Highly efficient production of an influenza H9N2 vaccine using MDCK suspension cells

The use of H9N2 subtype avian influenza vaccines is an effective approach for the control of the virus spread among the poultry and for the upgrading of vaccine manufacturing cell culture-based production platform could overcome the limitations of conventional egg-based platform and alternate it. The development of serum-free suspension cell culture could allow even higher virus productivity, where a suspension cell line with good performance and proper culture strategies are required. In this work, an adherent Mardin-Darby canine kidney (MDCK) cell line was adapted to suspension growth to cell concentration up to 12 × 106 cells/mL in a serum-free medium in batch cultures. Subsequently, the H9N2 influenza virus propagation in this MDCK cell line was evaluated with the optimization of infection conditions in terms of MOI and cell concentration for infection. Furthermore, various feed strategies were tested in the infection phase for improved virus titer and a maximum hemagglutinin titer of 13 log2 (HAU/50 μL) was obtained using the 1:2 medium dilution strategy. The evaluation of MDCK cell growth and H9N2 virus production in bioreactors with optimized operating conditions showed comparable cell performance and virus yield compared to shake flasks, with a high cell-specific virus yield above 13000 virions/cell. With the purified H9N2 virus harvested from the bioreactors, the MDCK cell-derived vaccine was able to induce high titers of neutralizing antibodies in chickens. Overall, the results demonstrate the promising application of the highly efficient MDCK cell-based production platform for the avian influenza vaccine manufacturing.

Serological and Nucleocapsid Gene Based Molecular Characterization of Canine Distemper Virus (CDV) Isolated from Dogs of Southern Gujarat, India

Background: The present study was undertaken to diagnose and characterize canine distemper virus (CDV) isolated from dogs of southern Gujarat, India. CDV is lethal disease of canines and felines. Total of 40 different samples were collected from 18 suspected stray dogs having different clinical signs which were processed for diagnosis and characterization of CDV.Methods: All samples were processed by employing different methods like, Immunochromatography based lateral flow test (LFA), IgG based indirect enzyme linked immunosorbent assay (i-ELISA), one step RT-PCR, nested one step RT-PCR and virus isolation in MDCK cell line. Restriction endonuclease (RE) analysis was used to characterize CDV Nucleocapsid (N) gene. Conclusion: Only 04 samples (02 nasal and 02 ocular swabs) of 02 dogs found positive for LFA, while 14 serum samples out of 17 samples of 18 dogs found positive for IgG antibody. As all dogs were unvaccinated, serum samples found positive in IgG based ELISA considered for confirmative positive for CDV infection. Whereas 13 samples of 10 dogs found positive for one step RT-PCR and nested one step RT-PCR. In RE digestion, characteristic two bands were found. All representative CDV positive samples of 10 dogs showed characteristic cytopathic effect in MDCK cell line. On age group wise percent positivity was found 71.42 % (05/07) in 0 - ≤6 months, while 77.77% (07/09) in 6- ≤12 months of age group, whereas, both samples were found positive in 12 months and above group. Overall 77.77% (14/18) dogs found positive for CDV infection. To the best of our knowledge, this is the first report on study of CDV infection in dogs from Gujarat state, India.

Highly efficient production of an influenza H9N2 vaccine using MDCK suspension cells

The use of H9N2 subtype avian influenza vaccines is an effective approach for the control of the virus spread among the poultry, and for the upgrading of vaccine manufacturing, cell culture-based production platform could overcome the limitations of conventional egg-based platform and alternate it. The development of serum-free suspension cell culture could allow even higher virus productivity, where a suspension cell line with good performance and proper culture strategies are required. In this work, an adherent Mardin–Darby canine kidney (MDCK) cell line was adapted to suspension growth to cell concentration up to 12 × 106 cells/mL in a serum-free medium in batch cultures. Subsequently, the H9N2 influenza virus propagation in this MDCK cell line was evaluated with the optimization of infection conditions in terms of MOI and cell concentration for infection. Furthermore, various feed strategies were tested in the infection phase for improved virus titer and a maximum hemagglutinin titer of 13 log2 (HAU/50 μL) was obtained using the 1:2 medium dilution strategy. The evaluation of MDCK cell growth and H9N2 virus production in bioreactors with optimized operating conditions showed comparable cell performance and virus yield compared to shake flasks, with a high cell-specific virus yield above 13,000 virions/cell. With the purified H9N2 virus harvested from the bioreactors, the MDCK cell-derived vaccine was able to induce high titers of neutralizing antibodies in chickens. Overall, the results demonstrate the promising application of the highly efficient MDCK cell-based production platform for the avian influenza vaccine manufacturing.

Highly Efficient Production of an Influenza H9N2 Vaccine Using MDCK Suspension Cells

H9N2 subtype avian influenza virus poses a constant threat to the poultry industry and the control of the disease leans upon the use of effective vaccines. As an alternative to the conventional chicken embryonated eggs, animal cell culture could overcome the limitations of egg supplies and upgrade the manufacturing of avian influenza vaccines for poultry. Development of serum-free suspension cell culture could allow even higher virus productivity, where a suspension cell line with good growth and production performance is required. In this work, an adherent MDCK cell line was adapted to suspension growth to cell concentration up to 12 × 106 cells/mL in a serum-free medium in batch cultures. Subsequently, the influenza virus propagation in this MDCK cell line was evaluated and was improved with the medium exchange at time of infection as well as optimization of infection conditions in terms of MOI and cell concentration for infection. Furthermore, various feed strategies were tested in the infection phase for improved virus titer and a maximum hemagglutinin titer of 13 log2 (HAU/50 μL) was obtained using the 1:2 medium dilution strategy. Evaluation of MDCK cell growth and H9N2 virus propagation in the bioreactors with optimized operating conditions showed comparable cell performance and virus yield compared to shake flasks, with a high cell-specific virus yield above 14000 virions/cell. With the purified H9N2 virus harvested from the bioreactor, the MDCK cell-derived vaccine was able to induce high titers of neutralizing antibodies in chickens. Overall, the results demonstrate the promising application of the highly efficient MDCK cell-based production platform for the avian influenza vaccine manufacturing.