Background Vaccines and vaccine boosting have blunted excess morbidity and mortality from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in older nursing home residents (NHR). However, the impact of repeated vaccination on the T-cell response based on biological sex and prior infection of NHR remain understudied.Methods We examined T-cell responses to SARS-CoV-2 mRNA vaccines in a cohort of NHR and healthcare workers (HCW) over 2 years. We used interferon-gamma ELIspot and flow cytometry to assess T-cell response before, 2 weeks, and 6 months after the initial series and each of 2 booster vaccines. We analyzed these data longitudinally with mixed-effect modeling and also examined subsets of our cohorts for additional changes in T-cell effector function.Results Prior SARS-CoV-2 infection and female sex contributed to higher T-cell response in NHR but not HCW. When looking across time points, NHR but not HCW with prior infection had significantly higher T-cell responses than infection-naive subjects. These patterns of response were maintained across multiple booster vaccinations.Conclusions These results suggest that the age, multimorbidity, and/or frailty of the NHR cohort may accentuate sex and infection status differences in T-cell response to mRNA vaccination. T-cells responses in nursing home residents (NHR) and health care workers (HCW) after SARS-CoV-2 mRNA vaccination show that both groups have vaccine-induced responses, but prior infection and female sex contribute to higher responses in NHR but not HCW.

Background: The adjuvanted recombinant zoster vaccine (RZV) is highly effective even in adults over 80 years old. The high efficacy of RZV is attributed to its highly reactogenic adjuvant, AS01, but limited studies have been done on AS01's activation of human immune cells. Methods: We stimulated peripheral blood mononuclear cells (PBMC) with AS01 and used flow cytometry and RNA Sequencing (RNAseq) to analyze the impacts on human primary cells. Results: We found that incubation of PBMC with AS01 activated monocytes to a greater extent than any other cell population, including dendritic cells. Both classical and non-classical monocytes demonstrated this activation. RNASeq showed that TNF-alpha and IL1R pathways were highly upregulated in response to AS01 exposure, even in older adults. Conclusions: In a PBMC co-culture, AS01 strongly activates human monocytes to upregulate costimulation markers and induce cytokines that mediate systemic inflammation. Understanding AS01's impacts on human cells opens possibilities to further address the reduced vaccine response associated with aging.

Background Despite wide use of adjuvanted influenza vaccine in nursing home residents (NHR), little immunogenicity data exist for this population. Methods We collected blood from NHR (n = 85) living in nursing homes participating in a cluster randomized clinical trial comparing MF59-adjuvanted trivalent inactivated influenza vaccine (aTIV) with nonadjuvanted vaccine (TIV) (parent trial, NCT02882100). NHR received either vaccine during the 2016-2017 influenza season. We assessed cellular and humoral immunity using flow cytometry and hemagglutinin inhibition, antineuraminidase (enzyme-linked lectin assay), and microneutralization assays. Results Both vaccines were similarly immunogenic and induced antigen-specific antibodies and T cells, but aTIV specifically induced significantly larger 28 days after vaccination (D28) titers against A/H3N2 neuraminidase than TIV. Conclusions NHRs respond immunologically to TIV and aTIV. From these data, the larger aTIV-induced antineuraminidase response at D28 may help explain the increased clinical protection observed in the parent clinical trial for aTIV over TIV in NHR during the A/H3N2-dominant 2016-2017 influenza season. Additionally, a decline back to prevaccination titers at 6 months after vaccination emphasizes the importance of annual vaccination against influenza. We find that standard and adjuvanted influenza vaccines both induce increases in humoral and cellular immunity that contract to baseline by D180. In the 2016-2017 influenza season, the primary difference between vaccines was humoral antineuraminidase immunity to A/H3N2.

Abstract Background In a study initiated in 2020, we recruited nursing home (NH) residents and collected blood samples serially after SARS-CoV-2 vaccinations. While sex differences in vaccine-induced immune response have been previously observed, similar vaccine efficacy across sex groups was reported for Moderna and Pfizer mRNA vaccines. Sex differences in immune response may diminish in very late life, so we sought to compare immune response to mRNA primary series and monovalent and bivalent boosters in NH residents, a frail elderly population. Methods We analyzed anti-Spike antibodies and neutralizing titers to Wuhan strain for sex differences in immune response following primary series and subsequent booster doses. We summarized demographics and infection history of NH subjects with available sample data at each vaccine dose. Omicron BA.5 assays were compared for the second monovalent and bivalent boosters. Comparisons were stratified by prior infection at the time of sample. Samples collected about breakthrough infections were excluded. Geometric mean titers were calculated for each post-vaccine time and compared across sex groups using t-tests on log-transformed titers. Results Despite new enrollment and loss to follow up, our cohort retained a sex balance ranging from 39% women, 61% men to an even split across four post-vaccine timepoints. Men and women had similarly advanced age and rates of prior COVID-19 over time (Table). Following the primary series, women with prior infection had significantly higher anti-Spike antibodies and higher neutralizing titers than men with prior infection (Figure 1). Following both monovalent booster doses and a bivalent booster dose, no sex differences were detected in these endpoints among those with prior infection. Neither were differences observed in infection naive subjects nor in titers for the Omicron BA.5 strain (Figure 2).Table 1:Demographics of NH residents with available samples by vaccine doseFigure 1:Post-vaccine Wuhan anti-Spike antibodies and neutralizing titers Post-vaccine Wuhan anti-Spike antibodies and neutralizing titers by vaccine dose and sex, with bars showing geometric mean titers and 95% confidence intervals. Significant differences in GMT indicated with * at p < 0.05.Figure 2:Post-vaccine Omicron BA.5 anti-Spike antibodies and neutralizing titers Post-vaccine Omicron BA.5 anti-Spike antibodies and neutralizing titers by vaccine dose and sex, with bars showing geometric mean titers and 95% confidence intervals. No sex differences detected. Conclusion Sex differences observed among NH residents with prior infection following primary series mRNA vaccination diminished with additional vaccine doses. Within NH residents, this may be a result of sex differences in attrition among the lowest responders. Further study is required to assess sex differences in the durability of immune response between doses and in T-cell response. Disclosures Stefan Gravenstein, MD, MPH, CDC: Grant/Research Support|Genentech: Advisor/Consultant|Genentech: Grant/Research Support|GSK: Advisor/Consultant|GSK: Honoraria|Janssen: Advisor/Consultant|Janssen: Honoraria|NIH: Grant/Research Support|Pfizer: Grant/Research Support|Pfizer: Honoraria|Sanofi: Advisor/Consultant|Sanofi: Grant/Research Support|Sanofi: Honoraria|Seqirus: Grant/Research Support|Seqirus: Honoraria David Canaday, MD, Pfizer: Grant/Research Support

Background: CD4+ T cells are a critical component of effective immune responses to varicella zoster virus (VZV), but their functional properties during the reactivation acute vs latent phase of infection remain poorly defined. Methods: Here we assessed the functional and transcriptomic properties of peripheral blood CD4+ T cells in persons with acute herpes zoster (HZ) compared to those with a prior history of HZ infection using multicolor flow cytometry and RNA sequencing. Results: We found significant differences between the polyfunctionality of VZV-specific total memory, effector memory, and central memory CD4+ T cells in acute vs prior HZ. VZV-specific CD4+ memory T-cell responses in acute HZ reactivation had higher frequencies of IFN-γ and IL-2 producing cells compared to those with prior HZ. In addition, cytotoxic markers were higher in VZV-specific CD4+ T cells than non-VZV-specific cells. Transcriptomic analysis of ex vivo total memory CD4+ T cells from these individuals showed differential regulation of T-cell survival and differentiation pathways, including TCR, cytotoxic T lymphocytes (CTL), T helper, inflammation, and MTOR signaling pathways. These gene signatures correlated with the frequency of IFN-γ and IL-2 producing cells responding to VZV. Conclusions: In summary, VZV-specific CD4+ T cells from acute HZ individuals had unique functional and transcriptomic features, and VZV-specific CD4+ T cells as a group had a higher expression of cytotoxic molecules including Perforin, Granzyme-B, and CD107a.

Vaccines are less effective in older adults, and efforts to improve vaccine efficacy generally show limited clinical improvement. However, the vaccine Shingrix, developed by GlaxoSmithKline, is FDA approved and recommended for adults over the age of 50 for the prevention of shingles. Shingrix contains the novel adjuvant AS01 and shows remarkably high (>90%) effectiveness at both preventing shingles and reducing the development of postherpetic neuralgia, even in adults over 80. We seek to examine AS01-induced activation of myeloid cells and determine their impacts on T cell memory differentiation and function in an effort to understand how this vaccine’s high clinical efficacy is produced. We use peripheral blood samples to examine AS01-induced innate immune cell activation, including interactions with T cells once activated. We find that AS01 induces costimulatory marker upregulation and cytokine production in myeloid dendritic cells and monocytes in a synergistic manner, with the most upregulation seen in monocytes. Naïve and memory CD4 T cells incubated with AS01-treated monocytes have increased frequencies of TH17 (CCR6+ CXCR3−) cells and decreased frequencies of TH1 cells compared to controls. Naïve CD4 T cells proliferate more when incubated with AS01-treated monocytes than with controls. AS01-treated monocytes additionally induce 2x less IFN-γ and IL-5 in response to mitogen (SEB) from CD4 T cells, suggesting a modulation of response. We propose that AS01 influences monocytes to diversify the existing memory T cell repertoire and skews naïve T cells towards more cytotoxic phenotypes such as TH17, contributing to greater clinical efficacy in preventing shingles in vivo. Supported by VA Merit grant (CX002060)

Background: Seasonal influenza vaccines approved and offered in the United States have varying reported degrees of effectiveness year over year and between manufacturers. Influenza vaccines produced from live virus may include single stranded RNA (ssRNA) that is a potent activator of the innate Toll-like receptor 7 (TLR-7) ligand. Plasmacytoid dendritic cells (pDC) can be activated by ssRNA to produce type I interferons such as IFN-alpha, which has been shown to have an adjuvant-like effect. Objective: Our aim was to determine if IFN-alpha induction in peripheral blood mononuclear cells (PBMCs) exposed to eight different commercial influenza vaccines is a pDC-dependent process mediated through TLR-7 signaling. Results: We demonstrate the ability of multiple vaccines to induce IFN-alpha in a TLR-7-dependent fashion. A number of vaccines however lacked IFN-alpha induction. The significance of these differences between vaccines is unclear, since all the approved vaccine formulations offer some degree of protection. Published by Elsevier Ltd.

Recently defined specialized subset of Treg expressing CXCR5+PD1+FoxP3+ termed as T follicular regulatory (Tfr) cells regulates T follicular helper (Tfh) mediated B cell high-affinity and isotype-switched antibody production. To date, much of our understanding of the suppression mechanism of Tfr cells has been obtained from animal studies. The mechanism of Tfr regulation of humoral immunity in humans is still understudied. Here, we used cells from human mesenteric lymph nodes to study how Tfr affect Tfh and B cells and what specific Tfr-induced changes occur on Tfh and B cells in vitro. We report that Tfr suppress both Tfh and B cell function and proliferation in a dose dependent manner. We find that Tfr downregulate CD40, CD80/CD86 and CD38 on B cells and CD28, OX40, ICOS and CD38 on Tfh cells. We also show that blocking immune checkpoint receptors inhibits Tfr suppression of Tfh and B cells and partially restores the Tfh mediated B cell antibody production. These findings reveal that Tfr regulate activation of Tfh and B cells by downregulating key costimulatory receptors and ligands that help induce activation in both Tfh and B cells. These changes ultimately lead to decreased proliferation of both B cells and Tfh and reduced production of immunoglobulin by B cells. This research was supported in part by NIH R01 and VA.