Yeast expressed recombinant Hemagglutinin protein of novel H1N1 elicits neutralising antibodies in rabbits and mice

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.

Figure 1

Vector map of the recombinant construct and Schematic diagram of genetic recombination in Pichia. Panel 1A: Plasmid map of recombinant yeast transfer vector pPIC9KH1N1HA. Panel 1B: Schematic diagram showing the genetic recombination event that result in the formation of Pichia transformant with multiple-copy integrants(The gene of interest (H1N1HA) is positioned between Eco RI and Not I sites under the control of AOX1 promoter, the secretory signal (SS), transcription termination (TT) signal sequences are on 5' and 3'end of the H1N1HA gene respectively. HIS 4 locus is carrying the Sal I recognition sequence and the integration of the transgene within the Pichia genome will be at His 4 locus).

Figure 2

PAGE analysis of the culture supernatants under denaturing and native conditions demonstrating the secreted expression of recombinant HA protein as HA monomers and HA trimers respectively. Panel 2A: Methanol induced culture supernatants under denaturing conditionLane M: Pre-stained Protein marker (Fermentas, USA, #SM 0671) Lane 1: Cell culture supernatant from Pichia multiple copy integrant Clone A Lane 2: Cell culture supernatant from Pichia multiple copy integrant Clone B Lane 3: Cell culture supernatant from Negative clone Panel 2B: Methanol induced culture supernatants under native conditionLane M: Pre-stained Protein marker (Fermentas, USA, #SM 1811) Lane 1: Cell culture supernatant from Pichia multiple copy integrant Clone B Lane 2: Cell culture supernatant from Pichia multiple copy integrant Clone A Lane 3: Cell culture supernatant from Negative clone.

Figure 3

FPLC purification of yeast derived HA protein via size exclusion chromatography.

Figure 4

Western Blot analysis of the culture using H1N1 HA specific antibodies confirming the secreted expression of recombinant HA protein supernatants under denaturing and native conditions. Panel 4A: Under denaturing condition: Lane M: Pre-stained Protein marker (Fermentas, USA, #SM 0671) Lane 1: Cell culture supernatant from Pichia multiple copy integrant Clone A Lane 2: Cell culture supernatant from Pichia multiple copy integrant Clone B Lane 3: Cell culture supernatant from negative clone Panel 4B: Under native condition: Lane M: Pre-stained Protein marker (Fermentas, USA, #SM 0671) Lane 1: Cell culture supernatant from Pichia multiple copy integrant Clone A Lane 2: Cell culture supernatant from Pichia multiple copy integrant Clone B Lane 3: Cell culture supernatant from negative clone.

Figure 5

Native PAGE analysis of the yeast derived H1N1HA digested with trypsin Lane M: Pre-stained Protein marker (Fermentas, USA, #SM 0671) Lane 1: Recombinant H1N1HA after digestion with trypsin Lane 2: Negative control (Recombinant H1N1HA mock digested).

Figure 6

Hemagglutination (HA) activity of H1N1 virus and Hemagglutination Inhibition (HI) activity of HA immunized sera using chicken RBCs.Panel 6A: Hemagglutination (HA) activity of H1N1 virus. Lane A: Two fold serial diluted MDCK derived H1N1 virus mixed with 0.5% chicken RBCs. Lane B: Negative control (PBS mixed with 0.5% chicken RBCs). Panel 6B: Hemagglutination Inhibition (HI) activity of HA immunized sera using chicken RBCs. Lane A: Negative control (None immunized mice serum mixed with 4HA units of H1N1) Lane B: Two fold serially diluted HA immunized representative mice serum mixed with 4HA units of H1N1 Lane C: Two fold serially diluted HA immunized representative rabbit serum mixed with 4HA units of H1N1.

Figure 7

Plaque Reduction Neutralization Test (PRNT) of HA immunized sera demonstrating virus neutralization activity against H1N1 virus. Panel 7A and 7B: Representative immunized rabbit serum showing virus neutralisation activity against H1N1 virus (panel A) compared to the non immunised control rabbit serum (panel B) Panel 7C:Graph depicting the PRNT titres (50% plaque reduction) of immune mice/rabbit sera samples in comparison to non immune sera samples.