Error-prone pcr-based mutagenesis strategy for rapidly generating high-yield influenza vaccine candidates

Abstract

Vaccination is the primary strategy for the prevention and control of influenza outbreaks. However, the manufacture of influenza vaccine requires a high-yield seed strain, and the conventional methods for generating such strains are time consuming. In this study, we developed a novel method to rapidly generate high-yield candidate vaccine strains by integrating error-prone PCR, site-directed mutagenesis strategies, and reverse genetics. We used this method to generate seed strains for the influenza A(H1N1)pdm09 virus and produced six high-yield candidate strains. We used a mouse model to assess the efficacy of two of the six candidate strains as a vaccine seed virus: both strains provided complete protection in mice against lethal challenge, thus validating our method. Results confirmed that the efficacy of these candidate vaccine seed strains was not affected by the yield-optimization procedure.

Keywords: Error-prone PCR; High-yield strain; Influenza A virus; Vaccine; Vaccine production.

Figure 1

Overall strategy for generating a hemagglutinin mutant library. Based on this method, chicken embryonic egg–propagated seed viruses with high yield can be selected for influenza vaccine candidate strains. The 130loop + 190helix or 190helix + 220loop in hemagglutinin gene of influenza A/California/04/09 (H1N1) virus are amplified by error-prone PCR (epPCR) by using a GeneMorph II Random Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) (step 1). epPCR products are then used as primers in the site-directed mutagenesis (step 2). After DpnI digestion at 37°C for 1 h (step 3), the PCR product is transformed into XL1-Blue Supercompetent Cells (Agilent Technologies, Santa Clara, CA), and the cells are inoculated onto LB (Luria Bertani) plates or into LB medium (step 4). The individual plasmid or plasmid library is then extracted (step 5) and used with 6 internal genes from A/Puerto Rico/8/1934(H1N1) and the neuraminidase gene from CA/04 to rescue viruses (step 6). Rescued viruses are inoculated into 10-day-old chicken embryonic eggs to generate high-yield vaccine seed. The steps are indicated by circled numbers in the figure.

Figure 2

Characterization of the high-yield influenza A(H1N1)pdm09 vaccine candidates generated by using the error-prone PCR–based mutagenesis strategy. Viruses were inoculated into Madin-Darby canine kidney cells at a multiplicity of infection of 0.001. (A) Growth curves for wild-type (WT) and mutant viruses (nos. 22, 58, 79, 81, 88, and 144) measured by 50% tissue culture infectious dose (TCID₅₀; values shown below columns) at various hours after infection (hpi). (B) Western blot showing the nucleoprotein expression level for the WT and mutant viruses (viruses shown above columns).

Figure 3

Quantification of wild-type (WT) and mutant viruses propagated in eggs as determined by using a hemagglutinin gene–specific quantitative reverse transcription PCR method. Results are expressed as the median (horizontal bars) RNA copy number (1 µl cDNA) ± SD (vertical bars).

Figure 4

Total protein quantification of wild-type (WT) and mutant viruses that purified from the allantoic fluids of 11-day-old embryonated chicken eggs. Results are expressed as the median (horizontal bars) protein concentration ±SD (vertical bars).

Figure 5

Protective effect of high-yield vaccine candidates in mice challenged with mouse-adapted influenza A/California/04/09 (H1N1) virus. Groups of control; mock-vaccinated; and mutant number 81–, 88–vaccinated mice were intranasally inoculated with 10 × the 50% lethal dose of virus, after which their body weights (A) and survival times (B) were monitored for 14 days. Results are shown as the mean ± SD in each group (A).

Figure 6

Histopathologic findings in hematoxylin and eosin–stained lung samples from vaccinated and mock-vaccinated mice challenged with mouse-adapted influenza A(H1N1)pdm09 virus. Four days after challenge, groups of mutant number 81–vaccinated (A), mutant number 88–vaccinated (B), and mock-vaccinated (C) mice were euthanized, and lungs were collected for histopathologic examination; non-vaccinated, non-challenged mice served as environmental controls (D). A, B, and D) Scale bar =100 µm; C) scale bar = 40 µm.

Figure 7

The three-dimensional structures of the hemagglutinin of the wild–type (WT) influenza A/California/04/09 (H1N1) virus and mutant viruses (nos. 22 and 81) in contact with human-like receptor analog 6SLN (panels A, C, and E) and avian-like receptor analog 3SLN (panels B, D, and F). The protein structures of hemagglutinin are shown in light grey; residue 133 is in orange. The side chains of interacting residues on the receptor-binding sites are labeled with residue names and locations. The single letter amino acid annotations were used together with H3 numbering for all binding residues. 6SLN is shown in cyan, and 3SLN is shown in magenta. Red dots indicate the oxygen atoms from all water molecules that are in contact with hemagglutinin side chains. The distances (in angstroms) between the water molecule and the nearest atom on both the protein and ligand sides are indicated by dashed lines. (A) WT hemagglutinin with lysine at position 133 in contact with 6SLN. (B) WT hemagglutinin with lysine at position 133 in contact with 3SLN. (C) K133N mutant hemagglutinin with asparagine at position 133 in contact with 6SLN. (D) K133N mutant hemagglutinin receptor-binding sites with asparagine at position 133 in contact with 3SLN. (E) Mution K133I at the hemagglutinin receptor-binding site with isoleucine at position 133 in contact with 6SLN. (F) Mutant K133I with isoleucine at position 133 in contact with 3SLN. Simulations on the hemagglutinin were performed by using the FoldX empirical force field (Schymkowitz et al., 2005), and the structure was visualized by using Chimera (Pettersen et al., 2004); PoseScore (Fan et al., 2011) was used to estimate the likeness of the WT and mutant protein–glycan binding avidities to that of the native virus.