Recombinant HA-based vaccine outperforms split and subunit vaccines in elicitation of influenza-specific CD4 T cells and CD4 T cell-dependent antibody responses in humans

Abstract

Although traditional egg-based inactivated influenza vaccines can protect against infection, there have been significant efforts to develop improved formats to overcome disadvantages of this platform. Here, we have assessed human CD4 T cell responses to a traditional egg-based influenza vaccine with recently available cell-derived vaccines and recombinant baculovirus-derived vaccines. Adults were administered either egg-derived Fluzone^®, mammalian cell-derived Flucelvax^® or recombinant HA (Flublok^®). CD4 T cell responses to each HA protein were assessed by cytokine EliSpot and intracellular staining assays. The specificity and magnitude of antibody responses were quantified by ELISA and HAI assays. By all criteria, Flublok vaccine exhibited superior performance in eliciting both CD4 T cell responses and HA-specific antibody responses, whether measured by mean response magnitude or percent of responders. Although the mechanism(s) underlying this advantage is not yet clear, it is likely that both qualitative and quantitative features of the vaccines impact the response.

Keywords: Immunology; Vaccines.

Fig. 1. Vaccine comparison study design.

Study subjects aged 18–49 years old were randomized into three cohorts, each receiving a licensed seasonal influenza vaccine. The vaccine formulations administered were egg-derived split-vaccine Fluzone quadrivalent (blue), mammalian cell-based Flucelvax (gold), recombinant HA made in the baculovirus system Flublok (red) and egg-derived split-vaccine Fluzone High (HD) dose (light blue). Subjects were enrolled for a single season in one of the vaccine groups. Over the course of 3 consecutive seasons 152 healthy subjects were divided into the first three vaccine groups as indicated in the left-hand portion of the Figure. In the third season, an additional 21 subjects were enrolled in the Fluzone HD group. Blood samples were taken at day 0 (D0) prior to vaccination, and day 7 (D7, for flow cytometry, which is peak for some subsets of CD4 cells at this time), day 14 (D14, for EliSpot assays that peak at this time point) and day 28 (D28, neutralizing antibody) post vaccination. Sera for HAI assays were sampled at day 0 and day 28.

Fig. 2. Human HA-specific CD4 T-cell responses post vaccination.

CD4-enriched cells from the vaccinated subjects were stimulated with overlapping pools of peptides from H1, H3 and HA-B proteins and evaluated for IFN-γ production using cytokine EliSpot assays. The responses for the 3 groups Fluzone (blue), Flucelvax (gold) or Flublok (red) are indicated. In a, the CD4 T cells expanded between day 0 and day 14 for each HA peptide pool tested were summed. The data are represented as the change in the response between day 0 and day 14 (D14–D0) with the median response indicated by a black line. In b, the expansion of CD4 T cells for each HA peptide set is shown for the HA protein that is indicated above each panel. H1 is shown on the left, H3 in the middle and HA-B on the right. The top panels illustrate the response of each individual, represented as a single dot with the spread in reactivity of the population and the median value indicated by a black line. The bottom bar graphs indicate the average response of the individuals shown in the top panel and the 95% confidence interval of each group for each HA. The p values shown were calculated by the Wilcoxon rank sum test.

Fig. 3. HA-specific CD4 T-cell responses post vaccination for individuals.

CD4-enriched populations of cells from the subjects vaccinated with Fluzone (top), Flucelvax (middle) or Flublok (bottom) were stimulated with pools of peptides from the HA indicated. H1-specific responses are shown in black bars, H3 in white and HA-B in grey. IFN-γ cytokine producing cells were quantified using EliSpot assays. The data are represented as a stacked bar for each vaccinated individual and the year of the study is indicated at the bottom of the graphs. A dotted line indicates the 100-spot cutoff used to estimate a positive CD4 T-cell response. The boxed number at the top of each panel is the percent response-based frequency using this cutoff for each vaccine.

Fig. 4. The more robust CD4 T-cell response to Flublok post vaccination is not determined by HA dose within the vaccine.

A group of 21 healthy subjects, aged 18–49 years old were vaccinated with high dose trivalent Fluzone (blue) and compared to those subjects in years 1 and 2 that received trivalent Flublok (red). CD4-enriched populations were stimulated with pools of peptides from H1, H3 and HA-B and evaluated for IFN-γ production using cytokine EliSpot assays. The data are represented as the change in the response between day 0 and day 14 (D14–D0), with the median response indicated by a black line. The specific HA reactivity is indicated above the panels, with H1 on the left, H3 in the middle and HA-B on the right. The p values shown were calculated by the Wilcoxon rank sum test.

Fig. 5. Polyfunctional CD4 T-cell response to vaccination with three vaccine platforms.

PBMC from subjects vaccinated with Fluzone (blue), Flucelvax (gold) or Flublok (red) were stimulated in vitro with pools of peptides derived from H1 (left) or H3 (right) for 16 h. Brefeldin A and monensin were added for the last 8 h of culture to block cytokine secretion. H1- or H3-specific CD4 T cells were identified based on expression of CD69 + and the intracellular cytokines IL-2 and IFN-γ. Shown is quantification of cells producing both cytokines, IFN-γ + IL2 + (left), IFN-γ + IL2- (middle) and IFN-γ-IL2 + (right), using the gating scheme shown in Supplementary Fig. 5. The data are represented as the gain in the number in antigen-reactive, cytokine-positive cells between day 0 and day 7. The average response is indicated by the grey bar and the individual responses are indicated by the scatter. The p values shown were calculated by the Wilcoxon rank sum test.

Fig. 6. Antibody responses to vaccination as measured by HAI assay.

The neutralizing serum antibody response at day 0 before vaccination and day 28 post vaccination was measured by HAI assays from subjects vaccinated with Fluzone (blue), Flucelvax (gold) or Flublok (red). Shown in a is the fold-change between day 0 and day 28 (D28/D0) for each of the HA strains contained in the vaccine, H1 (top left), H3 (top right), HA-B Brisbane (Victoria lineage, bottom left) and HA-B Phuket (Yamagata lineage, bottom right). The data from individuals are represented by the scatter and the geometric mean is indicated by a black line. A dotted line indicating the fourfold response, typically used to indicate a positive antibody response, is shown. The p values were calculated by the Wilcoxon rank sum test. The table in b indicates the percent responders for each vaccine, indicated on the rows to the left, and each HA, indicated on the columns above, based on a fourfold or greater HAI response.

Fig. 7. Total HA-specific antibody response quantified by ELISA.

Total HA-specific antibody responses of subjects vaccinated with Fluzone (blue), Flucelvax (gold) or Flublok (red) are shown. Anti-HA antibody levels collected before vaccination and day 14 post vaccination were measured by HA-specific IgG ELISA assays. The relative titers were quantified based on the dilution of antibodies required to reach a fixed OD405 signal on the linear portion of the antibody titration curve. Shown in panel a is the fold-change between day 0 and day 14 (D14/D0) for each of the HA strains contained in the vaccine, H1 (top left), H3 (top right), HA-B Brisbane (Victoria lineage, bottom left) and HA-B Florida (a surrogate for B/Phuket, Yamagata lineage, bottom right). Responses among individuals are represented by the scatter and the geometric mean is indicated by a black line. A dotted line indicates the fourfold response. The p values shown were calculated by the Wilcoxon rank sum test. In b, the table below, indicates the percent responders for each vaccine. The vaccine is indicated to the left and the specific HA protein on the columns above, based on a fourfold or greater response.