A humanized mouse model identifies key amino acids for low immunogenicity of H7N9 vaccines

Influenza vaccines of H7N9 subtype are consistently less immunogenic in humans than vaccines developed for other subtypes. Although prior immunoinformatic analysis identified T-cell epitopes in H7 hemagglutinin (HA) which potentially enhance regulatory T cell response due to conservation with the human genome, the links between the T-cell epitopes and low immunogenicity of H7 HA remains unknown due to the lack of animal models reproducing the response observed in humans. Here, we utilized a humanized mouse model to recapitulate the low immunogenicity of H7 HA. Our analysis demonstrated that modification of a single H7 epitope by changing 3 amino acids so that it is homologous with a known H3 immunogenic epitope sequence significantly improved the immunogenicity of the H7 HA in the humanized mouse model, leading to a greater than 4-fold increase in HA-binding IgG responses. Thus, we provide experimental evidence for the important contribution of this H7-specific T cell epitope in determining the immunogenicity of an influenza vaccine. Furthermore, this study delineates strategies that can be used for screening and selecting vaccine strains using immunoinformatics tools and a humanized mouse model

Comparison of the Mutation Rates of Human Influenza A and B Viruses

Human influenza A viruses evolve more rapidly than influenza B viruses. To clarify the cause of this difference, we have evaluated the mutation rate of the nonstructural gene as revealed by the genetic diversity observed during the growth of individual plaques in MDCK cells. Six plaques were studied, representing two strains each of type A and B viruses. A total of 813,663 nucleotides were sequenced, giving rates of 2.0 × 10(−6) and 0.6 × 10(−6) mutations per site per infectious cycle, which, when extended to 1 year, agree well with the published annual evolutionary rates

Influence of Acylation Sites of Influenza B Virus Hemagglutinin on Fusion Pore Formation and Dilation

The cytoplasmic tail (CT) of hemagglutinin (HA) of influenza B virus (BHA) contains at positions 578 and 581 two highly conserved cysteine residues (Cys578 and Cys581) that are modified with palmitic acid (PA) through a thioester linkage. To investigate the role of PA in the fusion activity of BHA, site-specific mutagenesis was performed with influenza B virus B/Kanagawa/73 HA cDNA. All of the HA mutants were expressed on Cos cells by an expression vector. The membrane fusion ability of the HA mutants at a low pH was quantitatively examined with lipid (octadecyl rhodamine B chloride) and aqueous (calcein) dye transfer assays and with the syncytium formation assay. Two deacylation mutants lacking a CT or carrying serine residues substituting for Cys578 and Cys581 promoted full fusion. However, one of the single-acylation-site mutants, C6, in which Cys581 is replaced with serine, promoted hemifusion but not pore formation. In contrast, four other single-acylation-site mutants that have a sole cysteine residue in the CT at position 575, 577, 579, or 581 promoted full fusion. The impaired pore-forming ability of C6 was improved by amino acid substitution between residues 578 and 582 or by deletion of the carboxy-terminal leucine at position 582. Syncytium-forming ability, however, was not adequately restored by these mutations. These facts indicated that the acylation was not significant in membrane fusion by BHA but that pore formation and pore dilation were appreciably affected by the particular amino acid sequence of the CT and the existence of a single acylation site in CT residue 578

Accumulation of Amino Acid Substitutions Promotes Irreversible Structural Changes in the Hemagglutinin of Human Influenza AH3 Virus during Evolution

In order to clarify the effect of an accumulation of amino acid substitutions on the hemadsorption character of the influenza AH3 virus hemagglutinin (HA) protein, we introduced single-point amino acid changes into the HA1 domain of the HA proteins of influenza viruses isolated in 1968 (A/Aichi/2/68) and 1997 (A/Sydney/5/97) by using PCR-based random mutation or site-directed mutagenesis. These substitutions were classified as positive or negative according to their effects on the hemadsorption activity. The rate of positive substitutions was about 50% for both strains. Of 44 amino acid changes that were identical in the two strains with regard to both the substituted amino acids and their positions in the HA1 domain, 22% of the changes that were positive in A/Aichi/2/68 were negative in A/Sydney/5/97 and 27% of the changes that were negative in A/Aichi/2/68 were positive in A/Sydney/5/97. A similar discordance rate was also seen for the antigenic sites. These results suggest that the accumulation of amino acid substitutions in the HA protein during evolution promoted irreversible structural changes and therefore that antigenic changes in the H3HA protein may not be limited

Restriction of Amino Acid Change in Influenza A Virus H3HA: Comparison of Amino Acid Changes Observed in Nature and In Vitro

We introduced 248 single-point amino acid changes into hemagglutinin (HA) protein of the A/Aichi/2/68 (H3N2) strain by a PCR random mutation method. These changes were classified as positive or negative according to their effect on hemadsorption activity. We observed following results. (i) The percentage of surviving amino acid changes on the HA1 domain that did not abrogate hemadsorption activity was calculated to be ca. 44%. In nature, it is estimated to be ca. 39.6%. This difference in surviving amino acid changes on the HA protein between natural isolates and in vitro mutants might be due to the immune pressure against the former. (ii) A total of 26 amino acid changes in the in vitro mutants matched those at which mainstream amino acid changes had occurred in the H3HA1 polypeptide from 1968 to 2000. Of these, 25 were positive. We suggest that the majority of amino acid changes on the HA protein during evolution might be restricted to those that were positive on the HA of A/Aichi/2/68. (iii) We constructed two-point amino acid changes on the HA protein by using positive mutants. These two-point amino acid changes with a random combination did not inhibit hemadsorption activity. It is possible that an accumulation of amino acid change might occur without order. (iv) From the analysis of amino acids participating in mainstream amino acid change, each antigenic site could be further divided into smaller sites. The amino acid substitutions in the gaps between these smaller sites resulted in mostly hemadsorption-negative changes. These gap positions may play an important role in maintaining the function of the HA protein, and therefore amino acid changes are restricted at these locations

Cell-Based Influenza A/H1N1pdm09 Vaccine Viruses Containing Chimeric Hemagglutinin with Improved Membrane Fusion Ability

The H1N1 influenza pandemic vaccine has been developed from the A/California/07/09 (Cal) virus and the well-known high-yield A/Puerto Rico/8/34 (PR8) virus by classical reassortment and reverse genetics (RG) in eggs. Previous studies have suggested that Cal-derived chimeric hemagglutinin (HA) and neuraminidase (NA) improve virus yields. However, the cell-based vaccine of the H1N1 pandemic virus has been less investigated. RG viruses that contained Cal-derived chimeric HA and NA could be rescued in Madin–Darby canine kidney cells that expressed α2,6-sialyltransferase (MDCK-SIAT1). The viral growth kinetics and chimeric HA and NA properties were analyzed. We attempted to generate various RG viruses that contained Cal-derived chimeric HA and NA, but half of them could not be rescued in MDCK-SIAT1 cells. When both the 3′- and 5′-terminal regions of Cal HA viral RNA were replaced with the corresponding regions of PR8 HA, the RG viruses were rescued. Our results were largely consistent with those of previous studies, in which the N- and C-terminal chimeric HA slightly improved virus yield. Importantly, the chimeric HA, compared to Cal HA, showed cell fusion ability at a broader pH range, likely due to amino acid substitutions in the transmembrane region of HA. The rescued RG virus with high virus yield harbored the chimeric HA capable of cell fusion at a broader range of pH

Growth capacities of PR8 viruses in NIID-MDCK cells.

<p>Replication kinetics of original PR8 (org-PR8), the passage-10 virus (P10), hg-PR8 and rg-hg-PR8 were compared in NIID-MDCK cells. NIID-MDCK cells were inoculated with the respective viruses at an m.o.i. of 0.0001. The supernatants were collected at 24, 48, and 72 hpi, and the virus titers were determined in a plaque formation assay. The vertical axis shows the mean PFUs ± SD at each time point for three or more independent experiments.</p

Western blotting analysis of the purified viral proteins.

<p>Purified viral concentrates of NIIDRG-10C, -10.1C, -10 and -10.1 were analyzed by SDS-PAGE. HA proteins were detected using a rabbit polyclonal antibody against recombinant HA protein of H7N9 (A/Shanghai/1/2013) (Sino Biological Inc. Beijing, China) and a donkey anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody by western blotting analysis. Purified viral proteins were treated (A) or untreated (B) with N-glycosidase F.</p

Gene constellations and the growth capacities of the reassortants between org-PR8 and hg-PR8 viruses.

<p>The gene constellations of org-PR8, P10, hg-PR8 and reassortant viruses between org-PR8 and hg-PR8 are indicated by black and white squares. Black squares indicate gene segments containing non-synonymous mutations compared with the sequences of the corresponding segment of org-PR8. NIID-MDCK cells were inoculated with these viruses at an m.o.i. of 0.0001. The peak virus titers were determined in a plaque formation assay. Significant differences in the peak virus titers were assessed with Bonferroni’s multiple-comparison test. (*<i>p<0</i>.<i>01</i> from org-PR8)</p

Total protein yields of reassortant viruses.

<p>Average total protein yields (TPYs) of the reassortants. The vertical axis shows the average yields ± SD for three independent experiments. The differences in the yields between the reassortants were statistically analyzed using Welch’s t-test. (*<i>p<0</i>.<i>05</i>)</p