Protection by universal influenza vaccine is mediated by memory CD4 T cells

Abstract

There is a diverse array of influenza viruses which circulate between different species, reassort and drift over time. Current seasonal influenza vaccines are ineffective in controlling these viruses. We have developed a novel universal vaccine which elicits robust T cell responses and protection against diverse influenza viruses in mouse and human models. Vaccine mediated protection was dependent on influenza-specific CD4+ T cells, whereby depletion of CD4+ T cells at either vaccination or challenge time points significantly reduced survival in mice. Vaccine memory CD4+ T cells were needed for early antibody production and CD8+ T cell recall responses. Furthermore, influenza-specific CD4+ T cells from vaccination manifested primarily Tfh and Th1 profiles with anti-viral cytokine production. The vaccine boosted H5-specific T cells from human PBMCs, specifically CD4+ and CD8+ T effector memory type, ensuring the vaccine was truly universal for its future application. These findings have implications for the development and optimization of T cell activating vaccines for universal immunity against influenza.

Keywords: IL-15; Influenza virus; T cells; Universal vaccine; Vaccinia.

Conflict of interest statement

Conflicts of interest

Wyeth/IL-15/5flu is incorporated in a patent (LPP, TAW) filed by the Office of Technology Transfer, NIH, US Department of Health and Human Services. There are no other conflicts to declare.

Copyright © 2018 Elsevier Ltd. All rights reserved.

Figures

Fig. 1.

Wyeth/IL-15/5flu does not protect T cell deficient mouse strains from H3N2 infection. (A) BALB/c, nude and SCID mice were vaccinated twice s.c. with Wyeth/IL-15/5flu (Vacc A) or Wyeth (Vacc C), and challenged with 1LD50 of H3N2 virus i.n.. (B) Viral loads of day 7 post infection lungs were determined by standard TCID50 on MDCK cells, for individual mouse viral loads and group mean (n = 3). (C) Data represents the fold reduction in viral load compared to negative (Vacc C) controls of the same mouse strain (n = 3), and (D) monitored for survival day 14 (n = 5). Experiment was repeated twice.

Fig. 2.

Adoptive T cell transfer of CD4+ or CD8+ T cells provides challenge for protection, whilst sera shows no protection. (A) Naïve BALB/c mice were given memory splenocytes from vaccinated mice purified by magnetic selection (B) for total CD3+, CD4+ or CD8+ T cells. (A) Mice were given T cells i.v., and immune serum was given i.p. 500 μl on 4 separate days. Mice were then infected with 1LD50 H3N2, lung viral load determined at day 7 (C) (n = 3), and fold reduction in viral load compared to PBS negative controls (D), and monitored for survival to day 14 (E) (n = 5). Experiment was repeated twice. (C) Data represents the individual viral loads and group mean (n = 3), (D) data represents the average viral load reduction compared to PBS negative control mice, and (E) the % survival by day 14 post infection (n = 5).

Fig. 3.

T cell depletion impairs CD4+ T cell help for memory and CD4+ T cells are needed at challenge for protection. (A) Mice were depleted for CD4+ and CD8+ at challenge, CD4+ T cells at challenge or vaccination, or CD8+ T cells at challenge. Prior to infection selected mice were given naïve splenocytes i.v. 9 days after the final depletion. (B) The efficiency of T cell depletion was assessed from the peripheral blood at day 7 and day 30 after the last dose of monoclonal antibody treatment, and % reduction compared to undepleted mice, as shown in representative FACS plots. (C) The % of CD4+ and CD8+ T cells of total DAPI+ cells in the peripheral blood was also assessed at -day 1 prior to challenge (n = 3 pooled). Mice were infected with 1LD50 H3N2, lung viral load determined at day 7 (n = 3) (D), fold reduction in viral load compared to negative controls (E), and monitored for survival day 14 (n = 5) (F). BAL NP147-specific IFN-γ+ CD8+ T cells were enumerated at day 7 post H3N2 infection (data represents n = 3, mean+/−stdev) (G), **p > 0.001 vs Vacc A controls (t-test). Experiment repeated at least twice.

Fig. 4.

The early local antibody response from vaccinated mice is CD4+ T cell dependent. (A) Vaccinated and unvaccinated mice were depleted for CD4+ T cells, and then were infected with 1LD50 H3N2, and the local mediastinal lymph node (mLN) harvested at day 7, for a further 3 day in vitro culture to harvest antibody lymphocyte secretions (ALS). The mLN of 3 mice per group was pooled per group and 1 × 106 cells cultured. The ALS supernatant was tested in triplicate by in ELISA for H3-HA (B) and NP (C) protein, for IgG1, IgM and IgA isotype. Data represents the pooled response of triplicates, mean+/−stdev, #p > 0.05 vs PBS controls (by t-test).

Fig. 5.

Vaccination increases Tfh and Th1 CD4+ T cell phenotype responses. BALB/c mice were vaccinated twice or received PBS, infected with H3N2 1LD50, and day 7 post infection BAL harvested. Cells were stained with a panel of antibodies representing CD4+ T cell phenotype profiles (A), stimulated with MHC-II restricted peptides, NP55 and HA140, for cytokine production of IFN-γ+ and IL-4, and analyzed by flow cytometry. (A) The CD4+ T cell panel was assessed for Tfh (CD4+ CXCR5+), Th1 (CD4+ IFN-γ+), Th2 (CD4+ IL-4+), Th17 (CD4+ IL-17A+) d and Treg (CD4+ CD25+ FoxP3+). Tfh cells were also assessed for PD1 and Bcl6 expression, and Th17 cells for RORγt which was low in the BAL, and not included in the analysis. (B) The cytokine specific and phenotype response was determined for NP55 and HA140 peptides (data represents n = 3, mean+/−stdev), and (C) their proportions. (D) Polyfunctional cytokine production was also assessed for NP55 responses in the BAL from day 7 H3N2 infection, for Th1 (IFN-γ, TNF-α and IL-2) and Th2 (IL-4) cytokines (data represents n = 3, mean+/−stdev). (E) CD4+ T cells gated for single, double and triple cytokine production. Experiment repeated at least twice, *p > 0.01 vs PBS controls (by t-test).

Fig. 6.

Human PBMCs restimulated with Vaccinia A proliferate, expanding influenza-specific CD4+ and CD8+ T effector memory subsets. (A) PBMCs from 5 healthy donors were expanded in vitro after stimulation with UV-inactivated H5N2, Vacc A or Vacc C viruses (MOI4) and pre-incubated with CellTrace Violet. Cells were incubated for 4 or 10 days, in the presence of IL-2 from day 4 to generate T cell lines. (A) On the day of the experiment (day 0), restimulated day 4 or 10 PBMCs and direct ex vivo day 0 PBMCs from matched donors received homologous stimulations or media stimulation (background negative control). (B) representative FACs plots show the gating strategy, live PBMCs were gated CD3+ dump−, CD4+/CD8+, cell trace and IFN-γ+, then IFN-γ+ cells for CCR7 and CD54RA for memory subsets. The # cell number of IFN-γ+ CD4+ T cells (C) and IFN-γ+ CD8+ T cells (D) (dots represents individual values (n = 5), bars represent mean+/−stdev), p > 0.01 vs day 0 responses (by t-test). The proportion memory phenotype, TEM (CCR7− CD45RA−), TEMRA (CCR7− CD45RA+), TCM (CCR7+ CD45RA−), TN (CCR7+ CD45RA+) of day 10 IFN-γ+ CD4+ (E) and CD8+ T cells (F) (mean n = 5). Experiment repeated at least twice.