Immune responses in pigs vaccinated with adjuvanted and non-adjuvanted A(H1N1)pdm/09 influenza vaccines used in human immunization programmes

Abstract

Following the emergence and global spread of a novel H1N1 influenza virus in 2009, two A(H1N1)pdm/09 influenza vaccines produced from the A/California/07/09 H1N1 strain were selected and used for the national immunisation programme in the United Kingdom: an adjuvanted split virion vaccine and a non-adjuvanted whole virion vaccine. In this study, we assessed the immune responses generated in inbred large white pigs (Babraham line) following vaccination with these vaccines and after challenge with A(H1N1)pdm/09 virus three months post-vaccination. Both vaccines elicited strong antibody responses, which included high levels of influenza-specific IgG1 and haemagglutination inhibition titres to H1 virus. Immunisation with the adjuvanted split vaccine induced significantly higher interferon gamma production, increased frequency of interferon gamma-producing cells and proliferation of CD4(-)CD8(+) (cytotoxic) and CD4(+)CD8(+) (helper) T cells, after in vitro re-stimulation. Despite significant differences in the magnitude and breadth of immune responses in the two vaccinated and mock treated groups, similar quantities of viral RNA were detected from the nasal cavity in all pigs after live virus challenge. The present study provides support for the use of the pig as a valid experimental model for influenza infections in humans, including the assessment of protective efficacy of therapeutic interventions.

Figure 1. Antibody titre after vaccination and challenge.

Pigs were vaccinated with the adjuvanted split (solide line), non-adjuvanted whole (dashed line) or mock (dotted line) vaccine. The non-adjuvanted whole vaccine was administered twice (at day 0 and day 21pv). At day 93pv, all pigs were challenged with Eng195. Serum samples were taken at various time-points post-vaccination and challenge, and IgG1 (A) and IgG2 (B) Ab titres were quantified by ELISA. Results are expressed as the mean Log EU value ± SEM. *, P<0.05.

Figure 2. Interferon gamma response after vaccination and challenge.

Pigs were vaccinated with the adjuvanted split (solid line), non-adjuvanted whole (dashed line) or mock (dotted line) vaccine. The non-adjuvanted whole vaccine was administered twice (at day 0 and day 21pv). At day 93pv, all pigs were challenged with Eng195. Blood samples were taken at various time-points post-vaccination and challenge. 2×10⁵ PBMC per well were cultured in vitro for 3 days in the presence of 10⁵EID₅₀/ml inactivated Cal07, and interferon gamma (IFNγ) production was quantified in the supernatants by ELISA (A) and IFNγ-producing cells were quantified in the cellular fraction by ELISPOT (B). Results are expressed as the mean pg/ml (A) and IFNγ-producing cells per 10⁶ stimulated PBMC (B) ± SEM. *, P = 0.05. **, P equal to or below 0.01.

Figure 3. T cell sub-population proliferation monitored by CFSE labelling in response to in vitro re-stimulation with H1N1 influenza.

CFSE-labelled PBMC were cultured in vitro in the presence of 10⁵EID₅₀/ml inactivated Eng195, uninfected allantoic fluid (mock antigen), PWM or in media only. After 5 days, cells were stained with anti-CD4 and anti-CD8 monoclonal antibodies and analysed by flow cytometry for CFSE intensity. Three T cell subsets were identified on the basis of their CD4 and CD8 expression, and an example of the CFSE pattern at day 7pv in response to inactivated Eng195 in these various subsets in a pig vaccinated with the adjuvanted split vaccine is shown (A). At day 28pv, the percentage of cells proliferating (CFSE_Low cells) in response to media alone, mock antigen or inactivated Eng195 was determined after gating on CD4⁺CD8⁻ (B), CD4⁺CD8⁺ (C) or CD4⁻CD8⁺ (D) cells. Results are expressed as the mean percentage of proliferating (CFSE_Low) cells ± SEM. n.s = Not statistically significant.

Figure 4. Body temperature and detection of viral RNA in the nasal cavity after infectious challenge.

Pigs were vaccinated with the adjuvanted split (solid line), non-adjuvanted whole (dashed line) or mock (dotted line) vaccine, and all pigs were subsequently challenged with infectious Eng195 influenza virus. (A) Rectal temperature (in °C) was monitored one day prior and everyday following challenge. Results are expressed as the mean value ± SEM for each vaccination group. *, P<0.01. (B) Nasal swabs were collected one day prior and everyday following challenge. Shedding was measured in all samples through the detection of viral RNA by a modified influenza A M gene real-time RT-PCR (RRT-PCR) assay. By the use of a standard curve (on each test plate) generated from a dilution series of infectious Eng195 virus, the relative equivalent unit (REU) of viral RNA was determined based upon the cycle threshold (Ct) value obtained for each sample. Results are expressed as the mean REU ± SEM for each vaccination group.