Gene reassortment has proved useful in improving yields of influenza A antigens of egg-based inactivated vaccines, but similar approaches have been difficult with influenza B antigens. Current regulations for influenza vaccine seed viruses limit the number of egg passages and as a result resultant yields from influenza B vaccine seed viruses are frequently inconsistent. Therefore, reliable approaches to enhance yields of influenza B vaccine seed viruses are required for efficient vaccine manufacture. In the present study three stable cold-adapted (ca) mutants, caF, caM and caB derived from seasonal epidemic strains, B/Florida/4/2006, B/Malaysia/2506/2004 and B/Brisbane/60/2008 were prepared, which produced high hemagglutinin antigen yields and also increased viral yields of reassortants possessing the desired 6:2 gene constellation. The results demonstrate that consistent improvements in yields of influenza B viruses can be obtained by cold adaptation following extended passage. Taken together, the three ca viruses were shown to have potential as donor viruses for the preparation of high-yielding influenza B vaccine viruses by reassortment.
Highly pathogenic avian influenza (HPAI) H5N1 is an ongoing global health concern due to its severe sporadic outbreaks in Asia, Africa and Europe, which poses a potential pandemic threat. The development of safe and cost-effective vaccine candidates for HPAI is considered the best strategy for managing the disease and addressing the pandemic preparedness. The most potential vaccine candidate is the antigenic determinant of influenza A virus, hemagglutinin (HA). The present research was aimed at developing optimized expression in Nicotiana benthamiana and protein purification process for HA from the Malaysian isolate of H5N1 as a vaccine antigen for HPAI H5N1. Expression of HA from the Malaysian isolate of HPAI in N. benthamiana was confirmed, and more soluble protein was expressed as truncated HA, the HA1 domain over the entire ectodomain of HA. Two different purification processes were evaluated for efficiency in terms of purity and yield. Due to the reduced yield, protein degradation and length of the 3-column purification process, the 2-column method was chosen for target purification. Purified HA1 was found immunogenic in mice inducing H5 HA-specific IgG and a hemagglutination inhibition antibody. This paper offers an alternative production system of a vaccine candidate against a locally circulating HPAI, which has a regional significance.