Large-scale microcarrier culture of HEK293T cells and Vero cells in single-use bioreactors.

Gene therapy and viral vaccine are becoming attractive therapeutic options for the treatment of different malignant diseases. Viral vector productions are often using static culture vessels and small volume stainless steel bioreactors (SSB). However, the yield of each vessel can be relatively low and multiple vessels often need to be operated simultaneously. This significantly increases labor intensity, production costs, contamination risks, and limits its ability to be scaled up, thus, creating challenges to meet the quantities required once the gene therapy or viral vaccine medicine goes into clinical phases or to market. Single-use bioreactor combining with microcarrier provides a good option for viral vector and vaccine production. The goal of the present studies was to develop the microcarrier bead-to-bead expansion and transfer process for HEK293T cells and Vero cells and scale-up the cultures to 50-200 l single-use bioreactors. Following microcarrier bead-to-bead transfer, the peak cell concentration of HEK293T cells reached 1.5 × 106 cells/ml in XDR-50 bioreactor, whereas Vero cells reached 3.1 × 106 cells/ml and 3.3 × 106 cells/ml in XDR-50 bioreactor and XDR-200 bioreactor, respectively. The average growth rates reached 0.61-0.68/day. The successful microcarrier-based scaleup of these two cell lines in single-use bioreactors demonstrates potential large-scale production capabilities of viral vaccine and vector for current and future vaccines and gene therapy.

Influence of a forest edge on acoustical propagation: experimental results.

Acoustic propagation through a forest edge can produce complicated pressure time histories because of scattering from the trees and changes in the microclimate and ground parameters of the two regions. To better understand these effects, a field experiment was conducted to measure low-frequency acoustic pulses propagating in an open field, a forest, and passing through a forest edge in both directions. Waveforms measured in the open field were simple impulses with very low scattering, whereas waveforms at the edge and within the forest had stronger reverberations after the direct arrival. The direct wave pulse shapes increased in duration in accordance with the path length in the forest, which had an effective flow resistivity 12 to 13 that of the grassy open field. The measurements exhibit different rates of attenuation in the two regions, with relatively lower attenuation in the open field than higher rates in the forest. Decay of SEL transmitted into the forest was 4 dB more per tenfold distance than for outbound transmission. Stronger attenuation in the 1-2 kHz range occurs when propagating into the forest. While the measured meteorological profiles revealed three distinct microclimates, meteorological effects are not sufficient to explain the apparent non-reciprocal propagation.

Inactivated yellow fever 17D vaccine: development and nonclinical safety, immunogenicity and protective activity.

In the last 10 years new concerns have arisen about safety of the live, attenuated yellow fever (YF) 17D vaccine, in particular viscerotropic adverse events, which have a case-fatality rate of 64%. A non-replicating cell culture-based vaccine would not cause these adverse events, and potentially could be used in persons with precautions or contraindications to use of the live vaccine, including age <9 months and >60 years, egg allergy, immune suppression, and pregnancy. We developed a whole virion vaccine from the 17D strain inactivated with beta-propiolactone, and adsorbed to aluminum hydroxide. The inactivated vaccine was highly immunogenic in mice, hamsters, and cynomolgus macaques. After a single dose in hamsters and macaques, neutralizing antibody titers were similar to those elicited by the live 17D vaccine (YF-VAX, Sanofi Pasteur). After two doses of inactivated vaccine, neutralizing antibody titers in hamsters were significantly higher than after a single dose of YF-VAX [geometric mean titer (GMT) 20,480 vs. 1940, respectively (P<0.001, ANOVA)]. Hamsters given a single dose or two doses of inactivated vaccine or a single dose of YF-VAX were fully protected against hepatitis, viremia, weight loss and death after challenge with YF virus (Jimenez strain). A clinical trial of the inactivated vaccine (XRX-001) has been initiated.