Key Points

CMV-seropositive older adults have more EMRA and terminally differentiated T cells.

CMV seropositivity does not prevent Ab maintenance after SARS-CoV-2 vaccination.

CMV seropositivity does not impede SARS-CoV-2 vaccine T cell memory recall responses.

Abstract

Chronic infection with human CMV may contribute to poor vaccine efficacy in older adults. We assessed the effects of CMV serostatus on Ab quantity and quality, as well as cellular memory recall responses, after two and three SARS-CoV-2 mRNA vaccine doses, in older adults in assisted living facilities. CMV serostatus did not affect anti-Spike and anti–receptor-binding domain IgG Ab levels, nor neutralization capacity against wild-type or β variants of SARS-CoV-2 several months after vaccination. CMV seropositivity altered T cell expression of senescence-associated markers and increased effector memory re-expressing CD45RA T cell numbers, as has been previously reported; however, this did not impact Spike-specific CD4+ T cell memory recall responses. CMV-seropositive individuals did not have a higher incidence of COVID-19, although prior infection influenced humoral immunity. Therefore, CMV seropositivity may alter T cell composition but does not impede the durability of humoral protection or cellular memory responses after SARS-CoV-2 mRNA vaccination in older adults.

Introduction

Aging is associated with an increased frequency of viral respiratory infections and postinfection sequelae (1), as well as reduced efficacy and longevity of protective immunity after vaccination (2). Early in the COVID-19 pandemic, age was identified to be the most significant factor contributing to morbidity and mortality (3), and it was unclear how effective vaccines against the novel SARS-CoV-2 virus would be in older adults. In this population, vaccines that target a memory response (e.g., herpes zoster) are often more immunogenic and effective at preventing illness than vaccines that target viral Ags that are antigenically distant from previous circulating strains (e.g., some seasonal influenza vaccines) (4–7). SARS-CoV-2 mRNA vaccines have been shown to be protective in older adults (8–10), as they are effective at generating cellular and Ab-mediated immunity (11–14). However, in older adults, immune responses after vaccination are generally quite heterogeneous, and immunity may wane faster than in younger adults (11, 13).

In older adults, both immunosenescence and inflammation are thought to influence the immunogenicity of vaccines and longevity of protective immune responses after vaccination (15, 16). Many studies have also implicated human CMV as a significant contributor to age-associated immune dysfunction. CMV is a common and persistent β-herpesvirus, found in ∼60–90% of adults worldwide, and seropositivity increases with age (17, 18). CMV infection is typically asymptomatic in immunocompetent individuals, but its accompanying chronic immune activation fundamentally alters immune cell composition and function. There is a consensus that CMV seropositivity has a long-term impact on the maturation and composition of immune cells, including increased numbers and prevalence of CD8+ T cells, with expansion of CMV-specific effector and memory cells at the expense of naive T cells (19, 20). Age-associated immunosenescence likewise contributes to similar changes within the T cell repertoire, reducing naive T cells and increasing memory T cell populations, which have impaired proliferation, differentiation, and effector functions (21, 22). A dysfunctional T cell repertoire may also have significant effects on B cell proliferation, differentiation, and maturation (23). Accordingly, CMV seropositivity has been implicated as an exacerbating factor in age-associated immune remodeling and interindividual immune diversity (24–27), as well as a modifying factor that may compromise infection outcomes and quality and longevity of immune protection after vaccination (28).

Although there is a paucity of data to date, CMV reactivation in older adults has been suggested to contribute to more severe COVID-19 (29–33), as CMV seropositivity has been associated with impaired Ab production and cellular memory recall responses (34–36). However, CMV seropositivity has also been reported to enhance cellular and Ab immune responses to unrelated bacteria or viruses, through diversification of CD8+ TCRs and augmentation of basal inflammation (37–40). CMV seropositivity has, in addition, been associated with a reduced humoral response to inactivated split influenza virus vaccines (41–44) and viral vector–based Ebola vaccines (45). However, a recent meta-analysis reported that there was insufficient evidence that CMV-seropositive individuals have decreased Ab production after influenza vaccination (46). Recent data also show that CMV seropositivity in young adults does not affect Ab or cellular responses after vaccination with the adenovirus-based vector vaccine ChAdOx1 nCoV-19 (47). These conflicting reports may suggest context-, time-, and age-dependent effects of chronic CMV infection on immune function. SARS-CoV-2 mRNA vaccines have been widely deployed in Canada, particularly in older adults. Whether CMV seropositivity impacts SARS-CoV-2 mRNA vaccine efficacy and durability of immunity is not yet known. In this study, we investigated effects of CMV serostatus on vaccine-associated humoral protection and cellular memory recall responses several months after two and three doses of SARS-CoV-2 mRNA vaccines in older adults. We found that CMV serostatus does not impede Ab or cellular responses to SARS-CoV-2 vaccination in older adults.

Materials and MethodsParticipant recruitment and blood collection

Participants in the COVID in Long-Term Care Study (https://covidinltc.ca) were recruited from assisted living facilities (17 nursing homes and 8 retirement homes) in Ontario, Canada, between March and December 2021. All protocols were approved by the Hamilton Integrated Research Ethics Board and other site-specific research ethics boards, and informed consent was obtained. Venous blood was drawn in anti-coagulant–free vacutainers for isolation of serum, as per standard protocols (48). Venous blood was drawn in heparin-coated vacutainers for immunophenotyping and T cell activation assays. Blood was collected at least 7 d after two and/or three mRNA vaccine doses. Participants received two doses of Moderna Spikevax (100 µg; mRNA-1273) or Pfizer Comirnaty (30 µg; BNT162b2) as per the recommended schedules, and a third mRNA vaccine dose in Fall 2021 at least 6 mo from the last dose, as per Province of Ontario guidelines (49). For this study, humoral and cellular data were retrospectively assessed in the context of CMV serostatus from a cohort of 186 participants, 65 y of age and older. Blood was drawn after two and three mRNA vaccine doses from 47 cohort participants. Participant cohort demographics are summarized in Table I.

Determination of CMV serostatus

CMV seropositivity was determined by ELISA with a human anti-CMV IgG ELISA kit (CMV) (no. ab108639, Abcam) as per the manufacturer’s instructions. Serum samples from a participant’s first blood draw were diluted 1:40 and assessed in duplicate. Samples with a CMV IgG index above or equal to the positive standard were classified as CMV seropositive, whereas samples with a CMV IgG index less than the positive standard were classified as CMV seronegative.

Whole-blood immunophenotyping

Circulating immune cell populations were quantitated in whole blood using fluorophore-conjugated mAbs by multicolor flow cytometry with a CytoFLEX LX (four lasers, Beckman Coulter), as per standard protocols (48, 50, 51). CountBright absolute counting beads (no. C36950, Invitrogen/Life Technologies) were used to determine absolute cell counts. Data were analyzed with FlowJo v10.8.1 (Tree Star), following a previously published gating strategy to identify T cell populations (50). Five main subsets of human CD8+ and CD4+ T cells (naive [N], central memory [CM], effector memory [EM], EM re-expressing CD45RA [EMRA], and terminally differentiated [TD]) were identified by their expression of CD45RA, CCR7, CD28, and/or CD57. CD8N and CD4N were classified as CD45RA+CCR7+, CD8CM and CD4CM as CD45RA−CCR7+, CD8EM and CD4EM as CD45RA−CCR7−, CD8EMRA and CD4EMRA as CD45RA+CCR7−, and CD8TD and CD4TD were classified as CD45RA+CCR7−CD28−CD57+, as per standard protocols (52).

Assessment of T cell memory responses to SARS-CoV-2 Spike by an activation-induced marker assay

Ag-specific T cell recall responses were evaluated by an activation-induced marker (AIM) assay as per established protocols (50). Each participant sample was stimulated with a Spike glycoprotein SARS-CoV-2 peptide pool (1 μg/ml) containing overlapping peptides of the complete immunodominant sequence domain (no. 130-126-701, Miltenyi Biotec) as well as influenza hemagglutinin (HA) peptides (4 µl of 0.12 μg/µl HA; AgriFlu, Afluria Tetra inactivated influenza vaccine 2020–2021 season, Seqirus, Maidenhead, U.K.). A negative medium control (unstimulated) and positive stimulation control (polyclonal stimulation with CytoStim at 0.5 µl/well; no. 130-092-173, Miltenyi Biotec) were included with each sample. For each of these four conditions, 100 µl of heparinized venous blood was incubated with an equal volume of IMDM with GlutaMAX supplement (no. 31980030, Invitrogen/Life Technologies) for 44 h in 96-well flat bottom plates at 37°C. Samples were stained with fluorophore-conjugated mAbs and assessed with a CytoFLEX LX (4 lasers, Beckman Coulter, Brea, CA) as previously described (50). Data were analyzed with FlowJo, following a previously published gating strategy to identify AIM+ T cells (50). Activated T cells (AIM positive) were identified by their coexpression of CD25 and CD134 (OX40) on CD4+ T cells (53, 54) and coexpression of CD69 and CD137 (4-1BB) on CD8+ T cells (55). Samples with a T cell count of at least 20 events and ≥2-fold above the unstimulated sample (negative control; i.e., stimulation index ≥ 2) were defined as AIM-positive. Expression of CXCR3 (CD183; Brilliant Violet 421, no. 353716, BioLegend), CCR4 (CD194; Brilliant Violet 605, no. 359418, BioLegend), and CCR6 (CD196; Brilliant Violet 785, no. 353422, BioLegend) was used to identify Th1 (CXCR3+CCR6−CCR4−), Th2 (CXCR3−CCR4+CCR6−), and Th17 (CXCR3−CCR4+CCR6+) AIM+CD4+ T cell subsets.

Measurements of anti–SARS-CoV-2 Abs and neutralizing capacity

Serum anti–SARS-CoV-2 Spike protein and receptor-binding domain (RBD) IgG, IgA, and IgM Abs were measured by a validated ELISA as previously described (50, 56), with assay cutoff 3 SD above the mean of a pre-COVID-19 population from the same geographic region. Ab neutralization capacity was assessed by cell culture assays with Vero E6 (ATCC CRL-1586) cells and live SARS-CoV-2, with data reported as geometric microneutralization titer at 50% (MNT50), which ranged from below detection (MNT50 = 5; 1:10 dilution) to MNT50 = 1280 (56). Ab neutralization was measured against the ancestral strain of SARS-CoV-2 and the β variant of concern (B.1.351). The β variant was obtained through BEI Resources (National Institute of Allergy and Infectious Diseases, National Institutes of Health: SARS-related coronavirus 2, isolate hCoV-19/South Africa/KRISP-K005325/2020, NR-54009, contributed by Alex Sigal and Tulio de Oliveira).

Determination of prior SARS-CoV-2 infection

Due to demand for and limits on availability of testing, participants were not consistently tested for COVID-19 by a nasopharyngeal swab PCR assay, even when they were symptomatic. Serological testing for anti-nucleocapsid SARS-CoV-2 IgG and IgA Abs was performed on all collected samples by the ELISA described above (50, 56), using assay wells coated with 2 µg/ml nucleocapsid Ag (Jackson ImmunoResearch Laboratories). Participants were identified to have had COVID-19 when they were seropositive for IgG or IgA anti-nucleocapsid Abs and had a documented positive nasopharyngeal PCR test prior to any blood collection, as summarized in Table I.

Statistical analysis

Statistical analyses were conducted using GraphPad Prism version 9 (GraphPad Software, San Diego, CA). Two-group comparisons of dose and CMV seropositivity or prior COVID-19 and CMV seropositivity were assessed by two-way ANOVA. Differences between CMV-seropositive and CMV-seronegative group Ab levels, Ab neutralization capacity, T cell immunophenotype, and T cell memory recall responses were assessed by a Student t test with Welch’s correction or a Mann–Whitney U test, according to data normality. The p values are reported as two-tailed, and p values <0.05 were considered significant.

ResultsParticipant demographics

Serum anti-CMV IgG Abs were measured by ELISA, and 69.4% (n = 129/186) of participants were found to be CMV seropositive. Age and sex distribution were similar between seropositive (median 85 ± 6.8 y, 67.6% female) and seronegative (median 86 ± 7.7 y, 69.8% female) participants (Table I). Blood samples were collected at a median of 179.8 d (CMV seronegative) and 175.4 d (CMV seronegative) after two doses of Moderna Spikevax (100 µg; mRNA-1273) or Pfizer Comirnaty (30 µg; BNT162b2) administered as per the manufacturer-recommended schedules. In Ontario, Canada, third dose vaccinations were recommended for older adults in congregate living beginning in August 2021 when they were >6 mo after their second vaccinations (57). Participants received third doses in August–September 2021, and blood samples were collected at a median of 82.0 d (CMV seronegative) or 83.7 d (CMV seropositive) after third doses. Participants were classified as having had a previous SARS-CoV-2 infection when they had a documented positive PCR test and/or were seropositive for IgG or IgA nucleocapsid Abs. A positive nasopharyngeal PCR test and/or serum anti-nucleocapsid IgG or IgA Abs were reported in 37.8% of CMV-seronegative participants and 34.4% of CMV-seropositive participants.

Table I.

Participant demographics

CMV seropositivity does not impede anti–SARS-CoV-2 Ab production or neutralization in older adults

To assess the effects of CMV serostatus on Ab responses after SARS-CoV-2 vaccination, serum anti–SARS-CoV-2 Spike and RBD IgG, IgA, and IgM Ab levels were measured by ELISA (Fig. 1, Table II). The number of responders (i.e., individuals with Abs above the threshold limit of detection) significantly increased between post-second and post-third dose measurements of anti-Spike IgG, IgA, and IgM Abs, as well as anti-RBD IgG Abs, but not anti-RBD IgA or IgM Abs (Table II). For example, 5–7 mo after second dose vaccinations, anti-Spike IgG Abs were detected in 88.7% of participants, and anti-RBD IgG Abs were detected in 63.5% of participants. Approximately 3 mo after the third vaccine dose, 97.3 and 94.6% of participants had detectable anti-Spike IgG and anti-RBD IgG, respectively. CMV seropositivity did not impact the frequency of responders for anti-Spike and anti-RBD IgG, IgA, and IgM Abs. Accordingly, two-group analyses showed a main effect of vaccine dose, but not CMV serostatus, on serum anti-Spike IgG (Fig. 1A) and anti-RBD IgG (Fig. 1D) Abs, anti-Spike IgA (Fig. 1B) and anti-RBD IgA (Fig. 1E) Abs, as well as anti-Spike IgM (Fig. 1C) Abs. There were no significant main effects of vaccine dose or CMV serostatus on anti-RBD IgM (Fig. 1F) Abs. Therefore, CMV seropositivity does not impede maintenance of antiviral Abs several months after two or three doses of SARS-CoV-2 mRNA vaccines.

FIGURE 1.

Abs and neutralization capacity by CMV serostatus after two and three COVID-19 vaccines in older adults. Serum SARS-CoV-2 anti-Spike and anti-RBD Abs were detected by ELISA, and Ab neutralization capacity was assessed by MNT50 with live SARS-CoV-2 virus, postdose 2 (PD2), and postdose 3 (PD3) vaccination in CMV-seronegative (−ve) and CMV-seropositive (+ve) individuals. (A–C) Anti-Spike Abs: IgG (A), IgA (B), and IgM (C). (D–F) Anti-RBD Abs: IgG (D), IgA (E), and IgM (F). (G and H) Ab neutralization capacity was assessed against ancestral (G) and β variant (H) SARS-CoV-2. CMV-PD2, n = 51; CMV-PD3, n = 23; CMV+PD2, n = 108; CMV+PD3, n = 51. Dotted lines indicate the threshold of detection. Each data point indicates an individual participant, with the center line at the median. Associations between CMV serostatus and vaccine dose were assessed by two-way ANOVA, with a Tukey’s test post hoc analysis of significant main effects. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Table II.

Responders to vaccination by CMV serostatus: Abs

To examine potential effects of CMV serostatus on Ab function, serum Ab neutralization capacity was assessed by MNT50 assays against live ancestral (wild-type) and β variant SARS-CoV-2 (Fig. 1G, 1H). Vaccines were designed against the wild-type virus, whereas the β variant contains mutations that confer increased transmissibility and immune evasion (58). Neutralization of ancestral and β variant SARS-CoV-2 ranged from below the detection limit to MNT50 = 1280, although mean neutralization was consistently higher against the ancestral virus compared with the β variant after two and three vaccine doses. Neutralization capacity was similar between CMV-seropositive and CMV-seronegative individuals against both ancestral and β variant SARS-CoV-2, although there was a main effect of dose on neutralization capacity. In particular, significant increases in serum Ab neutralization were observed against the β variant between second and third dose vaccinations. Anti-Spike and anti-RBD IgG levels moderately correlated with Ab neutralization capacity, irrespective of CMV serostatus (Table III). As well, modest correlations were generally observed between ancestral SARS-CoV-2 neutralization capacity and anti-Spike and anti-RBD IgA Abs in both CMV-seropositive and CMV-seronegative participants. Therefore, CMV seropositivity does not compromise maintenance of vaccine-elicited Ab neutralization of SARS-CoV-2 several months after two or three vaccine doses.

Table III.

Associations of Ab and neutralization responses after vaccination by CMV serostatus

As serum was collected from some participants after both second and third vaccine doses, these paired data were also assessed independently (Supplemental Fig. 1). Consistent with our pooled participant data, there were main effects of vaccine dose but not CMV serostatus on anti-Spike and anti-RBD IgG and IgA serum Ab levels. We did observe an interaction between CMV serostatus and vaccine dose on anti-RBD IgM measurements by intraindividual analysis, but most participants had Ab levels below the threshold. Intraindividual analyses also showed that the number of vaccine doses, but not CMV serostatus, had a significant effect on Ab neutralization capacity against wild-type and β variant SARS-CoV-2. Therefore, CMV seropositivity does not significantly impact intraindividual changes in Ab levels or neutralization capacity between two and three doses of SARS-CoV-2 mRNA vaccines.

We next considered postdose 2 and postdose 3 vaccination Ab measurements in the context of prior SARS-CoV-2 infection (Fig. 2). As summarized in Table I, the incidence of COVID-19 (prior to blood collections) was similar between CMV-seronegative and CMV-seropositive participants. We observed a main effect of prior SARS-CoV-2 infection on anti-Spike and anti-RBD IgG, IgA, and IgM Abs after two doses of mRNA vaccines, as well as anti-Spike and anti-RBD IgG and IgA, but not IgM, serum Abs after three vaccine doses. We observed an interaction between prior COVID-19 and CMV serostatus for anti-Spike and anti-RBD IgA Abs postdose 2 and postdose 3, and IgM Abs postdose 2. There was a main effect of CMV serostatus on anti-Spike IgM Abs, but most individuals had levels below the detection threshold. We in addition observed main effects of prior SARS-CoV-2 infection, but not CMV serostatus, on Ab neutralization of ancestral SARS-CoV-2 after two and three vaccine doses, and the β variant after two, but not three, vaccine doses. Collectively, these data indicate that CMV serostatus does not appear to have a major impact on the longevity of circulating anti-Spike or anti-RBD IgG Abs, or total serum Ab neutralization capacity, after SARS-CoV-2 infection or vaccination.

FIGURE 2.

Abs and neutralization capacity by CMV serostatus and prior SARS-CoV-2 infection after two and three COVID-19 vaccines in older adults. SARS-CoV-2 anti-Spike and anti-RBD Abs were measured in serum of CMV-seronegative (CMV−) and CMV-seropositive (CMV+) individuals by ELISA, and serum Ab neutralization capacity was assessed by MNT50 with live SARS-CoV-2 virus. Data are stratified by prior SARS-CoV-2 infection history. (A–F) Postdose 2: anti-Spike IgG (A), IgA (B), and IgM (C) Abs, and anti-RBD IgG (D), IgA (E), and IgM (F) Abs. (G–L) Postdose 3: anti-Spike IgG (G), IgA (H) and IgM (I) Abs, and anti-RBD IgG (J), IgA (K), and IgM (L) Abs. (M and N) Postdose 2 SARS-CoV-2 neutralization: ancestral (M) and β variant (N). (O and P) Postdose 3 SARS-CoV-2 neutralization: ancestral (O) and β variant (P). PD2: CMV−No, n = 33; CMV−Yes, n = 18; CMV+No, n = 77; CMV+Yes, n = 31. PD3: CMV−No, n = 13; CMV−Yes, n = 10; CMV+No, n = 27; CMV+Yes, n = 24. Dotted lines indicate the threshold of detection. Each data point indicates an individual participant, with the center line at the median. Associations between CMV serostatus and prior COVID-19 were assessed by two-way ANOVA, with a Tukey’s test post hoc analysis of significant main effects and interactions. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

CMV serostatus influences peripheral CD4+ and CD8+ T cell immunophenotype in older adults

To examine the impact of CMV seropositivity on the T cell repertoire, whole-blood CD4+ and CD8+ T cell composition was quantitated and the surface expression of CD28 and CD57 was measured by flow cytometry (Fig. 3, Supplemental Fig. 2). Chronic T cell activation and an altered T cell repertoire are characteristics of CMV seropositivity (20, 38, 59). Accordingly, CMV-seropositive individuals had significant changes to their peripheral blood T cell composition. We found no changes in numbers of circulating total leukocytes, total CD4+ T cells, or CD4N, CD4EM, CD8N, or CD8EM T cell populations by CMV serostatus. However, CMV seropositivity increased numbers of total CD8+ T cells, as well as CD4EMRA, CD4TD, CD8EMRA, and CD8TD T cells, and decreased numbers of CD4CM and CD8CM T cells.

FIGURE 3.

Effect of CMV serostatus on the circulating T cell repertoire in older adults. T cell populations in whole blood were assessed by flow cytometry in CMV-seronegative (−ve) and CMV-seropositive (+ve) individuals. (A) Relative prevalence of CD4+ and CD8+ T cell subsets by CMV serostatus (also see Supplemental Fig. 2). (B–D) Absolute cell counts of (B) total leukocytes, (C) total CD4+ T cells, and (D) CD8+ T cells. (E–I) Absolute cell counts of CD4+ T cells: (E) naive, (F) central memory, (G) effector memory, (H) EMRA, and (I) terminally differentiated. (J–N) Absolute cell counts of CD8+ T cells: (J) naive, (K) central memory, (L) effector memory, (M) EMRA, and (N) terminally differentiated. (O) Surface geometric mean expression of CD57 and CD28 on CD4+ and CD8+ T cells by CMV serostatus. Blood was assessed either postdose 2 or postdose 3 for each participant. CMV−ve, n = 56; CMV+ve, n = 128. Each data point in (B)–(N) indicates an individual participant, and data are presented as box-and-whisker plots, minimum to maximum, with the center line at the median. The surface marker expression in (O) was visualized by concatenating uncompensated events in FlowJo for each participant and indicated T cell population grouped according to CMV serostatus, and then geometric mean fluorescence intensity expression data of each CMV group were overlaid onto the same histogram plot. Associations between T cell subsets and CMV serostatus were assessed by a Student t test with Welch’s correction or a Mann–Whitney U test, according to normality. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

CD28 is a costimulatory molecule that contributes to TCR Ag–mediated activation of T cells, whereas CD57 is a marker of TD T cells as well as an indicator of immune senescence (60). Repeated T cell activation is associated with upregulation of CD57 and a reduction in CD28 expression (61–63). Consistent with these prior data, comparisons of CD28 and CD57 expression on T cell populations by CMV serostatus in our cohort of older adults (Fig. 3O) revealed increased CD57 expression and reduced CD28 expression on total CD4+ and CD8+ T cell populations, as well as more specifically CD4CM, CD4EM, CD4EMRA, CD8N, and CD8EMRA T cells, in CMV-seropositive individuals. Expression of CD28 was also decreased on CD8CM and CD8EM cells of CMV-seropositive individuals, although their expression of CD57 was not influenced by CMV serostatus. CMV serostatus did not alter CD57 or CD28 expression on CD4N T cells.

As even mild COVD-19 can have lasting effects on immune cell composition (50), we also considered combined effects of prior SARS-CoV-2 infection and CMV serostatus on T cell composition (Fig. 4, Supplemental Fig. 2). Prior SARS-CoV-2 infection was associated with lower total leukocyte counts, and an interaction was observed between CMV serostatus and prior COVID-19 that influenced the numbers of CD8TD cells. Otherwise, there were no significant main effects of prior SARS-CoV-2 infection on absolute cell numbers nor the prevalence of the assessed CD4+ or CD8+ T cell populations.

FIGURE 4.

Effects of CMV serostatus and prior COVID-19 on the circulating T cell repertoire in older adults. T cell populations in whole blood were assessed by flow cytometry in CMV-seronegative (CMV−) and CMV-seropositive (CMV+) individuals. Data are stratified by prior SARS-CoV-2 infection history. (A and B) Absolute cell counts of (A) total leukocytes and (B) total T cells. (C–H) Absolute cell counts of CD4+ T cells: (C) total, (D) naive, (E) central memory, (F) effector memory, (G) EMRA, and (H) terminally differentiated. (I–N) Absolute cell counts of CD8+ T cells: (I) total, (J) naive, (K) central memory, (L) effector memory, (M) EMRA, and (N) terminally differentiated. Blood was assessed either postdose 2 or postdose 3 for each participant. CMV−No, n = 35; CMV−Yes, n = 21; CMV+No, n = 84; CMV+Yes, n = 44. Each data point indicates an individual participant. Data are presented as box-and-whisker plots, minimum to maximum, with the center line at the median. Associations between CMV serostatus and prior COVID-19 were assessed by two-way ANOVA, with a Tukey’s test post hoc analysis of significant main effects and interactions. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

In summary, there are significant changes to the relative composition and phenotype of peripheral blood CD8+ T cell and CD4+ T cell subsets between CMV-seronegative and CMV-seropositive older adults, irrespective of prior COVID-19. The observed expansion of EMRA and TD T cells, as well as reduced surface expression of the costimulatory molecule CD28 on CD8N T cells in particular, may influence vaccine-specific T cell responses.

CMV serostatus influences CD4+ and CD8+ T cell SARS-CoV-2 Ag-induced recall responses in older adults

An AIM assay was used to examine T cell memory responses by stimulation with the SARS-CoV-2 Spike Ag after second and third dose SARS-CoV-2 mRNA vaccinations (Fig. 5, Table IV). SARS-CoV-2 vaccines are unusual in that healthy adults generate strong CD4+ T cell memory recall responses, but weaker CD8+ T cell memory responses (64). We also made similar observations in older adults. Most study participants had CD4+ T cell responses to SARS-CoV-2 Spike (postdose 2, 93.1%; postdose 3, 95.9%), but only 19.5% of participants had Spike-elicited CD8+ memory T cell responses after two vaccine doses, although this increased to 29.7% of participants after three vaccine doses (Table IV). CMV serostatus did not influence the number of individuals with SARS-CoV-2 Spike-AIM+CD4+ T cells or Spike-AIM+CD8+ T cells after second or third dose vaccinations. Grouped analyses revealed a significant main effect of CMV serostatus on the frequency of Spike-AIM+CD8+ T cells (Fig. 5A). However, despite greater variance of data in CMV-seropositive individuals, post hoc analyses by CMV serostatus were not significant postdose 2 or postdose 3. Intraindividual paired analyses also showed no main effects of vaccine dose or CMV serostatus on activation of Spike-AIM+CD8+ T cells (Supplemental Fig. 3A). Prior COVID-19 did not influence the prevalence of Spike-AIM+CD8+ T cells after two or three vaccine doses, although CMV seropositivity contributed to increased Spike-AIM+CD8+ T cell activation postdose 2 (Fig. 6). Grouped analyses on a population and intraindividual basis showed a main effect of vaccine dose (Fig. 5B, Supplemental Fig. 3B), but the prevalence of Spike-AIM+CD4+ T cells was likewise not different by CMV serostatus (Fig. 5B) or by prior COVID-19 (Fig. 6B, 6G). Therefore, CMV serostatus contributes to increased CD8+ T cell, but not CD4+ T cell, memory recall responses to the SARS-CoV-2 Spike protein several months after second and third dose vaccinations.

FIGURE 5.

CD4+ and CD8+ T cell AIM responses to SARS-CoV-2 Spike in older adults. T cell memory responses to SARS-CoV-2 Spike were assessed by AIM assay in CMV-seronegative (CMV−) and CMV-seropositive (CMV+) individuals postdose 2 (PD2) and postdose 3 (PD3) SARS-CoV-2 vaccination. (A) AIM+CD8+ T cells (expressing CD69 and CD137) were measured as a proportion of total CD8+ T cells. (B) AIM+CD4+ T cells (expressing CD25 and OX40) were measured as a proportion of total CD4+ T cells. (C–E) AIM+CD4+ T cell Th1 (C), Th2 (D), and Th17 (E) subsets. Each data point indicates an individual participant. For (A) and (B), data from all individuals are graphed irrespective of whether they meet cutoff requirements for a “positive” result. For (C)–(E), only data from individuals with positive Spike-AIM+CD4+ T cell memory recall responses were graphed (Table IV). CMV−PD2, n = 51; CMV−PD3, n = 23; CMV+PD2, n = 108; CMV+PD3, n = 51. Data are presented as box-and-whisker plots, minimum to maximum, with the center line at the median. Associations between CMV serostatus and vaccine dose were assessed by two-way ANOVA, with a Tukey’s test post hoc analysis of significant main effects. *p < 0.05, ***p < 0.001.