 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 degrees C) were noted in several of the TrD and vaccine inoculated animals on Day 6 following inoculation and mean temperatures for the V3526 i.t./i.s. 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 i.t./i.s. and three of 12 TC-83 inoculated animals, however, these signs resolved within 3 weeks for all V3526 i.t./i.s. 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 i.t./i.s. 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 i.t./i.s. 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.

Venezuelan equine encephalitis virus vaccine candidate (V3526) safety, immunogenicity and efficacy in horses.

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.

Environmental hazard assessment of Venezuelan equine encephalitis virus vaccine candidate strain V3526.

A hazard assessment of Venezuelan equine encephalitis (VEE) virus sub-types and vaccine candidates was performed according to standard risk assessment procedures. Data from published literature demonstrates a considerable degree of safety of V3526 when compared to TC-83 vaccine, the protective measure that has been used to protect laboratory workers for over four decades. V3526 is a new recombinant vaccine candidate that is a vastly different product with a diminished hazard to public health and the general environment. A weight-of-evidence (WOE)-based scheme was employed to assign weights for relevance, quality, and adequacy of evidence in published literature on medical pathology, epidemiology, pre-clinical investigational studies, and environmental studies. The results of this assessment indicated that V3526 has a low adverse impact on public health and the general environment. Although there are currently no human infectivity or pathogenicity data for V3526, existing evidence from published experimental animal studies reveals a diminished hazard for environmental transmission and distribution. Recently, the US Centers for Disease Control and Prevention (CDC) excluded V3526 from select agent requirements set forth under the Health and Human Services (HHS) regulations in Title 42 C.F.R. Part 73 and the US Department Agriculture (USDA) regulations set forth in Title 7 C.F.R. Part 331 and Title 9 C.F.R. Part 121. This paper summarizes the background, rationale, and hazard analysis used for assessing the environmental hazard of the VEE vaccine candidate strain V3526.