key: cord-0302118-uusc2gle authors: Breznik, J. A.; Huynh, A.; Zhang, A.; Bilaver, L.; Bhakta, H.; Stacey, H. D.; Ang, J. C.; Bramson, J. L.; Nazy, I.; Miller, M. S.; Denburg, J.; Costa, A. P.; Bowdish, D. M. E.; Group, COVID-in-LTC Study title: CMV seropositivity in older adults changes T cell functionality, but does not prevent antibody or cellular SARS-CoV-2 vaccine responses date: 2022-05-28 journal: nan DOI: 10.1101/2022.05.27.22275673 sha: e948eb647d26a5dc5d27f1061e1c25e4899ef467 doc_id: 302118 cord_uid: uusc2gle It has been previously reported that chronic infection with human cytomegalovirus (CMV) may contribute to poor vaccine responses against de novo antigens in older adults. We assessed effects of CMV serostatus on antibody quantity and quality, as well as cellular memory responses, after 2 and 3 SARS-CoV-2 mRNA vaccine doses, in older adults in congregate living facilities. CMV serostatus did not affect anti-Spike and anti-RBD IgG antibody levels, nor neutralization capacity against wildtype or beta variants of SARS-CoV-2. CMV seropositivity altered T cell expression of senescence-associated markers and increased TEMRA cell numbers, as has been previously reported; however, this did not impact the Spike-specific CD4+ T cell memory responses. CMV seropositive individuals did not have a higher incidence of COVID-19, though prior infection influenced humoral immunity. Therefore, CMV seropositivity may alter T cell composition but does not impede humoral or cellular memory responses after SARS-CoV-2 mRNA vaccination in older adults. Aging is associated with an increased frequency of viral respiratory infections and post-81 infection sequelae 1 , as well as reduced vaccine efficacy 2 . Early in the COVID-19 pandemic, age 82 was identified to be the most significant factor contributing to morbidity and mortality 3 , and it 83 was unclear how effective vaccines against the de novo SARS-CoV-2 virus would be in older 84 adults. Older adults often respond well to vaccines that target a memory response (e.g. Herpes 85 zoster), but vaccines against de novo antigens or antigens that are antigenically distant from 86 previous strains (e.g. some seasonal influenza vaccines) are often less immunogenic 4, 5 . Fortunately, SARS-CoV-2 mRNA vaccines have been shown to be protective in older adults 6, 7, 8 , 88 as they are effective at generating cellular and antibody-mediated immunity 9, 10, 11, 12 , though 89 responses are generally more heterogeneous and wane faster than in younger adults 9, 11 . In older adults, both immunosenescence and inflammation are thought to influence the 92 immunogenicity and longevity of vaccine responses 13, 14 . Many studies have also implicated 93 human cytomegalovirus (CMV) as a significant contributor to age-associated immune 94 dysfunction. CMV is a common and persistent β-herpesvirus, found in ~60-90% of adults 95 worldwide, and seropositivity increases with age 15, 16 . CMV infection is typically asymptomatic 96 in immunocompetent individuals, but its accompanying chronic immune activation 97 fundamentally alters immune cell composition and function. There is a consensus that CMV 98 Whole Blood Immunophenotyping 149 Circulating immune cell populations were quantitated in whole blood using fluorophore-150 conjugated monoclonal antibodies by multicolour flow cytometry with a CytoFLEX LX (4 laser, 151 Beckman Coulter), as per standard protocols 46, 47, 48 . Count-Bright™ absolute counting beads 152 (Invitrogen Life Technologies, Carlsbad, CA, USA) were used to determine absolute cell counts. were identified by their expression of CD45RA, CCR7, CD28 and/or CD57. CD8N and CD4N 157 were classified as CD45RA + CCR7 + ; CD8CM and CD4CM as CD45RA -CCR7 + ; CD8EM and 158 CD4EM as CD45RA -CCR7 -, CD8EMRA and CD4EMRA as CD45RA + CCR7 -; and CD8TD and CD4TD 159 as CD45RA + CCR7 -CD28 -CD57 + , as per standard protocols 68 . 162 Antigen-specific T cell recall responses were assessed as per established protocols 47 using a CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 28, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 plates at 37 °C. Samples were stained with fluorophore-conjugated monoclonal antibodies and 173 assessed with a CytoFLEX LX (4 laser, Beckman Coulter, Brea, CA, USA) as previously 174 described 47 . Data was analyzed with FlowJo V10.1 (TreeStar, Inc.) as previously published 47 . Antigen-specific T cells (AIM-positive) were identified by co-expression of CD25 and CD134 176 (OX40) on CD4 + T cells 49, 50 , and co-expression of CD69 and CD137 (4-1BB) on CD8 + T cells 51 . Samples with a T cell frequency of at least 20 events and >2-fold above an unstimulated sample 178 (negative control; i.e. stimulation index >2), were defined as AIM-positive. Expression of 179 CXCR3, CCR4 and/or CCR6 was used to identify CD4 + Th1 (CXCR3 + CCR6 -CCR4 -), Th2 180 (CXCR3 -CCR4 + CCR6 -), and Th17 (CXCR3 -CCR4 + CCR6 + ) CD4 + T cell subsets. Serum anti-SARS-CoV-2 spike (S) protein and receptor binding domain (RBD) IgG, IgA and 184 IgM antibodies were measured by a validated ELISA as previously described 47, 52 , with assay 185 cut-off 3 standard deviations above the mean of a pre-COVID-19 population from the same 186 geographic region. Data are reported as a ratio of observed optical density (OD) to the 187 determined assay cut-off OD. Antibody neutralization capacity was assessed by cell culture 188 assays with Vero E6 (ATCC CRL-1586) cells and live SARS-CoV-2, with data reported as 189 geometric microneutralization titers at 50% (MNT50), which ranged from below detection 190 (MNT50 = 5; 1:10 dilution) to MNT50 = 1280 52 . Antibody neutralization was measured against 191 the ancestral strain of SARS-CoV-2 and the beta variant of concern (B.1.351). The beta variant 192 was obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate hCoV-193 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) August 2021, if they were more than 6 months post-2 nd vaccinations 53 . Participants received 3 rd 217 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 28, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 225 in older adults 226 To assess effects of CMV serostatus on antibody responses after SARS-CoV-2 227 vaccination, serum anti-SARS-CoV-2 Spike and RBD IgG, IgA, and IgM antibody levels were 228 measured by ELISA ( Figure 1 ; Table 2 ). The number of responders (i.e. individuals with 229 antibodies above the threshold limit of detection) significantly increased between post-2 nd and Spike and anti-RBD IgG, IgA, and IgM antibodies. Accordingly, two-group analyses showed a 237 main effect of vaccine dose, but not CMV serostatus, on serum anti-Spike IgG ( Figure 1A ) and 238 anti-RBD ( Figure 1D ) antibodies, as well as anti-Spike IgA ( Figure 1B ) and anti-Spike IgM 239 ( Figure 1C) antibodies. There were no significant main effects of vaccine dose or CMV 240 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. As serum was collected from some participants after both 2 nd and 3 rd vaccine doses, these 262 paired data were also assessed independently (Supplementary Figure 1) . Consistent with our 263 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Figure 3) . Collectively, these 284 data indicate that CMV serostatus does not appear to have a major impact on anti-Spike or anti-285 RBD IgG antibody production, or total serum antibody neutralization capacity. Moreover, CMV 286 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 28, 2022. ; https://doi.org/10.1101/2022.05.27.22275673 doi: medRxiv preprint seropositivity in the older adult cohort did not impair IgG antibody production or neutralization 287 in response to SARS-CoV-2 vaccination. 290 To examine the impact of CMV seropositivity on the T cell repertoire, whole blood CD4 + 291 and CD8 + T cell composition was quantitated, and their surface expression of CD28 and CD57 CD28 is a co-stimulatory molecule that contributes to TCR-antigen-mediated activation 301 of T cells, while CD57 is a marker of terminally differentiated T cells as well as an indicator of 302 immune senescence 56 . Repeated T cell activation is associated with upregulation of CD57 and a 303 reduction in CD28 expression 57, 58, 59 . Consistent with these prior data, comparisons of CD28 and 304 CD57 expression on T cell populations by CMV serostatus in our cohort of older adults ( Figure 305 2O) revealed increased CD57 expression and reduced CD28 expression on total CD4 + and CD8 + 306 T cell populations, as well as more specifically CD4CM, CD4EM, CD4EMRA, CD8N, and CD8EMRA 307 T cells. Expression of CD28 was also decreased on CD8CM and CD8EM cells of CMV 308 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 28, 2022. ; https://doi.org/10.1101/2022.05.27.22275673 doi: medRxiv preprint seropositive individuals, though expression of CD57 was not influenced by CMV serostatus. CMV serostatus did not affect CD57 or CD28 expression on CD4N T cells. 324 An activation-induced marker (AIM) assay was used to examine T cell memory 325 responses after second and third dose SARS-CoV-2 mRNA vaccinations ( Figure 3 ; Table 4 ). SARS-CoV-2 vaccines are unusual in that healthy adults generate strong CD4 + T cell memory 327 recall responses, but weaker CD8 + T cell memory responses 60 . We also made similar 328 observations in older adults. Most study participants had CD4 + T cell responses to SARS-CoV-2 329 Spike (post-dose 2: 93.1%; post-dose 3: 95.9%), but only 19.5% of participants had Spike-330 elicited CD8 + memory T cell responses after 2 vaccine doses, though this increased to 29.7% of 331 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Figure 7 ). Therefore, CMV serostatus contributes to increased CD8 + T cell, but not CD4 + T cell, 344 memory responses to SARS-CoV-2 Spike antigen. Figure 7) . Interestingly, when we considered these data in context 354 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. showed distinct Th1 skewed responses, though this was not observed after HA stimulation. These data collectively suggest that CMV serostatus alters the functional composition of memory 369 T cells, with antigen-specific effects, though CMV serostatus but does not alter the ability of 370 older adults to generate CD4 + or CD8 + T cell memory, nor the incidence of activation after 371 SARS-CoV-2 vaccination. whether the CMV seropositivity is associated with a reduction in naïve CD4 + and CD8 + T cells. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 28, 2022. ; https://doi.org/10.1101/2022.05.27.22275673 doi: medRxiv preprint with observations from a previous study that found CMV serostatus did not alter the ability of 402 older adults to generate memory responses to the newly emergent (at the time) West Nile virus 46 . 403 We observed in our cohort of older adults that T cell memory responses were not different by 404 CMV serostatus. Furthermore, it has been reported that T cell memory responses to the SARS- CoV-2 Spike protein are boosted in convalescent younger adults after vaccination 65, 66 . We 406 identified an increase in Spike-specific CD4 + T cell memory responses between 2 and 3 mRNA 407 vaccine doses in our older adult cohort, but we did not observe increased CD8 + or CD4 + T cell 408 memory responses in convalescent older adults after vaccination. This may in part be because CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 28, 2022. ; https://doi.org/10.1101/2022.05.27.22275673 doi: medRxiv preprint is associated with Th1-biased CD4 + T cell responses, which are not further modified by any prior 425 SARS-CoV-2 infection. Our observations also suggest that Spike-specific memory T cell 426 functional responses change between 2 and 3 vaccine doses in older adults, congruent with 427 observations of changes in memory T cell phenotype between vaccine doses in younger adults 65 . Our data also show that CMV seropositivity does not prevent production of anti-Spike or CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 28, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 remodelling of innate and adaptive immunity in older adults may contribute to the pathogenesis 448 and severity of SARS-CoV-2 infection. CMV serostatus may in addition impact humoral or 449 cellular responses to breakthrough infections with current or emerging variants of concern. 450 451 In conclusion, our data shows that CMV serostatus alters the T cell repertoire but does CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 28, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 28, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 28, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 antibodies. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 28, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 28, 2022. ; https://doi.org/10.1101/2022.05.27.22275673 doi: medRxiv preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 28, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Associations between CMV serostatus and vaccine dose were assessed by two-way ANOVA, 772 with Tukey's test post-hoc analysis. **p<0.01, ***p<0.001. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 28, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 test, according to normality. **p<0.01, ***p<0.001, ***p<0.0001. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 28, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Characteristics of Anti-SARS-CoV-2 Antibodies in Recovered COVID-639 Guidance on booster COVID-19 vaccine doses in Canada -642 SARS-COV-2 Variants: Differences and Potential of Immune 645 T cell responses to cytomegalovirus CD8+ CD28− and CD8+ CD57+ T cells 651 and their role in health and disease Cytomegalovirus-specific CD4+ T cells in healthy carriers are 654 continuously driven to replicative exhaustion Properties of end-stage human T cells 658 defined by CD45RA re-expression Expression of CD57 defines replicative senescence and antigen-661 induced apoptotic death of CD8+ T cells mRNA vaccine-induced T cells respond identically to SARS-CoV-2 665 variants of concern but differ in longevity and homing properties depending on prior 666 infection status A brief history of T cell help to B cells Virus-Specific Memory T-Cell Frequencies, but Not With an Impaired T-Cell Response 673 to Acute Influenza Virus Infection SARS-CoV-2-Specific T Cells Exhibit Phenotypic Features of Helper 683 Function, Lack of Terminal Differentiation, and High Proliferation Potential Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans 687 with COVID-19 Disease and Unexposed Individuals Cytomegalovirus infection in Gambian infants leads to profound CD8 T-cell differentiation Better Response to Influenza Virus Vaccination in Physically 693 Immunoglobulins as Well as Improvements in the Inflammatory and CD8+ T-Cell 695 Profiles Antigen-Specific Adaptive Immunity to SARS-CoV-2 Acute COVID-19 and Associations with Age and Disease Severity Effects of aging, cytomegalovirus infection, and EBV infection on human 702 B cell repertoires Viral epitope profiling of COVID-19 patients reveals cross-reactivity 705 and correlates of severity High incidence of Epstein-Barr virus, cytomegalovirus, and human-708 herpes virus-6 reactivations in critically ill patients with COVID-19 Cytomegalovirus latent infection is associated with an increased risk of 712 COVID-19-related hospitalization An inflammatory cytokine signature predicts COVID-19 severity 715 and survival leukocytes; (C) total CD4 + T cells; (D) CD8 + T cells. Absolute cell 747 counts of CD4 + T cells: (E) naïve; (F) central memory; (G) effector memory; (H) EMRA; (I) 748 terminally differentiated L) effector memory; (M) EMRA; (N) terminally differentiated. (O) Surface geometric mean 750 expression of CD57 and CD28 on CD4 + and CD8 + T cells by CMV serostatus 752 minimum to maximum, where the center line indicates the median. The surface marker 753 expression in O was visualized by concatenating uncompensated events in FlowJo for each 754 participant and indicated T cell population grouped according to CMV serostatus, and then 755 geometric mean fluorescence intensity expression data of each CMV group was overlaid onto the 756 same histogram plot. Associations between T cell subsets and CMV serostatus Student's t test with Welch's correction or Mann-Whitney U test, according to normality International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity