Key Determinants of Cell-Mediated Immune Responses: A Randomized Trial of High Dose Vs. Standard Dose Split-Virus Influenza Vaccine in Older Adults

Janet E McElhaney, Chris P Verschoor, Laura Haynes, Graham Pawelec, Mark Loeb, Melissa K Andrew, George A Kuchel, Janet E McElhaney, Chris P Verschoor, Laura Haynes, Graham Pawelec, Mark Loeb, Melissa K Andrew, George A Kuchel

Abstract

Background: Efforts to improve influenza vaccine effectiveness in older adults have resulted in some successes, such as the introduction of high-dose split-virus influenza vaccine (HD-SVV), yet studies of cell-mediated immune responses to these vaccines remain limited. We have shown that the activity of the cytolytic mediator, granzyme B (GrB), in peripheral blood mononuclear cells (PBMC) challenged with influenza A/H3N2 virus, correlates with protection against influenza following standard dose vaccination (SD-SVV) in older adults. Further, the interferon-γ (IFNγ) to interleukin-10 (IL-10) ratio can be a correlate of protection depending on the timing of vaccination relative to exposure to influenza.

Methods: In a double-blind trial (ClinicalTrials.gov NCT02297542) older adults (≥65 years, n=582) were randomized to receive SD-SVV or HD-SVV (Fluzone®) from 2014/15 to 2017/18. Young adults (20-40 years, n=79) received SD-SVV. At 0, 4, 10 and 20 weeks post-vaccination, serum antibody titers, IFNγ, IL-10, and inducible GrB (iGrB) were measured in ex vivo influenza virus-challenged PBMC. iGrB is defined as the fold change in GrB activity from baseline levels (bGrB) in circulating T cells. Responses of older adults were compared to younger controls, while specifically for older adults we analyzed effects of age, sex, cytomegalovirus (CMV) serostatus, frailty, and vaccine dose.

Results: Prior to vaccination, younger adults produced significantly higher IFNγ, IL-10 and iGrB levels, but with no difference in the IFNγ:IL-10 ratio. Relative to SD-SVV recipients, older HD-SVV recipients exhibited significantly lower IFNγ:IL-10 ratios at 4 weeks post-vaccination. In contrast, IFNγ and iGrB levels were higher in younger SD vs. older SD or HD recipients; only the HD group showed a significant IFNγ response to vaccination compared to the SD groups while all three groups showed a significant iGrB response to vaccination. In a regression analysis, female sex and HD-SVV were associated with higher IL-10 levels, while SD-SVV was associated with lower iGrB levels. Prior season influenza vaccination showed a decline in iGrB levels but an increase in IFNγ and IL-10 levels, which correlated with influenza A/H3N2 hemagglutination inhibition antibody titers.

Conclusions: Overall, HD-SVV amplified the IL-10 response consistent with enhanced antibody responses, with little effect on the iGrB response relative to SD-SVV in either younger or older adults. These results suggest that enhanced protection with HD-SVV is largely antibody-mediated.

Keywords: cell-mediated immune responses; cytomegalovirus; frailty index; granzyme B; interferon-gamma; interleukin-10; older adults; split-virus influenza vaccine.

Conflict of interest statement

JEM reports payments to her institution for her participation in advisory, scientific, or data safety and monitoring boards from Sanofi, GSK, Pfizer, Merck, ResTORbio, and Medicago, outside the submitted work and as a site lead for clinical trials sponsored by VBI and Jansen, unrelated to the current study. ML reports a grant from Seqirus and honoraria for advisory activities from Seqirus, Sanofi, Medicago, and Pfizer. MA reports grants from Sanofi, GSK, Pfizer and the Canadian Frailty Network for work unrelated to the current study, and honoraria for advisory activities from Sanofi, Seqirus and Pfizer. GK reports honoraria from ResTORbio, Janssen Pharmaceuticals and Spring Discovery.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Cell-mediated immune (CMI) responses to live A/H3N2 virus in ex vivo stimulated PBMCs for the four study time points (in weeks). IFNγ (A), IL-10 (B), IFNγ:IL-10 ratio (C), and inducible GrB (iGrB levels) (D), and basal GrB (bGrB) (E) are presented for young (standard dose; red squares, dashed line) and older (standard dose = blue squares, solid line; high dose = green circles, solid line) adults. The concentration of IFNγ, IL-10 and bGrB are presented as the geometric mean and 95% confidence interval, while the natural log(Ln) IFNγ:IL-10 ratio, and iGrB are presented as the mean and 95% confidence interval.
FIGURE 2
FIGURE 2
The fold change from pre-to 4 weeks post-vaccination in measures of the cell-mediated immune response to live A/H3N2 virus in ex vivo stimulated PBMCs for IFNγ, IL-10, IFNγ:IL-10, and iGrB. The mean and 95% confidence interval of the natural log(Ln) fold-change is presented for young (standard dose; red squares) and older (standard dose = blue squares; high dose = green circles) adults; the red dashed line (fold-change = 0) indicates no response to the vaccine.
FIGURE 3
FIGURE 3
Factors associated with the of cell-mediated immune (CMI) response to vaccination (A) The effect of different characteristics of older adult vaccinees are shown for each of the CMI measures in an unadjusted (red dots) and adjusted (blue dots) linear regression analysis, and includes frailty as a categorical variable. For (B), the association between each CMI measure and frailty as standardized continuous variable in both unadjusted and adjusted analyses is presented. The regression coefficient and 95% confidence interval is presented, which represents a 1-standard deviation (SD) change in each measure for a given change in a participant factor, [ie. relative to the reference (ref), or per SD change]. Points and error bars above the red, dashed line indicate a significant positive correlation with a CMI measure, whereas those below the red, dashed line indicate a significant inverse correlation. Note that due to the scale of the y-axis, certain confidence intervals are not visible beyond the point estimate.
FIGURE 4
FIGURE 4
The trend in cell-mediated immune (CMI) responses to live A/H3N2 virus and hemagglutination inhibition (HAI) antibody titers over the course of the study in older adults. For the absolute CMI/HAI measures (A) and fold-change in HAI/CMI measures from pre-to 4 weeks post-vaccination (B), the estimated marginal mean and 95% confidence interval of the standardized value (mean = 0, standard deviation = 1) in each year, which was derived from a linear mixed model accounting for repeated measures within participant, is presented on the y-axis. The slope (β) and significance (asterisks) of the regression coefficient for year in these models is presented along the top of each plot. ***p < 0.001; **p < 0.01; *p < 0.05.

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