Randomized controlled ferret study to assess the direct impact of 2008-09 trivalent inactivated influenza vaccine on A(H1N1)pdm09 disease risk

Abstract

During spring-summer 2009, several observational studies from Canada showed increased risk of medically-attended, laboratory-confirmed A(H1N1)pdm09 illness among prior recipients of 2008-09 trivalent inactivated influenza vaccine (TIV). Explanatory hypotheses included direct and indirect vaccine effects. In a randomized placebo-controlled ferret study, we tested whether prior receipt of 2008-09 TIV may have directly influenced A(H1N1)pdm09 illness. Thirty-two ferrets (16/group) received 0.5 mL intra-muscular injections of the Canadian-manufactured, commercially-available, non-adjuvanted, split 2008-09 Fluviral or PBS placebo on days 0 and 28. On day 49 all animals were challenged (Ch0) with A(H1N1)pdm09. Four ferrets per group were randomly selected for sacrifice at day 5 post-challenge (Ch+5) and the rest followed until Ch+14. Sera were tested for antibody to vaccine antigens and A(H1N1)pdm09 by hemagglutination inhibition (HI), microneutralization (MN), nucleoprotein-based ELISA and HA1-based microarray assays. Clinical characteristics and nasal virus titers were recorded pre-challenge then post-challenge until sacrifice when lung virus titers, cytokines and inflammatory scores were determined. Baseline characteristics were similar between the two groups of influenza-naïve animals. Antibody rise to vaccine antigens was evident by ELISA and HA1-based microarray but not by HI or MN assays; virus challenge raised antibody to A(H1N1)pdm09 by all assays in both groups. Beginning at Ch+2, vaccinated animals experienced greater loss of appetite and weight than placebo animals, reaching the greatest between-group difference in weight loss relative to baseline at Ch+5 (7.4% vs. 5.2%; p = 0.01). At Ch+5 vaccinated animals had higher lung virus titers (log-mean 4.96 vs. 4.23pfu/mL, respectively; p = 0.01), lung inflammatory scores (5.8 vs. 2.1, respectively; p = 0.051) and cytokine levels (p>0.05). At Ch+14, both groups had recovered. Findings in influenza-naïve, systematically-infected ferrets may not replicate the human experience. While they cannot be considered conclusive to explain human observations, these ferret findings are consistent with direct, adverse effect of prior 2008-09 TIV receipt on A(H1N1)pdm09 illness. As such, they warrant further in-depth investigation and search for possible mechanistic explanations.

Figure 1. Study protocol.

Randomized blinded placebo-controlled experiment of Canadian manufactured, commercially-available 2008–09 trivalent inactivated influenza vaccine (TIV: Fluviral) on A(H1N1)pdm09 disease risk in ferrets.

Figure 2. HA1 microarray serological values by study antigens, group and day.

Box plots display median (dash) and mean (dot) of log₁₀-transformed HA1 protein microarray signal values. The box extends to the 25^th/75^th percentiles and whiskers extend to minimum/maximum values. H1-07 indicates A/Brisbane/59/2007 (H1N1)-like; H3-07 indicates A/Brisbane/10/2007 (H3N2)-like; H1-09 indicates A/California/7/2009 (H1N1)pdm09-like (Table S3; grey-shaded). Sample size as follows: Pre-immunization Vaccine = 15, Placebo = 16 (3 ferrets each per group pre-shipment serum was substituted owing to insufficient day 0 available); Day 28 Vaccine = 14, Placebo = 15; Day 49 Vaccine = 12, Placebo = 11; Day 54 Vaccine = 2, Placebo = 4; Day 63 Vaccine = 9, Placebo = 8. **indicates statistical significance at p<0.01 and *indicates statistical significance at p<0.05 in comparing vaccine to placebo group at the designated time point. ΔΔ indicates statistical significance at p<0.01 and Δ indicates statistical significance at p<0.05 in comparing values within study groups at days 28, 49, 54 and 63 relative to pre-immunization, colour coded by vaccine (red) or placebo (blue). □□ indicates statistical significance at p<0.01 and □ indicates statistical significance at p<0.05 in comparing day 63 to day 49 within groups, colour coded per above by study group.

Figure 3. Clinical outcomes including weight loss, nasal wash and lung virus titers by study group and day.

Clinical outcomes are displayed including: (A) Mean percentage weight relative to baseline by study group and day, with standard errors. (B) Nasal wash virus titers by study group and day. (C) Lung homogenate virus titers at day 5 post-challenge. Box plots (B and C) display mean (dot) and median (line) virus titres as log pfu/mL. Per usual, the box extends to the 25^th/75^th percentiles and whiskers extend to minimum/maximum values. Ch refers to challenge day and Ch+1, Ch+2 etc indicate day post-challenge (i.e. day one post-challenge, day two post-challenge etc). Ch+5 indicates day five post-challenge on which four animals per group were randomly selected for sacrifice. Statistically significant between-group differences are as specified.

Figure 4. Ferret lung histology at day 5 post-challenge (Ch+5).

Salient lung histologic features (hematoxylin eosin stain micrometric scale in lower left of each panel) including vaccine and placebo ferrets with highest and lowest combined Ch+5 inflammatory scores within their group: (A) Ferret #81 (placebo; inflammatory score 0.5) indicating very mild/minimal peri-bronchial inflammation; (B) Ferret #63 (vaccinated; inflammatory score 0.5) indicating very mild/minimal peri-vascular inflammation; (C) Ferret #58 (placebo; inflammatory score 4.0) indicating moderate bronchial and mild peri-bronchial/peri-vascular inflammation; (D) Ferret #69 (vaccinated; inflammatory score 11.5) indicating severe bronchopneumonia. The increased micrometric scale in panel D reflects that the pathologic changes are marked and diffuse, best rendered at low magnification, whereas the mild to moderate and more focal changes in panels A to C necessitate photomicrographs at higher magnification. Corresponding histopathology scores are shown in Table S9.

Figure 5. Lung cytokine values at days 5 and 14 post-challenge with A(H1N1)pdm09 by study group.

Per usual, box plots display mean (dot) and median (dash) virus titers with box extending to the 25^th/75^th percentiles and whiskers extending to minimum/maximum values. Cytokine values relative to control are displayed at (A) day 5 post challenge (Ch+5) on which four animals per group were randomly selected for sacrifice and (B) day 14 post-challenge (Ch+14) on which the remaining 12 animals per group were sacrificed. Thus, at Ch+5, n = 4 (except IFN alpha for which n = 2 for placebo group) and at Ch+14, n = 12.