Abstract
Currently available vaccines for the pandemic Influenza A (H1N1) 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA) based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI) activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat.
Highlights
Influenza viruses belonging to the Orthomyxoviridae family are enveloped viruses with segmented negative sense RNA genome surrounded by a helical symmetry shell
In light of the above facts, we have investigated the feasibility of using Pichia pastoris as an expression host for recombinant production of H1N1 HA protein and elicitation of neutralising antibodies against the same protein in BALB/c mice and rabbits
Generation of recombinant Pichia pastoris with multi copy integrants of H1N1 HA gene Full length H1N1 HA synthetic gene of A/California/04/ 2009 was inserted at EcoRI -Not I sites into pPICK9K yeast transfer vector under AOX1 promoter in fusion with S.cervecea alpha secretory signal at N-terminus
Summary
Influenza viruses belonging to the Orthomyxoviridae family are enveloped viruses with segmented negative sense RNA genome surrounded by a helical symmetry shell. Since the protein product may be typically obtained as insoluble, mis-folded inclusion bodies, subsequent solubilization and re-folding steps are required [9,10]. This incorrect folding can be a result of inadequate intracellular chaperone concentrations or the reducing environment of the cytoplasm [11]. E. coli is not generally suitable for use in expression studies with proteins that contain a high level of disulphide connectivity or proteins that require other types of post-translational modifications such as glycosylation [12,13]. E.coli expressed proteins tend to retain their amino-terminal methionine, which may affect protein stability as reported earlier [14,15]
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