Superior immunogenicity of inactivated whole virus H5N1 influenza vaccine is primarily controlled by Toll-like receptor signalling

Abstract

In the case of an influenza pandemic, the current global influenza vaccine production capacity will be unable to meet the demand for billions of vaccine doses. The ongoing threat of an H5N1 pandemic therefore urges the development of highly immunogenic, dose-sparing vaccine formulations. In unprimed individuals, inactivated whole virus (WIV) vaccines are more immunogenic and induce protective antibody responses at a lower antigen dose than other formulations like split virus (SV) or subunit (SU) vaccines. The reason for this discrepancy in immunogenicity is a long-standing enigma. Here, we show that stimulation of Toll-like receptors (TLRs) of the innate immune system, in particular stimulation of TLR7, by H5N1 WIV vaccine is the prime determinant of the greater magnitude and Th1 polarization of the WIV-induced immune response, as compared to SV- or SU-induced responses. This TLR dependency largely explains the relative loss of immunogenicity in SV and SU vaccines. The natural pathogen-associated molecular pattern (PAMP) recognized by TLR7 is viral genomic ssRNA. Processing of whole virus particles into SV or SU vaccines destroys the integrity of the viral particle and leaves the viral RNA prone to degradation or involves its active removal. Our results show for a classic vaccine that the acquired immune response evoked by vaccination can be enhanced and steered by the innate immune system, which is triggered by interaction of an intrinsic vaccine component with a pattern recognition receptor (PRR). The insights presented here may be used to further improve the immune-stimulatory and dose-sparing properties of classic influenza vaccine formulations such as WIV, and will facilitate the development of new, even more powerful vaccines to face the next influenza pandemic.

Figure 1. TLRs contribute to the efficacy of H5N1 WIV vaccine.

Four weeks after immunization of wild-type, TLR7^−/−, and MyD88^−/−/TRIF^−/− mice with WIV, SV, or SU vaccine (5 µg HA), serum HI titres (A) and H5N1-specific IgG titres (B) were determined for the individual mice. Titres below the detection limit were assigned with half the value of the lowest detectable serum dilution, which was 8 in the HI assay and 100 in the IgG ELISA. Significant (p<0.05) and highly significant (p<0.01) differences between wild-type mice and mutant mice receiving the same vaccine are indicated by * and **, respectively. GMT indicates geometric mean titter.

Figure 2. Induction of IFNγ-producing T cells by H5N1 WIV vaccine depends on TLR7 signalling.

Spleen cells of wild-type mice and mutant mice immunized with WIV, SV, or SU vaccine were re-stimulated in vitro with SU vaccine, and numbers of IFNγ-producing cells were determined by Elispot assay. Bars represent the average values of triplicate determinations per mouse for each mouse type and immunization group (n = 8; MyD88^−/−/TRIF^−/−/SU, n = 7), with standard deviation. Significant (p<0.05) and highly significant (p<0.01) differences between wild-type mice and mutant mice receiving the same vaccine are indicated by * and **, respectively.

Figure 3. H5N1 WIV vaccine induces Th1-type antibody responses via TLR7 signalling.

Serum titres of H5N1-specific IgG1 subtype (Th2-type antibody), and IgG2c, IgG2b, and IgG3 subtypes (Th1-type antibodies) were determined by ELISA. Geometric mean titres are plotted for each group of wild-type mice or mutant mice (n = 8; MyD88^−/−/TRIF^−/−/SU, n = 7) immunized with WIV, SV, or SU vaccine. Significant (p<0.05) and highly significant (p<0.01) differences between wild-type mice and mutant mice receiving the same vaccine are indicated by * and **, respectively.

Figure 4. Induction of IFNα by WIV is TLR7-dependent in bone-marrow derived pDCs, but not in spleen-derived pDCs.

Bone-marrow cells cultured with FLT3L (containing 20–30% pDCs) (A), or pDC-enriched spleen cell cultures (containing 62%–68% pDCs) (B) of wild-type mice (black bars) and TLR7^−/− mice (white bars) were incubated overnight with WIV, SV, or SU vaccine. IFNα was measured in cell supernatants by sandwich ELISA. Bars represent average values of triplicate determinations with standard deviation, and are representative of three independent experiments.