Sex-dependent niche responses modulate steady-state and regenerative hematopoiesis.

Hematopoietic stem cells (HSCs) adapt to organismal blood production needs by balancing self-renewal and differentiation, adjusting to physiological demands and external stimuli. Although sex differences have been implicated in differential hematopoietic function in males versus females, the mediators responsible for these effects require further study. Here, we characterized hematopoiesis at a steady state and during regeneration following hematopoietic stem cell transplantation (HST). RNA sequencing of lineage(-) bone marrow cells from C57/Bl6 mice revealed a broad transcriptional similarity between the sexes. However, we identified distinct sex differences in key biological pathways, with female cells showing reduced expression of signatures involved in inflammation and enrichment of genes related to glycolysis, hypoxia, and cell cycle regulation, suggesting a more quiescent and less inflammatory profile compared with male cells. To determine the functional impacts of the observed transcriptomic differences, we performed sex-matched and mismatched transplantation studies of lineage(-) donor cells. During short-term 56-day HST recovery, we found a male donor cell proliferative advantage, coinciding with elevated serum TNF-α, and a male recipient engraftment advantage, coinciding with increased serum CXCL12. Together, we show that sex-specific cell responses, marked by differing expression of pathways regulating metabolism, hypoxia, and inflammation, shape normal and regenerative hematopoiesis, with implications for the clinical understanding of hematopoietic function.

Pre-vaccination transcriptomic profiles of immune responders to the MUC1 peptide vaccine for colon cancer prevention.

Self-antigens abnormally expressed on tumors, such as MUC1, have been targeted by therapeutic cancer vaccines. We recently assessed in two clinical trials in a preventative setting whether immunity induced with a MUC1 peptide vaccine could reduce high colon cancer risk in individuals with a history of premalignant colon adenomas. In both trials, there were immune responders and non-responders to the vaccine. Here we used PBMC pre-vaccination and 2 weeks after the first vaccine of responders and non-responders selected from both trials to identify early biomarkers of immune response involved in long-term memory generation and prevention of adenoma recurrence. We performed flow cytometry, phosflow, and differential gene expression analyses on PBMCs collected from MUC1 vaccine responders and non-responders pre-vaccination and two weeks after the first of three vaccine doses. MUC1 vaccine responders had higher frequencies of CD4 cells pre-vaccination, increased expression of CD40L on CD8 and CD4 T-cells, and a greater increase in ICOS expression on CD8 T-cells. Differential gene expression analysis revealed that iCOSL, PI3K AKT MTOR, and B-cell signaling pathways are activated early in response to the MUC1 vaccine. We identified six specific transcripts involved in elevated antigen presentation, B-cell activation, and NF-kB1 activation that were directly linked to finding antibody response at week 12. Finally, a model using these transcripts was able to predict non-responders with accuracy. These findings suggest that individuals who can be predicted to respond to the MUC1 vaccine, and potentially other vaccines, have greater readiness in all immune compartments to present and respond to antigens. Predictive biomarkers of MUC1 vaccine response may lead to more effective vaccines tailored to individuals with high risk for cancer but with varying immune fitness.

Adjuvant AS01 activates human monocytes for costimulation and systemic inflammation.

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-ɑ 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.

Activated NK Cells with Pro-inflammatory Features are Associated with Atherogenesis in Perinatally HIV-Acquired Adolescents.

Human immunodeficiency virus (HIV) is associated with persistent immune activation and dysfunction in people with HIV despite treatment with antiretroviral therapy (ART). Modulation of the immune system may be driven by: low-level HIV replication, co-pathogens, gut dysbiosis /translocation, altered lipid profiles, and ART toxicities. In addition, perinatally acquired HIV (PHIV) and lifelong ART may alter the development and function of the immune system. Our preliminary data and published literature suggest reprogramming innate immune cells may accelerate aging and increase the risk for future end-organ complications, including cardiovascular disease (CVD). The exact mechanisms, however, are currently unknown. Natural killer (NK) cells are a highly heterogeneous cell population with divergent functions. They play a critical role in HIV transmission and disease progression in adults. Recent studies suggest the important role of NK cells in CVDs; however, little is known about NK cells and their role in HIV-associated cardiovascular risk in PHIV adolescents. Here, we investigated NK cell subsets and their potential role in atherogenesis in PHIV adolescents compared to HIV-negative adolescents in Uganda. Our data suggest, for the first time, that activated NK subsets in PHIV adolescents may contribute to atherogenesis by promoting plasma oxidized low-density lipoprotein (Ox-LDL) uptake by vascular macrophages.

A transcriptional evaluation of the melanoma and squamous cell carcinoma TIL compartment reveals an unexpected spectrum of exhausted and functional T cells.

Introduction: Significant heterogeneity exists within the tumor-infiltrating CD8 T cell population, and exhausted T cells harbor a subpopulation that may be replicating and may retain signatures of activation, with potential functional consequences in tumor progression. Dysfunctional immunity in the tumor microenvironment is associated with poor cancer outcomes, making exploration of these exhausted T cell subpopulations critical to the improvement of therapeutic approaches. Methods: To investigate mechanisms associated with terminally exhausted T cells, we sorted and performed transcriptional profiling of CD8+ tumor-infiltrating lymphocytes (TILs) co-expressing the exhaustion markers PD-1 and TIM-3 from large-volume melanoma tumors. We additionally performed immunologic phenotyping and functional validation, including at the single-cell level, to identify potential mechanisms that underlie their dysfunctional phenotype. Results: We identified novel dysregulated pathways in CD8+PD-1+TIM-3+ cells that have not been well studied in TILs; these include bile acid and peroxisome pathway-related metabolism and mammalian target of rapamycin (mTOR) signaling pathways, which are highly correlated with immune checkpoint receptor expression. Discussion: Based on bioinformatic integration of immunophenotypic data and network analysis, we propose unexpected targets for therapies to rescue the immune response to tumors in melanoma.

Loss of function of ribosomal protein L13a blocks blastocyst formation and reveals a potential nuclear role in gene expression.

Ribosomal proteins play diverse roles in development and disease. Most ribosomal proteins have canonical roles in protein synthesis, while some exhibit extra-ribosomal functions. Previous studies in our laboratory revealed that ribosomal protein L13a (RPL13a) is involved in the translational silencing of a cohort of inflammatory proteins in myeloid cells. This prompted us to investigate the role of RPL13a in embryonic development. Here we report that RPL13a is required for early development in mice. Crosses between Rpl13a+/- mice resulted in no Rpl13a-/- offspring. Closer examination revealed that Rpl13a-/- embryos were arrested at the morula stage during preimplantation development. RNA sequencing analysis of Rpl13a-/- morulae revealed widespread alterations in gene expression, including but not limited to several genes encoding proteins involved in the inflammatory response, embryogenesis, oocyte maturation, stemness, and pluripotency. Ex vivo analysis revealed that RPL13a was localized to the cytoplasm and nucleus between the two-cell and morula stages. RNAi-mediated depletion of RPL13a phenocopied Rpl13a-/- embryos and knockdown embryos exhibited increased expression of IL-7 and IL-17 and decreased expression of the lineage specifier genes Sox2, Pou5f1, and Cdx2. Lastly, a protein-protein interaction assay revealed that RPL13a is associated with chromatin, suggesting an extra ribosomal function in transcription. In summary, our data demonstrate that RPL13a is essential for the completion of preimplantation embryo development. The mechanistic basis of the absence of RPL13a-mediated embryonic lethality will be addressed in the future through follow-up studies on ribosome biogenesis, global protein synthesis, and identification of RPL13a target genes using chromatin immunoprecipitation and RNA-immunoprecipitation-based sequencing.

IL-21-IgFc immunotherapy alters transcriptional landscape of lymph node cells leading to enhanced flu vaccine response in aging and SIV infection.

Aging people living with HIV (PWH) frequently manifest impaired antibody (Ab) responses to seasonal flu vaccination which has been attributed to ongoing inflammation and immune activation. We have recently reported a similar scenario in old simian immunodeficiency virus (SIV) infected rhesus macaques (RM) with controlled viremia and have been able to compensate for this deficiency by immunotherapy with interleukin (IL)-21-IgFc. To understand the underlying mechanisms of IL-21-induced immunomodulation leading to enhanced flu vaccine response in aging and SIV, we have investigated draining lymph node (LN) cells of IL-21-treated and -untreated animals at postvaccination. We observed IL-21-induced proliferation of flu-specific LN memory CD4 T cells, expansion of B cells expressing IL-21 receptor (IL-21R), and modest expansion of T follicular helper cells (Tfh) co-expressing T-cell immunoreceptor with Ig and ITIM domains (TIGIT) and DNAX accessory molecule (DNAM-1). Transcriptional analysis of LN cells of IL-21-treated animals revealed significant inhibition of germinal center (GC) Tfh and B-cell interferon signaling pathways along with enhanced B-cell development and antigen presentation pathways. We conclude that IL-21 treatment at the time of flu vaccination in aging SIV-infected animals modulates the inductive LN GC activity, to reverse SIV-associated LN Tfh and B-cell dysfunction. IL-21 is a potential candidate molecule for immunotherapy to enhance flu vaccine responses in aging PWH who have deficient antibody responses.

Upregulation of IFN-stimulated genes persists beyond the transitory broad immunologic changes of acute HIV-1 infection.

Chronic immune activation during HIV-1 infection contributes to morbidity and mortality in people living with HIV. To elucidate the underlying biological pathways, we evaluated whole blood gene expression trajectories from before, through acute, and into chronic HIV-1 infection. Interferon-stimulated genes, including MX1, IFI27 and ISG15, were upregulated during acute infection, remained elevated into chronic infection, and were strongly correlated with plasma HIV-1 RNA as well as TNF-α and CXCL10 cytokine levels. In contrast, genes involved in cellular immune responses, such as CD8A, were upregulated during acute infection before reaching a peak and returning to near pre-infection levels in chronic infection. Our results indicate that chronic immune activation during HIV-1 infection is characterized by persistent elevation of a narrow set of interferon-stimulated genes and innate cytokines. These findings raise the prospect of devising a targeted intervention to restore healthy immune homeostasis in people living with HIV-1.

Upregulation of PTPRC and Interferon Response Pathways in HIV-1 Seroconverters Prior to Infection.

Human immunodeficiency virus 1 (HIV-1) exposed seronegative (HESN) individuals may have unique characteristics that alter susceptibility to HIV-1 infection. However, identifying truly exposed HESN is challenging. We utilized stored data and biospecimens from HIV-1 serodifferent couple cohorts, in which couples' HIV-1 exposures were quantified based on unprotected sex frequency and viral load of the partner with HIV-1. We compared peripheral blood gene expression between 15 HESN and 18 seroconverters prior to infection. We found PTPRC (encoding CD45 antigen) and interferon-response pathways had significantly higher expression among individuals who went on to become seropositive and thus may be a signature for increased acquisition risk.

Simulated galactic cosmic ray exposure activates dose-dependent DNA repair response and down regulates glucosinolate pathways in arabidopsis seedlings.

Outside the protection of Earth's magnetic field, organisms are constantly exposed to space radiation consisting of energetic protons and other heavier charged particles. With the goal of crewed Mars exploration, the production of fresh food during long duration space missions is critical for meeting astronauts' nutritional and psychological needs. However, the biological effects of space radiation on plants have not been sufficiently investigated and characterized. To that end, 10-day-old Arabidopsis seedlings were exposed to simulated Galactic Cosmic Rays (GCR) and assessed for transcriptomic changes. The simulated GCR irradiation was carried out in the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Lab (BNL). The exposures were conducted acutely for two dose points at 40 cGy or 80 cGy, with sequential delivery of proton, helium, oxygen, silicon, and iron ions. Control and irradiated seedlings were then harvested and preserved in RNAlater at 3 hrs post irradiation. Total RNA was isolated for transcriptomic analyses using RNAseq. The data revealed that the transcriptomic responses were dose-dependent, with significant upregulation of DNA repair pathways and downregulation of glucosinolate biosynthetic pathways. Glucosinolates are important for plant pathogen defense and for the taste of a plant, which are both relevant to growing plants for spaceflight. These findings fill in knowledge gaps of how plants respond to radiation in beyond-Earth environments.

COVID-19 vaccine booster dose needed to achieve Omicron-specific neutralisation in nursing home residents.

BACKGROUND: Nursing home (NH) residents have borne a disproportionate share of SARS-CoV-2 morbidity and mortality. Vaccines have limited hospitalisation and death from earlier variants in this vulnerable population. With the rise of Omicron and future variants, it is vital to sustain and broaden vaccine-induced protection. We examined the effect of boosting with BNT162b2 mRNA vaccine on humoral immunity and Omicron-specific neutralising activity among NH residents and healthcare workers (HCWs). METHODS: We longitudinally enrolled 85 NH residents (median age 77) and 48 HCWs (median age 51), and sampled them after the initial vaccination series; and just before and 2 weeks after booster vaccination. Anti-spike, anti-receptor binding domain (RBD) and neutralisation titres to the original Wuhan strain and neutralisation to the Omicron strain were obtained. FINDINGS: Booster vaccination significantly increased vaccine-specific anti-spike, anti-RBD, and neutralisation levels above the pre-booster levels in NH residents and HCWs, both in those with and without prior SARS-CoV-2 infection. Omicron-specific neutralisation activity was low after the initial 2 dose series with only 28% of NH residents' and 28% HCWs' titres above the assay's lower limit of detection. Omicron neutralising activity following the booster lifted 86% of NH residents and 93% of HCWs to the detectable range. INTERPRETATION: With boosting, the vast majority of HCWs and NH residents developed detectable Omicron-specific neutralising activity. These data provide immunologic evidence that strongly supports booster vaccination to broaden neutralising activity and counter waning immunity in the hope it will better protect this vulnerable, high-risk population against the Omicron variant. FUNDING: NIH AI129709-03S1, U01 CA260539-01, CDC 200-2016-91773, and VA BX005507-01.

Host transcriptomic signatures of tuberculosis can predict immune reconstitution inflammatory syndrome in HIV patients.

Immune reconstitution inflammatory syndrome (IRIS) can be a complication of antiretroviral therapy (ART) in patients with advanced HIV, but its pathogenesis is uncertain. In tuberculosis (TB) endemic countries, IRIS is often associated with mycobacterial infections or Bacille-Calmette-Guerin (BCG) vaccination in children. With no predictive or confirmatory tests at present, IRIS remains a diagnosis of exclusion. We tested whether RISK6 and Sweeney3, validated immune-based blood transcriptomic signatures for TB, could predict or diagnose IRIS in HIV+ children and adults. Transcripts were measured by RT-qPCR in BCG-vaccinated children and by microarray in HIV+ adults with TB including TB meningitis (TBM). Signature scores before ART initiation and up to IRIS diagnosis were compared between participants who did or did not develop IRIS. In children, RISK6 and Sweeney3 discriminated IRIS cases from non-IRIS controls before ART, and at diagnosis. In adults with TB, RISK6 discriminated IRIS cases from controls after half-week on ART and at TB-IRIS onset. In adults with TBM, only Sweeney3 discriminated IRIS cases from controls before ART, while both signatures distinguished cases from controls at TB-IRIS onset. Parsimonious whole blood transcriptomic signatures for TB showed potential to predict and diagnose IRIS in HIV+ children and adults.

Significant Reduction in Vaccine-Induced Antibody Levels and Neutralization Activity Among Healthcare Workers and Nursing Home Residents 6 Months Following Coronavirus Disease 2019 BNT162b2 mRNA Vaccination.

Antibody decline occurred from 2 weeks to 6 months post-BNT162b2 mRNA vaccination in nursing home (NH) residents and healthcare workers. Antispike, receptor-binding domain, and neutralization levels dropped >81% irrespective of prior infection. Notably, 69% of infection-naive NH residents had neutralizing antibodies at or below the assay's limit of detection.

Differential CD4+ T-Cell Cytokine and Cytotoxic Responses Between Reactivation and Latent Phases of Herpes Zoster Infection.

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.

Significantly elevated antibody levels and neutralization titers in nursing home residents after SARS-CoV-2 BNT162b2 mRNA booster vaccination.

Nursing home (NH) residents have experienced significant morbidity and mortality to SARS-CoV-2 throughout the pandemic. Vaccines initially curbed NH resident morbidity and mortality, but antibody levels and protection have declined with time since vaccination, prompting introduction of booster vaccination. This study assesses humoral immune response to booster vaccination in 85 NH residents and 44 health care workers (HCW) that we have followed longitudinally since initial SARS-CoV-2 BNT162b2 mRNA vaccination. The findings reveal that booster vaccination significantly increased anti-spike, anti-receptor binding domain, and neutralization titers above the pre-booster levels in almost all NH residents and HCW to significantly higher levels than shortly after the completion of the initial vaccine series. These data support the CDC recommendation to offer vaccine boosters to HCWs and NH residents on an immunological basis. Notably, even the older, more frail and more multi-morbid NH residents have sizable antibody increases with boosting.

Does a lack of vaccine side effects correlate with reduced BNT162b2 mRNA vaccine response among healthcare workers and nursing home residents?

BACKGROUND: The BNT162b2 SARS-CoV-2 mRNA vaccination has mitigated the burden of COVID-19 among residents of long-term care facilities considerably, despite being excluded from the vaccine trials. Data on reactogenicity (vaccine side effects) in this population are limited. AIMS: To assess reactogenicity among nursing home (NH) residents. To provide a plausible proxy for predicting vaccine response among this population. METHODS: We enrolled and sampled NH residents and community-dwelling healthcare workers who received the BNT162b2 mRNA vaccine, to assess local or systemic reactogenicity and antibody levels (immunogenicity). RESULTS: NH residents reported reactions at a much lower frequency and lesser severity than the community-dwelling healthcare workers. These reactions were mild and transient with all subjects experiencing more local than systemic reactions. Based on our reactogenicity and immunogenicity data, we developed a linear regression model predicting log-transformed anti-spike, anti-receptor-binding domain (RBD), and neutralizing titers, with a dichotomous variable indicating the presence or absence of reported reactions which revealed a statistically significant effect, with estimated shifts in log-transformed titers ranging from 0.32 to 0.37 (all p < 0.01) indicating greater immunogenicity in subjects with one or more reported reactions of varying severity. DISCUSSION: With a significantly lower incidence of post-vaccination reactions among NH residents as reported in this study, the BNT162b2 mRNA vaccine appears to be well-tolerated among this vulnerable population. If validated in larger populations, absence of reactogenicity could help guide clinicians in prioritizing vaccine boosters. CONCLUSIONS: Reactogenicity is significantly mild among nursing home residents and overall, subjects who reported post-vaccination reactions developed higher antibody titers.

Sex and age bias viral burden and interferon responses during SARS-CoV-2 infection in ferrets.

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) hospitalizations and deaths disportionally affect males and older ages. Here we investigated the impact of male sex and age comparing sex-matched or age-matched ferrets infected with SARS-CoV-2. Differences in temperature regulation was identified for male ferrets which was accompanied by prolonged viral replication in the upper respiratory tract after infection. Gene expression analysis of the nasal turbinates indicated that 1-year-old female ferrets had significant increases in interferon response genes post infection which were delayed in males. These results provide insight into COVID-19 and suggests that older males may play a role in viral transmission due to decreased antiviral responses.

Germinal Center T follicular helper (GC-Tfh) cell impairment in chronic HIV infection involves c-Maf signaling.

We have recently demonstrated that the function of T follicular helper (Tfh) cells from lymph nodes (LN) of HIV-infected individuals is impaired. We found that these cells were unable to provide proper help to germinal center (GC)-B cells, as observed by altered and inefficient anti-HIV antibody response and premature death of memory B cells. The underlying molecular mechanisms of this dysfunction remain poorly defined. Herein, we have used a unique transcriptional approach to identify these molecular defects. We consequently determined the transcriptional profiles of LN GC-Tfh cells following their interactions with LN GC-B cells from HIV-infected and HIV-uninfected individuals, rather than analyzing resting ex-vivo GC-Tfh cells. We observed that proliferating GC-Tfh cells from HIV-infected subjects were transcriptionally different than their HIV-uninfected counterparts, and displayed a significant downregulation of immune- and GC-Tfh-associated pathways and genes. Our results strongly demonstrated that MAF (coding for the transcription factor c-Maf) and its upstream signaling pathway mediators (IL6R and STAT3) were significantly downregulated in HIV-infected subjects, which could contribute to the impaired GC-Tfh and GC-B cell functions reported during infection. We further showed that c-Maf function was associated with the adenosine pathway and that the signaling upstream c-Maf could be partially restored by adenosine deaminase -1 (ADA-1) supplementation. Overall, we identified a novel mechanism that contributes to GC-Tfh cell impairment during HIV infection. Understanding how GC-Tfh cell function is altered in HIV is crucial and could provide critical information about the mechanisms leading to the development and maintenance of effective anti-HIV antibodies.

A follicular regulatory Innate Lymphoid Cell population impairs interactions between germinal center Tfh and B cells.

Innate Lymphoid Cells (ILCs) are immune cells typically found on mucosal surfaces and in secondary lymphoid organs where they regulate the immune response to pathogens. Despite their key role in the immune response, there are still fundamental gaps in our understanding of ILCs. Here we report a human ILC population present in the follicles of tonsils and lymph nodes termed follicular regulatory ILCs (ILCFR) that to our knowledge has not been previously identified. ILCFR have a distinct phenotype and transcriptional program when compared to other defined ILCs. Surprisingly, ILCFR inhibit the ability of follicular helper T (Tfh) cells to provide B cell help. The localization of ILCFR to the germinal centers suggests these cells may interfere with germinal center B cell (GC-B) and germinal center Tfh cell (GC-Tfh) interactions through the production of transforming growth factor beta (TGF-ß. Intriguingly, under conditions of impaired GC-Tfh-GC-B cell interactions, such as human immunodeficiency virus (HIV) infection, the frequency of these cells is increased. Overall, we predict a role for ILCFR in regulating GC-Tfh-GC-B cell interactions and propose they expand in chronic inflammatory conditions.

Reduced BNT162b2 Messenger RNA Vaccine Response in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-Naive Nursing Home Residents.

After BNT162b2 messenger RNA vaccination, antibody levels to spike, receptor-binding domain, and virus neutralization were examined in 149 nursing home residents and 110 healthcare worker controls. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-naive nursing home residents' median post-second vaccine dose antibody neutralization titers are one-quarter that of SARS-CoV-2-naive healthcare workers.