SARS-CoV-2 inflammation durably imprints memory CD4 T cells.

Memory CD4 T cells are critical to human immunity, yet it is unclear whether viral inflammation during memory formation has long-term consequences. Here, we compared transcriptional and epigenetic landscapes of Spike (S)-specific memory CD4 T cells in 24 individuals whose first exposure to S was via SARS-CoV-2 infection or mRNA vaccination. Nearly 2 years after memory formation, S-specific CD4 T cells established by infection remained enriched for transcripts related to cytotoxicity and for interferon-stimulated genes, likely because of a chromatin accessibility landscape altered by inflammation. Moreover, S-specific CD4 T cells primed by infection had reduced proliferative capacity in vitro relative to vaccine-primed cells. Furthermore, the transcriptional state of S-specific memory CD4 T cells was minimally altered by booster immunization and/or breakthrough infection. Thus, infection-associated inflammation durably imprints CD4 T cell memory, which affects the function of these cells and may have consequences for long-term immunity.

Integrative multi-omics profiling in human decedents receiving pig heart xenografts.

In a previous study, heart xenografts from 10-gene-edited pigs transplanted into two human decedents did not show evidence of acute-onset cellular- or antibody-mediated rejection. Here, to better understand the detailed molecular landscape following xenotransplantation, we carried out bulk and single-cell transcriptomics, lipidomics, proteomics and metabolomics on blood samples obtained from the transplanted decedents every 6 h, as well as histological and transcriptomic tissue profiling. We observed substantial early immune responses in peripheral blood mononuclear cells and xenograft tissue obtained from decedent 1 (male), associated with downstream T cell and natural killer cell activity. Longitudinal analyses indicated the presence of ischemia reperfusion injury, exacerbated by inadequate immunosuppression of T cells, consistent with previous findings of perioperative cardiac xenograft dysfunction in pig-to-nonhuman primate studies. Moreover, at 42 h after transplantation, substantial alterations in cellular metabolism and liver-damage pathways occurred, correlating with profound organ-wide physiological dysfunction. By contrast, relatively minor changes in RNA, protein, lipid and metabolism profiles were observed in decedent 2 (female) as compared to decedent 1. Overall, these multi-omics analyses delineate distinct responses to cardiac xenotransplantation in the two human decedents and reveal new insights into early molecular and immune responses after xenotransplantation. These findings may aid in the development of targeted therapeutic approaches to limit ischemia reperfusion injury-related phenotypes and improve outcomes.

Multimodal single-cell datasets characterize antigen-specific CD8+ T cells across SARS-CoV-2 vaccination and infection.

The immune response to SARS-CoV-2 antigen after infection or vaccination is defined by the durable production of antibodies and T cells. Population-based monitoring typically focuses on antibody titer, but there is a need for improved characterization and quantification of T cell responses. Here, we used multimodal sequencing technologies to perform a longitudinal analysis of circulating human leukocytes collected before and after immunization with the mRNA vaccine BNT162b2. Our data indicated distinct subpopulations of CD8+ T cells, which reliably appeared 28 days after prime vaccination. Using a suite of cross-modality integration tools, we defined their transcriptome, accessible chromatin landscape and immunophenotype, and we identified unique biomarkers within each modality. We further showed that this vaccine-induced population was SARS-CoV-2 antigen-specific and capable of rapid clonal expansion. Moreover, we identified these CD8+ T cell populations in scRNA-seq datasets from COVID-19 patients and found that their relative frequency and differentiation outcomes were predictive of subsequent clinical outcomes.

Immune response, phenotyping and molecular graft surveillance in kidney transplant recipients following severe acute respiratory syndrome coronavirus 2 vaccination.

BACKGROUND: Understanding immunogenicity and alloimmune risk following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination in kidney transplant recipients is imperative to understanding the correlates of protection and to inform clinical guidelines. METHODS: We studied 50 kidney transplant recipients following SARS-CoV-2 vaccination and quantified their anti-spike protein antibody, donor-derived cell-free DNA (dd-cfDNA), gene expression profiling (GEP), and alloantibody formation. RESULTS: Participants were stratified using nucleocapsid testing as either SARS-CoV-2-naïve or experienced prior to vaccination. One of 34 (3%) SARS-CoV-2 naïve participants developed anti-spike protein antibodies. In contrast, the odds ratio for the association of a prior history of SARS-CoV-2 infection with vaccine response was 18.3 (95% confidence interval 3.2, 105.0, p < 0.01). Pre- and post-vaccination levels did not change for median dd-cfDNA (0.23% vs. 0.21% respectively, p = 0.13), GEP scores (9.85 vs. 10.4 respectively, p = 0.45), calculated panel reactive antibody, de-novo donor specific antibody status, or estimated glomerular filtration rate. CONCLUSIONS: SARS-CoV-2 vaccines do not appear to trigger alloimmunity in kidney transplant recipients. The degree of vaccine immunogenicity was associated most strongly with a prior history of SARS-CoV-2 infection.

High-throughput interrogation of immune responses using the Human Immune Profiling Pipeline.

The current abundance of immunotherapy clinical trials presents an opportunity to learn about the underlying mechanisms and pharmacodynamic effects of novel drugs on the human immune system. Here, we present a protocol to study how these immune responses impact clinical outcomes using large-scale high-throughput immune profiling of clinical cohorts. We describe the Human Immune Profiling Pipeline, which comprises an end-to-end solution from flow cytometry results to computational approaches and unsupervised patient clustering based on lymphocyte landscape. For complete details on the use and execution of this protocol, please refer to Lyudovyk et al. (2022).1.

Human T follicular helper clones seed the germinal center-resident regulatory pool.

The mechanisms by which FOXP3+ T follicular regulatory (Tfr) cells simultaneously steer antibody formation toward microbe or vaccine recognition and away from self-reactivity remain incompletely understood. To explore underappreciated heterogeneity in human Tfr cell development, function, and localization, we used paired TCRVA/TCRVB sequencing to distinguish tonsillar Tfr cells that are clonally related to natural regulatory T cells (nTfr) from those likely induced from T follicular helper (Tfh) cells (iTfr). The proteins iTfr and nTfr cells differentially expressed were used to pinpoint their in situ locations via multiplex microscopy and establish their divergent functional roles. In silico analyses and in vitro tonsil organoid tracking models corroborated the existence of separate Treg-to-nTfr and Tfh-to-iTfr developmental trajectories. Our results identify human iTfr cells as a distinct CD38+, germinal center-resident, Tfh-descended subset that gains suppressive function while retaining the capacity to help B cells, whereas CD38- nTfr cells are elite suppressors primarily localized in follicular mantles. Interventions differentially targeting specific Tfr cell subsets may provide therapeutic opportunities to boost immunity or more precisely treat autoimmune diseases.

Multimodal characterization of antigen-specific CD8 + T cells across SARS-CoV-2 vaccination and infection.

The human immune response to SARS-CoV-2 antigen after infection or vaccination is defined by the durable production of antibodies and T cells. Population-based monitoring typically focuses on antibody titer, but there is a need for improved characterization and quantification of T cell responses. Here, we utilize multimodal sequencing technologies to perform a longitudinal analysis of circulating human leukocytes collected before and after BNT162b2 immunization. Our data reveal distinct subpopulations of CD8 + T cells which reliably appear 28 days after prime vaccination (7 days post boost). Using a suite of cross-modality integration tools, we define their transcriptome, accessible chromatin landscape, and immunophenotype, and identify unique biomarkers within each modality. By leveraging DNA-oligo-tagged peptide-MHC multimers and T cell receptor sequencing, we demonstrate that this vaccine-induced population is SARS-CoV-2 antigen-specific and capable of rapid clonal expansion. Moreover, we also identify these CD8 + populations in scRNA-seq datasets from COVID-19 patients and find that their relative frequency and differentiation outcomes are predictive of subsequent clinical outcomes. Our work contributes to our understanding of T cell immunity, and highlights the potential for integrative and multimodal analysis to characterize rare cell populations.

mRNA COVID-19 vaccine elicits potent adaptive immune response without the persistent inflammation seen in SARS-CoV-2 infection.

SARS-CoV-2 infection and vaccination elicit potent immune responses. Our study presents a comprehensive multimodal single-cell dataset of peripheral blood of patients with acute COVID-19 and of healthy volunteers before and after receiving the SARS-CoV-2 mRNA vaccine and booster. We compared host immune responses to the virus and vaccine using transcriptional profiling, coupled with B/T cell receptor repertoire reconstruction. COVID-19 patients displayed an enhanced interferon signature and cytotoxic gene upregulation, absent in vaccine recipients. These findings were validated in an independent dataset. Analysis of B and T cell repertoires revealed that, while the majority of clonal lymphocytes in COVID-19 patients were effector cells, clonal expansion was more evident among circulating memory cells in vaccine recipients. Furthermore, while clonal αß T cell responses were observed in both COVID-19 patients and vaccine recipients, dramatic expansion of clonal γδT cells was found only in infected individuals. Our dataset enables comparative analyses of immune responses to infection versus vaccination, including clonal B and T cell responses. Integrating our data with publicly available datasets allowed us to validate our findings in larger cohorts. To our knowledge, this is the first dataset to include comprehensive profiling of longitudinal samples from healthy volunteers pre/post SARS-CoV-2 vaccine and booster.

Inflammation durably imprints memory CD4+ T cells.

Adaptive immune responses are induced by vaccination and infection, yet little is known about how CD4+ T cell memory differs when primed in these two contexts. Notably, viral infection is generally associated with higher levels of systemic inflammation than is vaccination. To assess whether the inflammatory milieu at the time of CD4+ T cell priming has long-term effects on memory, we compared Spike-specific memory CD4+ T cells in 22 individuals around the time of the participants' third SARS-CoV-2 mRNA vaccination, with stratification by whether the participants' first exposure to Spike was via virus or mRNA vaccine. Multimodal single-cell profiling of Spike-specific CD4+ T cells revealed 755 differentially expressed genes that distinguished infection- and vaccine-primed memory CD4+ T cells. Spike-specific CD4+ T cells from infection-primed individuals had strong enrichment for cytotoxicity and interferon signaling genes, whereas Spike-specific CD4+ T cells from vaccine-primed individuals were enriched for proliferative pathways by gene set enrichment analysis. Moreover, Spike-specific memory CD4+ T cells established by infection had distinct epigenetic landscapes driven by enrichment of IRF-family transcription factors, relative to T cells established by mRNA vaccination. This transcriptional imprint was minimally altered following subsequent mRNA vaccination or breakthrough infection, reflecting the strong bias induced by the inflammatory environment during initial memory differentiation. Together, these data suggest that the inflammatory context during CD4+ T cell priming is durably imprinted in the memory state at transcriptional and epigenetic levels, which has implications for personalization of vaccination based on prior infection history.

mRNA COVID-19 vaccine elicits potent adaptive immune response without the acute inflammation of SARS-CoV-2 infection.

SARS-CoV-2 infection and vaccination elicit potent immune responses. Our study presents a comprehensive multimodal single-cell analysis of blood from COVID-19 patients and healthy volunteers receiving the SARS-CoV-2 vaccine and booster. We profiled immune responses via transcriptional analysis and lymphocyte repertoire reconstruction. COVID-19 patients displayed an enhanced interferon signature and cytotoxic gene upregulation, absent in vaccine recipients. B and T cell repertoire analysis revealed clonal expansion among effector cells in COVID-19 patients and memory cells in vaccine recipients. Furthermore, while clonal αß T cell responses were observed in both COVID-19 patients and vaccine recipients, expansion of clonal γδ T cells was found only in infected individuals. Our dataset enables side-by-side comparison of immune responses to infection versus vaccination, including clonal B and T cell responses. Our comparative analysis shows that vaccination induces a robust, durable clonal B and T cell responses, without the severe inflammation associated with infection.

Vaccine-Acquired SARS-CoV-2 Immunity versus Infection-Acquired Immunity: A Comparison of Three COVID-19 Vaccines.

Around the world, rollout of COVID-19 vaccines has been used as a strategy to end COVID-19-related restrictions and the pandemic. Several COVID-19 vaccine platforms have successfully protected against severe SARS-CoV-2 infection and subsequent deaths. Here, we compared humoral and cellular immunity in response to either infection or vaccination. We examined SARS-CoV-2 spike-specific immune responses from Pfizer/BioNTech BNT162b2, Moderna mRNA-1273, Janssen Ad26.COV2.S, and SARS-CoV-2 infection approximately 4 months post-exposure or vaccination. We found that these three vaccines all generate relatively similar immune responses and elicit a stronger response than natural infection. However, antibody responses to recent viral variants are diminished across all groups. The similarity of immune responses from the three vaccines studied here is an important finding in maximizing global protection as vaccination campaigns continue.

PD-1 directed immunotherapy alters Tfh and humoral immune responses to seasonal influenza vaccine.

Anti-programmed death-1 (anti-PD-1) immunotherapy reinvigorates CD8 T cell responses in patients with cancer but PD-1 is also expressed by other immune cells, including follicular helper CD4 T cells (Tfh) which are involved in germinal centre responses. Little is known, however, about the effects of anti-PD-1 immunotherapy on noncancer immune responses in humans. To investigate this question, we examined the impact of anti-PD-1 immunotherapy on the Tfh-B cell axis responding to unrelated viral antigens. Following influenza vaccination, a subset of adults receiving anti-PD-1 had more robust circulating Tfh responses than adults not receiving immunotherapy. PD-1 pathway blockade resulted in transcriptional signatures of increased cellular proliferation in circulating Tfh and responding B cells compared with controls. These latter observations suggest an underlying change in the Tfh-B cell and germinal centre axis in a subset of immunotherapy patients. Together, these results demonstrate dynamic effects of anti-PD-1 therapy on influenza vaccine responses and highlight analytical vaccination as an approach that may reveal underlying immune predisposition to adverse events.

Increased resistance of SARS-CoV-2 Omicron variant to neutralization by vaccine-elicited and therapeutic antibodies.

BACKGROUND: SARS-CoV-2 vaccines currently authorized for emergency use have been highly successful in preventing infection and lessening disease severity. The vaccines maintain effectiveness against earlier SARS-CoV-2 Variants of Concern but the heavily mutated, highly transmissible Omicron variant presents an obstacle both to vaccine protection and monoclonal antibody therapies. METHODS: Pseudotyped lentiviruses were incubated with serum from vaccinated and boosted donors or therapeutic monoclonal antibody and then applied to target cells. After 2 days, luciferase activity was measured in a microplate luminometer. Resistance mutations of the Omicron spike were identified using point-mutated spike protein pseudotypes and mapped onto the three-dimensional spike protein structure. FINDINGS: Virus with the Omicron spike protein was 26-fold resistant to neutralization by recovered donor sera and 26-34-fold resistance to Pfizer BNT162b2 and Moderna vaccine-elicited antibodies following two immunizations. A booster immunization increased neutralizing titres against Omicron. Neutralizing titres against Omicron were increased in the sera with a history of prior SARS-CoV-2 infection. Analysis of the therapeutic monoclonal antibodies showed that the Regeneron and Eli Lilly monoclonal antibodies were ineffective against the Omicron pseudotype while Sotrovimab and Evusheld were partially effective. INTERPRETATION: The results highlight the benefit of a booster immunization to protect against the Omicron variant and demonstrate the challenge to monoclonal antibody therapy. The decrease in neutralizing titres against Omicron suggest that much of the vaccine efficacy may rely on T cells. FUNDING: The work was funded by grants from the NIH to N.R.L. (DA046100, AI122390 and AI120898) and 55 to M.J.M. (UM1AI148574).

Neutralization of SARS-CoV-2 Variants by mRNA and Adenoviral Vector Vaccine-Elicited Antibodies.

The increasing prevalence of SARS-CoV-2 variants has raised concerns regarding possible decreases in vaccine effectiveness. Here, neutralizing antibody titers elicited by mRNA-based and adenoviral vector-based vaccines against variant pseudotyped viruses were measured. BNT162b2 and mRNA-1273-elicited antibodies showed modest neutralization resistance against Beta, Delta, Delta plus and Lambda variants whereas Ad26.COV2.S-elicited antibodies from a significant fraction of vaccinated individuals had less neutralizing titer (IC50 <50). The data underscore the importance of surveillance for breakthrough infections that result in severe COVID-19 and suggest a potential benefit by second immunization following Ad26.COV2.S to increase protection from current and future variants.

High-titer neutralization of Mu and C.1.2 SARS-CoV-2 variants by vaccine-elicited antibodies of previously infected individuals.

Recently identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants Mu and C.1.2 have spike proteins with mutations that may confer resistance to natural and vaccine-elicited antibodies. Analysis of neutralizing antibody titers in the sera of vaccinated individuals without previous history of infection and from convalescent individuals show partial resistance of the viruses. In contrast, sera from individuals with a previous history of SARS-CoV-2 infection who were subsequently vaccinated neutralize variants with titers 4- to 11-fold higher, providing a rationale for vaccination of individuals with previous infection. The heavily mutated C.1.2 spike is the most antibody neutralization-resistant spike to date; however, the avidity of C.1.2 spike protein for angiotensin-converting enzyme 2 (ACE2) is low. This finding suggests that the virus evolved to escape the humoral response but has a decrease in fitness, suggesting that it may cause milder disease or be less transmissible. It may be difficult for the spike protein to evolve to escape neutralizing antibodies while maintaining high affinity for ACE2.

Robust immune responses are observed after one dose of BNT162b2 mRNA vaccine dose in SARS-CoV-2-experienced individuals.

The use of coronavirus disease 2019 (COVID-19) vaccines will play the major role in helping to end the pandemic that has killed millions worldwide. COVID-19 vaccines have resulted in robust humoral responses and protective efficacy in human trials, but efficacy trials excluded individuals with a prior diagnosis of COVID-19. As a result, little is known about how immune responses induced by mRNA vaccines differ in individuals who recovered from COVID-19. Here, we evaluated longitudinal immune responses to two-dose BNT162b2 mRNA vaccination in 15 adults who had experienced COVID-19, compared to 21 adults who did not have prior COVID-19. Consistent with prior studies of mRNA vaccines, we observed robust cytotoxic CD8+ T cell responses in both cohorts after the second dose. Furthermore, SARS-CoV-2­naive individuals had progressive increases in humoral and antigen-specific antibody-secreting cell (ASC) responses after each dose of vaccine, whereas SARS-CoV-2­experienced individuals demonstrated strong humoral and antigen-specific ASC responses to the first dose but these responses were not further enhanced after the second dose of the vaccine at the time points studied. Together, these data highlight the relevance of immunological history for understanding vaccine immune responses and may have implications for personalizing mRNA vaccination regimens used to prevent COVID-19, including for the deployment of booster shots.

Evaluation of Immune Response and Disease Status in Systemic Lupus Erythematosus Patients Following SARS-CoV-2 Vaccination.

OBJECTIVE: To evaluate seroreactivity and disease flares after COVID-19 vaccination in a multiethnic/multiracial cohort of patients with systemic lupus erythematosus (SLE). METHODS: Ninety SLE patients and 20 healthy controls receiving a complete COVID-19 vaccine regimen were included. IgG seroreactivity to the SARS-CoV-2 spike receptor-binding domain (RBD) and SARS-CoV-2 microneutralization were used to evaluate B cell responses; interferon-γ (IFNγ) production was measured by enzyme-linked immunospot (ELISpot) assay in order to assess T cell responses. Disease activity was measured by the hybrid SLE Disease Activity Index (SLEDAI), and flares were identified according to the Safety of Estrogens in Lupus Erythematosus National Assessment-SLEDAI flare index. RESULTS: Overall, fully vaccinated SLE patients produced significantly lower IgG antibodies against SARS-CoV-2 spike RBD compared to fully vaccinated controls. Twenty-six SLE patients (28.8%) generated an IgG response below that of the lowest control (<100 units/ml). In logistic regression analyses, the use of any immunosuppressant or prednisone and a normal anti-double-stranded DNA antibody level prior to vaccination were associated with decreased vaccine responses. IgG seroreactivity to the SARS-CoV-2 spike RBD strongly correlated with the SARS-CoV-2 microneutralization titers and correlated with antigen-specific IFNγ production determined by ELISpot. In a subset of patients with poor antibody responses, IFNγ production was similarly diminished. Pre- and postvaccination SLEDAI scores were similar in both groups. Postvaccination flares occurred in 11.4% of patients; 1.3% of these were severe. CONCLUSION: In a multiethnic/multiracial study of SLE patients, 29% had a low response to the COVID-19 vaccine which was associated with receiving immunosuppressive therapy. Reassuringly, severe disease flares were rare. While minimal protective levels remain unknown, these data suggest that protocol development is needed to assess the efficacy of booster vaccination.

Rapid induction of antigen-specific CD4+ T cells is associated with coordinated humoral and cellular immunity to SARS-CoV-2 mRNA vaccination.

SARS-CoV-2 mRNA vaccines have shown remarkable clinical efficacy, but questions remain about the nature and kinetics of T cell priming. We performed longitudinal antigen-specific T cell analyses on healthy SARS-CoV-2-naive and recovered individuals prior to and following mRNA prime and boost vaccination. Vaccination induced rapid antigen-specific CD4+ T cell responses in naive subjects after the first dose, whereas CD8+ T cell responses developed gradually and were variable in magnitude. Vaccine-induced Th1 and Tfh cell responses following the first dose correlated with post-boost CD8+ T cells and neutralizing antibodies, respectively. Integrated analysis revealed coordinated immune responses with distinct trajectories in SARS-CoV-2-naive and recovered individuals. Last, whereas booster vaccination improved T cell responses in SARS-CoV-2-naive subjects, the second dose had little effect in SARS-CoV-2-recovered individuals. These findings highlight the role of rapidly primed CD4+ T cells in coordinating responses to the second vaccine dose in SARS-CoV-2-naive individuals.

Convalescent-Phase Sera and Vaccine-Elicited Antibodies Largely Maintain Neutralizing Titer against Global SARS-CoV-2 Variant Spikes.

The increasing prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with spike protein mutations raises concerns that antibodies elicited by natural infection or vaccination and therapeutic monoclonal antibodies will become less effective. We show that convalescent-phase sera neutralize pseudotyped viruses with the B.1.1.7, B.1.351, B.1.1.248, COH.20G/677H, 20A.EU2, and mink cluster 5 spike proteins with only a minor loss in titer. Similarly, antibodies elicited by Pfizer BNT162b2 vaccination neutralized B.1.351 and B.1.1.248 with only a 3-fold decrease in titer, an effect attributable to E484K. Analysis of the Regeneron monoclonal antibodies REGN10933 and REGN10987 showed that REGN10933 has lost neutralizing activity against the B.1.351 and B.1.1.248 pseudotyped viruses, and the cocktail is 9- to 15-fold decreased in titer. These findings suggest that antibodies elicited by natural infection and by the Pfizer vaccine will maintain protection against the B.1.1.7, B.1.351, and B.1.1.248 variants but that monoclonal antibody therapy may be less effective for patients infected with B.1.351 or B.1.1.248 SARS-CoV-2. IMPORTANCE The rapid evolution of SARS-CoV-2 variants has raised concerns with regard to their potential to escape from vaccine-elicited antibodies and anti-spike protein monoclonal antibodies. We report here on an analysis of sera from recovered patients and vaccinated individuals and on neutralization by Regeneron therapeutic monoclonal antibodies. Overall, the variants were neutralized nearly as well as the wild-type pseudotyped virus. The B.1.351 variant was somewhat resistant to vaccine-elicited antibodies but was still readily neutralized. One of the two Regeneron therapeutic monoclonal antibodies seems to have lost most of its activity against the B.1.351 variant, raising concerns that the combination therapy might be less effective for some patients. The findings should alleviate concerns that vaccines will become ineffective but suggest the importance of continued surveillance for potential new variants.