Production of Inactivated Influenza H5N1 Vaccines from MDCK Cells in Serum-Free Medium

BACKGROUND: Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK) cells grown in a serum-free (SF) medium microcarrier cell culture system. PRINCIPAL FINDING: The current study has evaluated the performance of cell adaptation switched from serum-containing (SC) medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3 × 10(6) cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA) units/50 µL and 7.1 ± 0.3 × 10(8) pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera. CONCLUSIONS: The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production

A Sabin 1 poliovirus-based vaccine vector transfects Vero cells with high efficiency

Over the past 40 years, live oral poliovirus (PV) vaccines have contributed to the eradication of wild PV in most countries. These live vaccine strains have a high safety record and can stimulate both cellular and humoral immune responses. As both of these factors are critical characteristics of a good vaccine, we aimed to modify the oral PV vaccines to create a powerful vaccine vector for extraneous antigen expression. In this study, we amplified three separate fragments from the Sabin 1 virus genome by RT-PCR and cloned them into the pGEM-TEasy vector. A cassette containing engineered protease cleavage sites and a polylinker was introduced into one of these fragments (f1) in front of the translation start site. This construction facilitated the insertion of foreign genes into the vector and the subsequent release of their co-translated antigens after digestion by endogenous protease. We also placed a ribozyme (Rz) sequence between the T7 promoter and viral genomic DNA so that in vitro transcription and Rz cleavage recreated the authentic 5′ end of the PV genome RNA. Poly(A)40 tails were added to the 3′ end of the genome to stabilize the transcribed RNA. The three PV genome fragments and their derivatives were cloned into various types of vectors that were transfected into Vero cells. Virus rescue experiments demonstrated that both the Rz and poly(A)40 elements were required for high transfection efficiency of the vector-derived RNAs