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

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 granzyme B (GrB) activity in influenza A/H3N2 challenged peripheral blood mononuclear cells (PBMC) 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.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-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, and in older adults, we analyzed effects of age, sex, cytomegalovirus (CMV) serostatus, frailty, and vaccine dose.Results: Prior to vaccination, younger compared to older adults produced significantly higher IFNγ, IL-10, and iGrB levels. 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; all three groups showed a significant iGrB response to vaccination. In a regression analysis, frailty was associated with lower IFNγ levels, whereas female sex and HD-SVV with higher IL-10 levels. Age and SD-SVV were associated with lower iGrB levels. The effect of prior season influenza vaccination was decreased iGrB levels, and increased IFNγ and IL-10 levels, which correlated with influenza A/H3N2 hemagglutination inhibition antibody titers.Conclusion: 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.Clinical Trial Registration: ClinicalTrials.gov (NCT02297542).

Granzyme B: A Double-Edged Sword in the Response to Influenza Infection in Vaccinated Older Adults

“…Following our discovery of GrB activity in circulating T cells, we found that this bGrB activity increased with age, frailty (Verschoor et al, 2021b) and cytomegalovirus (CMV) seropositivity, and correlated with the frequency of late/ terminally-differentiated, potentially senescent, CD8 + T cells (Haq et al, 2017;McElhaney et al, 2020). A recent study showed that CMV seropositivity in older adults was associated with 1) an increased frequency of CD8 + T cells expressing senescence-associated cell surface markers; 2) lower frequencies of influenza-specific memory T cells; and 3) an enhanced inflammatory response to influenza infection, but no difference in the memory T cell response to influenza infection when compared to seronegative individuals (van den Berg et al, 2021).…”

“…We have also calculated the amount of GrB activity induced with ex vivo stimulation (iGrB) as log (iGrB) log (ex vivo GrB)-log (bGrB) according to our published results from this clinical trial, which showed an age-related decline in iGrB levels in older compared to young adults included as a control group (Verschoor et al, 2021b). Although there were too few LCII cases to determine differences in iGrB levels in H3N2-LCII and No-LCII at 4-weeks post-vaccination, we did observe a poor iGrB response to vaccination in the H3N2-LCII subset but a significant increase in iGrB levels following H3N2 infection (Figure 4), consistent with re-stimulation of CTL memory following infection.…”

“…Further, the decline in vaccine effectiveness with age corresponds to a decreased frequency of GrB + Perf + CD8 + T cells and related cytolytic activity following influenza vaccination in older compared to young adults (Zhou and McElhaney, 2011;Zhou et al, 2016). We then discovered that overall GrB activity in circulating T cells of older adults was substantial and that the level of this "basal" GrB (bGrB) activity: 1) increases with age and in frail compared to non-frail older adults (Verschoor et al, 2021b); 2) is inversely proportional to the frequency of memory CD8 + T cells; and 3) is proportional to the frequency of circulating lateor terminally-differentiated CD8 + T cells some of which may be truly senescent (Haq et al, 2017); our unpublished data has shown a similar correlation between bGrB activity and the frequency of circulating CD8 + T cells that express GrB in the absence of Perf. Further, upon ex vivo infection with influenza A/H3N2 virus, ∼50% of the total CD8 + T cell population express GrB in the absence of Perf (Kumar et al, 2017) and can play an important role in immune pathology resulting in the loss of tissue function and integrity (Choy et al, 2004;Tschopp et al, 2006;Granville, 2010;Hendel et al, 2010;Russo et al, 2018;Turner et al, 2019;Matsubara et al, 2020).…”

“…In this 4-year randomized trial, we tested the ability of our standard operating procedures for ex vivo stimulation of PBMC with standardized preparations of live influenza virus and a validated assay of GrB activity, to predict risk for LCII in vaccinated older adults. We have previously shown that bGrB activity is increased in older compared to young adults and that the fold-increase in GrB activity above this baseline (bGrB) activity, induced with ex vivo challenge is diminished in older compared to young adults (Verschoor et al, 2021b). In this study, we explored bGrB activity pre-vaccination and GrB activity in ex vivo virus-challenged PBMC at 4-weeks post-vaccination as predictors of a subsequent LCII, and how ex vivo stimulated GrB activity changed following LCII at 20-weeks postvaccination.…”

Markers of systemic inflammation are positively associated with influenza vaccine antibody responses with a possible role for ILT2(+)CD57(+) NK-cells

“…This is also true with respect to acute care and outcomes related to SARS-COV-2 infection [ 6 ], and for certain symptoms of what is commonly referred to as “long-COVID” [ 7 , 8 ]. As observed with SARS-COV-2 [ 9 ], antibody [ 10 ] and cell-mediated [ 11 ] responses to the seasonal influenza vaccine wane with age, as does the effectiveness to prevent hospitalization, although this varies depending on subtype, season, and overall health status [ 12 – 14 ]. Of the three major influenza (sub)types included in the seasonal vaccine, A/H3N2 breakthrough tends to result in more hospitalizations [ 15 ] and mortality [ 16 ] in older adults, partly due to limited vaccine-induced antibody production [ 17 , 18 ], and especially in years of vaccine mismatch [ 19 ].…”