Flublok Quadrivalent Vaccine Adjuvanted with R-DOTAP Elicits a Robust and Multifunctional CD4 T Cell Response That Is of Greater Magnitude and Functional Diversity Than Conventional Adjuvant Systems

Abstract

It is clear that new approaches are needed to promote broadly protective immunity to viral pathogens, particularly those that are prone to mutation and escape from antibody-mediated immunity. CD4+ T cells, known to target many viral proteins and highly conserved peptide epitopes, can contribute greatly to protective immunity through multiple mechanisms. Despite this potential, CD4+ T cells are often poorly recruited by current vaccine strategies. Here, we have analyzed a promising new adjuvant (R-DOTAP), as well as conventional adjuvant systems AddaVax with or without an added TLR9 agonist CpG, to promote CD4+ T cell responses to the licensed vaccine Flublok containing H1, H3, and HA-B proteins. Our studies, using a preclinical mouse model of vaccination, revealed that the addition of R-DOTAP to Flublok dramatically enhances the magnitude and functionality of CD4+ T cells specific for HA-derived CD4+ T cell epitopes, far outperforming conventional adjuvant systems based on cytokine EliSpot assays and multiparameter flow cytometry. The elicited CD4+ T cells specific for HA-derived epitopes produce IL-2, IFN-γ, IL-4/5, and granzyme B and have multifunctional potential. Hence, R-DOTAP, which has been verified safe by human studies, can offer exciting opportunities as an immune stimulant for next-generation prophylactic recombinant protein-based vaccines.

Keywords: CD4+ T cells; adjuvants; hemagglutinin; influenza; vaccination.

Figure 1

Mapping of H3 CD4 T cell epitopes in B6 mice. Shown in (A) is peptide matrix, with all peptides that collectively span H3 (H3N2 Perth2009) contained in discreet pools of 9-10 peptides indicated by R1 though R10 or C1 through C10. After priming B6 mice by infection with A/Switzerland 2013 H3N2 at 900 PFU/mouse, day 12 post infection CD4 T cells isolated from the spleen were tested for recognition of the peptides in each pool using a cytokine EliSpot assay (B) for IL-2 (top) or IFN-γ (bottom). Peptides in pools C8 and R10 were the most stimulatory followed by C7 and R9. The candidate peptides are highlighted in (A), with the major candidates boxed in red outline. Peptide 18 and 50 were eliminated in a subsequent assay, shown in (C) in IL-2 (left) and IFN-γ (right) EliSpots. Shown in (D), red box, is the likely core of the dominant H3 peptide, contained primarily in peptide 36.

Figure 2

Cytokine EliSpot assays test the specificity of CD4 T cells elicited by Flublok adjuvanted with R-DOTAP. Mice were vaccinated with Flublok adjuvanted with R-DOTAP in the rear footpad and at day 9 the draining lymph node cells were enriched for CD4 T cells. Candidate peptides or peptide pools were tested for their ability to stimulate the CD4 T cells for production of IL-2 (left) or IFN-γ (right). The points represent the data from two independent assays and the average is shown the by the grey bar. H3N+ is a pool of peptides from the amino terminal half of the H3 Perth 2009 with added H3 strain variant peptides and the H3C+ is the comparable pool of peptides from the COOH terminus of H3, demonstrating that the defined H3 35/36 peptide comprises the primary epitope specificity. Also shown are the CD4 T cell responses to the 3 HA-B peptides previously defined.

Figure 3

CD4 T cell magnitude and phenotype post-vaccination with Flublok with R-DOTAP or comparator adjuvant systems. Different cohorts of mice were vaccinated with Flublok adjuvanted with Addavax (turquoise), Addavax+CpG (blue), R-DOTAP (orange) or no adjuvant (purple) as indicated in the key. Elicited CD4 T cells specific for HA-B or H3 (as indicated below each panel) were quantified by EliSpots for IFN-γ, IL-2, Granzyme B, or IL-4/5, as indicated above each panel. The frequency of reactive CD4 T cells per million cells is shown in (A) and the sum of the frequency per mouse is indicated in (B), which accounts for the yield of CD4 T cells elicited by the different adjuvant systems. Shown are the average (Mean) responses with error bars indicated the standard error of the mean. Statistical values were calculated using two-way ANOVA.

Figure 4

Flow cytometry analyses to quantify vaccine adjuvant-elicited CD4 T cells. After administration of Flublok adjuvanted with either AddaVax+CpG (AdVx+CpG), indicated in blue, or R-DOTAP, indicated in orange (6 mice per group) draining lymph node cells were sampled for expression of CD44 (as an indicator of antigen experience) or cytokine production, as described in Material and Methods, after stimulation with the pool of HA-B peptides. ((A), left) shows the Flow cytometry plot from a representative sample that illustrates the gating scheme used to quantify CD44 expressing CD4 T cells and the frequency of antigen experienced CD44 CD4 T cells ((A), right). In (B) the frequency of total antigen-experienced (CD44hi), cytokine producing cells were quantified by first gating on CD4, CD44 high and then the sum of those that produced either IFN-γ, IL-2 or IL-4/5. (C) CD44 high, CD4 T cells elicited by AddaVax+CpG (blue) or R-DOTAP (orange), were quantified for expression of the individual indicated cytokines. Shown are the individual mice (circles), mean response (bars) and error bars indicating standard error of the mean. Statistical analysis was done using the Mann Whitney unpaired nonparametric t test.

Figure 5

Subsets of vaccine-elicited CD4 T cells that expressed one or more cytokines. Two cohorts of mice (n = 6/group) were subcutaneously vaccinated with Flublok, adjuvanted with either AddaVax with CpG (AdVx+CpG; blue) or R-DOTAP (orange). Cells isolated from the draining lymph nodes were stimulated with the HA-B major epitope pool and subsequently treated to surface and intracellular staining. In (A), antigen experienced (CD44hi) cells expressing IFN-γ +, IL-2+ or IL-4/5+ CD4 T cells were identified and then Boolean combination gates were applied to determine the frequency of single, double or triple cytokine producers. Data is shown here as the mean ± SEM, with symbols to represent individual animals. Statistical analysis used the Mann-Whitney, multiple, unpaired, non-parametric t tests). (B), the frequency of total cytokine producers, listed below each pie, was normalized to 100% and each subpopulation of cytokine producers is presented as a fraction of the total. The frequency of IFN-γ +/IL-2+/IL-4/5+ triple producers and the IFN-γ +/IL-4/5+ double producers were undetectable and therefore excluded from this analysis.

Figure 6

Vaccine induced CD4 T cells with cytotoxic potential and polyfunctionality. CD4 T cells elicited by Flublok adjuvanted with either AddaVax+CpG (blue) or R-DOTAP (orange) were stimulated with the HA-B peptide pool. They were sequentially gated on CD4+, CD44+ and then analyzed for cytotoxic activity, based expression of the degranulation marker CD107A. In (A), CD107a+ cells were gated for expression of Granzyme B (GrzB) or for cells expressing both, as indicated beneath each bar. (B). CD4+CD44+ cells CD107a+ cells were examined for the expression of IL-2, IFN-γ, IL-4/5 or combinations of these cytokines, using Boolean gating. The fraction of CD107a positive cells that were also making cytokines is shown first as a black slice of the pie in grey. The cytokine profiles of these cells were then quantified for the cytokines indicated in the excised pie diagram in color. The colors of these slices represent the cytokines produced by the CD107a positive cells. The statistical tests in A represents six replicate animals from each group using multiple unpaired t tests with Welch’s correction.