Germinal center responses to SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals.

Vaccine-mediated immunity often relies on the generation of protective antibodies and memory B cells, which commonly stem from germinal center (GC) reactions. An in-depth comparison of the GC responses elicited by SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals has not yet been performed due to the challenge of directly probing human lymph nodes. Herein, through a fine-needle aspiration-based approach, we profiled the immune responses to SARS-CoV-2 mRNA vaccines in lymph nodes of healthy individuals and kidney transplant recipients (KTXs). We found that, unlike healthy subjects, KTXs presented deeply blunted SARS-CoV-2-specific GC B cell responses coupled with severely hindered T follicular helper cell, SARS-CoV-2 receptor binding domain-specific memory B cell, and neutralizing antibody responses. KTXs also displayed reduced SARS-CoV-2-specific CD4 and CD8 T cell frequencies. Broadly, these data indicate impaired GC-derived immunity in immunocompromised individuals and suggest a GC origin for certain humoral and memory B cell responses following mRNA vaccination.

Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine.

We examined antibody and memory B cell responses longitudinally for ∼9-10 months after primary 2-dose SARS-CoV-2 mRNA vaccination and 3 months after a 3rd dose. Antibody decay stabilized between 6 and 9 months, and antibody quality continued to improve for at least 9 months after 2-dose vaccination. Spike- and RBD-specific memory B cells remained durable over time, and 40%-50% of RBD-specific memory B cells simultaneously bound the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells were efficiently reactivated by a 3rd dose of wild-type vaccine and correlated with the corresponding increase in neutralizing antibody titers. In contrast, pre-3rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit the added protection afforded by repeat short interval boosting. These data provide insight into the quantity and quality of mRNA-vaccine-induced immunity over time through 3 or more antigen exposures.

Prior vaccination promotes early activation of memory T cells and enhances immune responses during SARS-CoV-2 breakthrough infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of vaccinated individuals is increasingly common but rarely results in severe disease, likely due to the enhanced potency and accelerated kinetics of memory immune responses. However, there have been few opportunities to rigorously study early recall responses during human viral infection. To better understand human immune memory and identify potential mediators of lasting vaccine efficacy, we used high-dimensional flow cytometry and SARS-CoV-2 antigen probes to examine immune responses in longitudinal samples from vaccinated individuals infected during the Omicron wave. These studies revealed heightened spike-specific responses during infection of vaccinated compared to unvaccinated individuals. Spike-specific cluster of differentiation (CD)4 T cells and plasmablasts expanded and CD8 T cells were robustly activated during the first week. In contrast, memory B cell activation, neutralizing antibody production and primary responses to nonspike antigens occurred during the second week. Collectively, these data demonstrate the functionality of vaccine-primed immune memory and highlight memory T cells as rapid responders during SARS-CoV-2 infection.

SARS-CoV-2 reservoir in post-acute sequelae of COVID-19 (PASC).

Millions of people are suffering from Long COVID or post-acute sequelae of COVID-19 (PASC). Several biological factors have emerged as potential drivers of PASC pathology. Some individuals with PASC may not fully clear the coronavirus SARS-CoV-2 after acute infection. Instead, replicating virus and/or viral RNA-potentially capable of being translated to produce viral proteins-persist in tissue as a 'reservoir'. This reservoir could modulate host immune responses or release viral proteins into the circulation. Here we review studies that have identified SARS-CoV-2 RNA/protein or immune responses indicative of a SARS-CoV-2 reservoir in PASC samples. Mechanisms by which a SARS-CoV-2 reservoir may contribute to PASC pathology, including coagulation, microbiome and neuroimmune abnormalities, are delineated. We identify research priorities to guide the further study of a SARS-CoV-2 reservoir in PASC, with the goal that clinical trials of antivirals or other therapeutics with potential to clear a SARS-CoV-2 reservoir are accelerated.

Shared and distinct biological circuits in effector, memory and exhausted CD8+ T cells revealed by temporal single-cell transcriptomics and epigenetics.

Naïve CD8+ T cells can differentiate into effector (Teff), memory (Tmem) or exhausted (Tex) T cells. These developmental pathways are associated with distinct transcriptional and epigenetic changes that endow cells with different functional capacities and therefore therapeutic potential. The molecular circuitry underlying these developmental trajectories and the extent of heterogeneity within Teff, Tmem and Tex populations remain poorly understood. Here, we used the lymphocytic choriomeningitis virus model of acute-resolving and chronic infection to address these gaps by applying longitudinal single-cell RNA-sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analyses. These analyses uncovered new subsets, including a subpopulation of Tex cells expressing natural killer cell-associated genes that is dependent on the transcription factor Zeb2, as well as multiple distinct TCF-1+ stem/progenitor-like subsets in acute and chronic infection. These data also revealed insights into the reshaping of Tex subsets following programmed death 1 (PD-1) pathway blockade and identified a key role for the cell stress regulator, Btg1, in establishing the Tex population. Finally, these results highlighted how the same biological circuits such as cytotoxicity or stem/progenitor pathways can be used by CD8+ T cell subsets with highly divergent underlying chromatin landscapes generated during different infections.

Immunological imprinting shapes the specificity of human antibody responses against SARS-CoV-2 variants.

The spike glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to accumulate substitutions, leading to breakthrough infections of vaccinated individuals. It remains unclear if exposures to antigenically distant SARS-CoV-2 variants can overcome memory B cell biases established by initial SARS-CoV-2 encounters. We determined the specificity and functionality of antibody and B cell responses following exposure to BA.5 and XBB variants in individuals who received ancestral SARS-CoV-2 mRNA vaccines. BA.5 exposures elicited antibody responses that targeted epitopes conserved between the BA.5 and ancestral spike. XBB exposures also elicited antibody responses that primarily targeted epitopes conserved between the XBB.1.5 and ancestral spike. However, unlike BA.5, a single XBB exposure elicited low frequencies of XBB.1.5-specific antibodies and B cells in some individuals. Pre-existing cross-reactive B cells and antibodies were correlated with stronger overall responses to XBB but weaker XBB-specific responses, suggesting that baseline immunity influences the activation of variant-specific SARS-CoV-2 responses.

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.

Concanamycin A counteracts HIV-1 Nef to enhance immune clearance of infected primary cells by cytotoxic T lymphocytes.

Nef is an HIV-encoded accessory protein that enhances pathogenicity by down-regulating major histocompatibility class I (MHC-I) expression to evade killing by cytotoxic T lymphocytes (CTLs). A potent Nef inhibitor that restores MHC-I is needed to promote immune-mediated clearance of HIV-infected cells. We discovered that the plecomacrolide family of natural products restored MHC-I to the surface of Nef-expressing primary cells with variable potency. Concanamycin A (CMA) counteracted Nef at subnanomolar concentrations that did not interfere with lysosomal acidification or degradation and were nontoxic in primary cell cultures. CMA specifically reversed Nef-mediated down-regulation of MHC-I, but not CD4, and cells treated with CMA showed reduced formation of the Nef:MHC-I:AP-1 complex required for MHC-I down-regulation. CMA restored expression of diverse allotypes of MHC-I in Nef-expressing cells and inhibited Nef alleles from divergent clades of HIV and simian immunodeficiency virus, including from primary patient isolates. Lastly, we found that restoration of MHC-I in HIV-infected cells was accompanied by enhanced CTL-mediated clearance of infected cells comparable to genetic deletion of Nef. Thus, we propose CMA as a lead compound for therapeutic inhibition of Nef to enhance immune-mediated clearance of HIV-infected cells.

Class 1-Selective Histone Deacetylase (HDAC) Inhibitors Enhance HIV Latency Reversal while Preserving the Activity of HDAC Isoforms Necessary for Maximal HIV Gene Expression.

Combinations of drugs that affect distinct mechanisms of HIV latency aim to induce robust latency reversal leading to cytopathicity and elimination of the persistent HIV reservoir. Thus far, attempts have focused on combinations of protein kinase C (PKC) agonists and pan-histone deacetylase inhibitors (HDIs) despite the knowledge that HIV gene expression is regulated by class 1 histone deacetylases. We hypothesized that class 1-selective HDIs would promote more robust HIV latency reversal in combination with a PKC agonist than pan-HDIs because they preserve the activity of proviral factors regulated by non-class 1 histone deacetylases. Here, we show that class 1-selective agents used alone or with the PKC agonist bryostatin-1 induced more HIV protein expression per infected cell. In addition, the combination of entinostat and bryostatin-1 induced viral outgrowth, whereas bryostatin-1 combinations with pan-HDIs did not. When class 1-selective HDIs were used in combination with pan-HDIs, the amount of viral protein expression and virus outgrowth resembled that of pan-HDIs alone, suggesting that pan-HDIs inhibit robust gene expression induced by class 1-selective HDIs. Consistent with this, pan-HDI-containing combinations reduced the activity of NF-κB and Hsp90, two cellular factors necessary for potent HIV protein expression, but did not significantly reduce overall cell viability. An assessment of viral clearance from in vitro cultures indicated that maximal protein expression induced by class 1-selective HDI treatment was crucial for reservoir clearance. These findings elucidate the limitations of current approaches and provide a path toward more effective strategies to eliminate the HIV reservoir.IMPORTANCE Despite effective antiretroviral therapy, HIV evades eradication in a latent form that is not affected by currently available drug regimens. Pharmacologic latency reversal that leads to death of cellular reservoirs has been proposed as a strategy for reservoir elimination. Because histone deacetylases (HDACs) promote HIV latency, HDAC inhibitors have been a focus of HIV cure research. However, many of these inhibitors broadly affect multiple classes of HDACs, including those that promote HIV gene expression (class 1 HDACs). Here, we demonstrate that targeted treatment with class 1-selective HDAC inhibitors induced more potent HIV latency reversal than broadly acting agents. Additionally, we provide evidence that broadly acting HDIs are limited by inhibitory effects on non-class 1 HDACs that support the activity of proviral factors. Thus, our work demonstrates that the use of targeted approaches to induce maximum latency reversal affords the greatest likelihood of reservoir elimination.

Quiescence Promotes Latent HIV Infection and Resistance to Reactivation from Latency with Histone Deacetylase Inhibitors.

Human immunodeficiency virus type 1 (HIV-1) establishes transcriptionally silent latent infections in resting memory T cells and hematopoietic stem and progenitor cells (HSPCs), which allows the virus to persist in infected individuals despite antiretroviral therapy. Developing in vitro models of HIV-1 latency that recapitulate the characteristics of latently infected cells in vivo is crucial to identifying and developing effective latency-reversing therapies. HSPCs exist in a quiescent state in vivo, and quiescence is correlated with latent infections in T cells. However, current models for culturing HSPCs and for infecting T cells in vitro require that the cells be maintained in an actively proliferating state. Here we describe a novel culture system in which primary human HSPCs cultured under hypothermic conditions are maintained in a quiescent state. We show that these quiescent HSPCs are susceptible to predominantly latent infection with HIV-1, while actively proliferating and differentiating HSPCs obtain predominantly active infections. Furthermore, we demonstrate that the most primitive quiescent HSPCs are more resistant to spontaneous reactivation from latency than more differentiated HSPCs and that quiescent HSPCs are resistant to reactivation by histone deacetylase inhibitors or P-TEFb activation but are susceptible to reactivation by protein kinase C (PKC) agonists. We also demonstrate that inhibition of HSP90, a known regulator of HIV transcription, recapitulates the quiescence and latency phenotypes of hypothermia, suggesting that hypothermia and HSP90 inhibition may regulate these processes by similar mechanisms. In summary, these studies describe a novel model for studying HIV-1 latency in human primary cells maintained in a quiescent state.IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) establishes a persistent infection for which there remains no feasible cure. Current approaches are unable to clear the virus despite decades of therapy due to the existence of latent reservoirs of integrated HIV-1, which can reactivate and contribute to viral rebound following treatment interruption. Previous clinical attempts to reactivate the latent reservoirs in an individual so that they can be eliminated by the immune response or viral cytopathic effect have failed, indicating the need for a better understanding of the processes regulating HIV-1 latency. Here we characterize a novel in vitro model of HIV-1 latency in primary hematopoietic stem and progenitor cells isolated from human cord blood that may better recapitulate the behavior of latently infected cells in vivo This model can be used to study mechanisms regulating latency and potential therapeutic approaches to reactivate latent infections in quiescent cells.

Immunological imprinting shapes the specificity of human antibody responses against SARS-CoV-2 variants.

The spike glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to accumulate substitutions, leading to breakthrough infections of vaccinated individuals and prompting the development of updated booster vaccines. Here, we determined the specificity and functionality of antibody and B cell responses following exposure to BA.5 and XBB variants in individuals who received ancestral SARS-CoV-2 mRNA vaccines. BA.5 exposures elicited antibody responses that primarily targeted epitopes conserved between the BA.5 and ancestral spike, with poor reactivity to the XBB.1.5 variant. XBB exposures also elicited antibody responses that targeted epitopes conserved between the XBB.1.5 and ancestral spike. However, unlike BA.5, a single XBB exposure elicited low levels of XBB.1.5-specific antibodies and B cells in some individuals. Pre-existing cross-reactive B cells and antibodies were correlated with stronger overall responses to XBB but weaker XBB-specific responses, suggesting that baseline immunity influences the activation of variant-specific SARS-CoV-2 responses.

Structure-Activity Relationships of Natural and Semisynthetic Plecomacrolides Suggest Distinct Pathways for HIV-1 Immune Evasion and Vacuolar ATPase-Dependent Lysosomal Acidification.

The human immunodeficiency virus (HIV)-encoded accessory protein Nef enhances pathogenicity by reducing major histocompatibility complex I (MHC-I) cell surface expression, protecting HIV-infected cells from immune recognition. Nef-dependent downmodulation of MHC-I can be reversed by subnanomolar concentrations of concanamycin A (1), a well-known inhibitor of vacuolar ATPase, at concentrations below those that interfere with lysosomal acidification or degradation. We conducted a structure-activity relationship study that assessed 76 compounds for Nef inhibition, 24 and 72 h viability, and lysosomal neutralization in Nef-expressing primary T cells. This analysis demonstrated that the most potent compounds were natural concanamycins and their derivatives. Comparison against a set of new, semisynthetic concanamycins revealed that substituents at C-8 and acylation of C-9 significantly affected Nef potency, target cell viability, and lysosomal neutralization. These findings provide important progress toward understanding the mechanism of action of these compounds and the identification of an advanced lead anti-HIV Nef inhibitory compound.

Automated Cytometric Gating with Human-Level Performance Using Bivariate Segmentation.

Recent advances in cytometry technology have enabled high-throughput data collection with multiple single-cell protein expression measurements. The significant biological and technical variance between samples in cytometry has long posed a formidable challenge during the gating process, especially for the initial gates which deal with unpredictable events, such as debris and technical artifacts. Even with the same experimental machine and protocol, the target population, as well as the cell population that needs to be excluded, may vary across different measurements. To address this challenge and mitigate the labor-intensive manual gating process, we propose a deep learning framework UNITO to rigorously identify the hierarchical cytometric subpopulations. The UNITO framework transformed a cell-level classification task into an image-based semantic segmentation problem. For reproducibility purposes, the framework was applied to three independent cohorts and successfully detected initial gates that were required to identify single cellular events as well as subsequent cell gates. We validated the UNITO framework by comparing its results with previous automated methods and the consensus of at least four experienced immunologists. UNITO outperformed existing automated methods and differed from human consensus by no more than each individual human. Most critically, UNITO framework functions as a fully automated pipeline after training and does not require human hints or prior knowledge. Unlike existing multi-channel classification or clustering pipelines, UNITO can reproduce a similar contour compared to manual gating for each intermediate gating to achieve better interpretability and provide post hoc visual inspection. Beyond acting as a pioneering framework that uses image segmentation to do auto-gating, UNITO gives a fast and interpretable way to assign the cell subtype membership, and the speed of UNITO will not be impacted by the number of cells from each sample. The pre-gating and gating inference takes approximately 2 minutes for each sample using our pre-defined 9 gates system, and it can also adapt to any sequential prediction with different configurations.

An extended SARS-CoV-2 mRNA vaccine prime-boost interval enhances B cell immunity with limited impact on T cells.

Spacing the first two doses of SARS-CoV-2 mRNA vaccines beyond 3-4 weeks raised initial concerns about vaccine efficacy. While studies have since shown that long-interval regimens induce robust antibody responses, their impact on B and T cell immunity is poorly known. Here, we compare SARS-CoV-2 naive donors B and T cell responses to two mRNA vaccine doses administered 3-4 versus 16 weeks apart. After boost, the longer interval results in a higher magnitude and a more mature phenotype of RBD-specific B cells. While the two geographically distinct cohorts present quantitative and qualitative differences in T cell responses at baseline and after priming, the second dose led to convergent features with overall similar magnitude, phenotype, and function of CD4+ and CD8+ T cell responses at post-boost memory time points. Therefore, compared to standard regimens, a 16-week interval has a favorable impact on the B cell compartment but minimally affects T cell immunity.

Prior vaccination enhances immune responses during SARS-CoV-2 breakthrough infection with early activation of memory T cells followed by production of potent neutralizing antibodies.

SARS-CoV-2 infection of vaccinated individuals is increasingly common but rarely results in severe disease, likely due to the enhanced potency and accelerated kinetics of memory immune responses. However, there have been few opportunities to rigorously study early recall responses during human viral infection. To better understand human immune memory and identify potential mediators of lasting vaccine efficacy, we used high-dimensional flow cytometry and SARS-CoV-2 antigen probes to examine immune responses in longitudinal samples from vaccinated individuals infected during the Omicron wave. These studies revealed heightened Spike-specific responses during infection of vaccinated compared to unvaccinated individuals. Spike-specific CD4 T cells and plasmablasts expanded and CD8 T cells were robustly activated during the first week. In contrast, memory B cell activation, neutralizing antibody production, and primary responses to non-Spike antigens occurred during the second week. Collectively, these data demonstrate the functionality of vaccine-primed immune memory and highlight memory T cells as rapid responders during SARS-CoV-2 infection.

Hematopoietic Stem and Progenitor Cells (HSPCs).

Cord blood is a readily available source of hematopoietic stem and progenitor cells (HSPCs) which can be infected with HIV-1 in vitro to produce inducible latently infected cells for reactivation studies. Infected HSPCs can also be found in the setting of clinically undetectable viremia in vivo. Here we describe an in vitro infection model utilizing cord blood derived HSPCs, as well as methods for isolating and characterizing provirus from bone marrow HSPCs from suppressed patients.

SARS-CoV-2 infections elicit higher levels of original antigenic sin antibodies compared with SARS-CoV-2 mRNA vaccinations.

It is important to determine if severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and SARS-CoV-2 mRNA vaccinations elicit different types of antibodies. Here, we characterize the magnitude and specificity of SARS-CoV-2 spike-reactive antibodies from 10 acutely infected health care workers with no prior SARS-CoV-2 exposure history and 23 participants who received SARS-CoV-2 mRNA vaccines. We found that infection and primary mRNA vaccination elicit S1- and S2-reactive antibodies, while secondary vaccination boosts mostly S1 antibodies. Using absorption assays, we found that SARS-CoV-2 infections elicit a large proportion of original antigenic sin-like antibodies that bind efficiently to the spike of common seasonal human coronaviruses but poorly to the spike of SARS-CoV-2. In converse, vaccination modestly boosts antibodies reactive to the spike of common seasonal human coronaviruses, and these antibodies cross-react more efficiently to the spike of SARS-CoV-2. Our data indicate that SARS-CoV-2 infections and mRNA vaccinations elicit fundamentally different antibody responses.

A boost with SARS-CoV-2 BNT162b2 mRNA vaccine elicits strong humoral responses independently of the interval between the first two doses.

Due to the recrudescence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections worldwide, mainly caused by the Omicron variant of concern (VOC) and its sub-lineages, several jurisdictions are administering an mRNA vaccine boost. Here, we analyze humoral responses induced after the second and third doses of an mRNA vaccine in naive and previously infected donors who received their second dose with an extended 16-week interval. We observe that the extended interval elicits robust humoral responses against VOCs, but this response is significantly diminished 4 months after the second dose. Administering a boost to these individuals brings back the humoral responses to the same levels obtained after the extended second dose. Interestingly, we observe that administering a boost to individuals that initially received a short 3- to 4-week regimen elicits humoral responses similar to those observed in the long interval regimen. Nevertheless, humoral responses elicited by the boost in naive individuals do not reach those present in previously infected vaccinated individuals.

Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine.

Despite a clear role in protective immunity, the durability and quality of antibody and memory B cell responses induced by mRNA vaccination, particularly by a 3 rd dose of vaccine, remains unclear. Here, we examined antibody and memory B cell responses in a cohort of individuals sampled longitudinally for ∼9-10 months after the primary 2-dose mRNA vaccine series, as well as for ∼3 months after a 3 rd mRNA vaccine dose. Notably, antibody decay slowed significantly between 6- and 9-months post-primary vaccination, essentially stabilizing at the time of the 3 rd dose. Antibody quality also continued to improve for at least 9 months after primary 2-dose vaccination. Spike- and RBD-specific memory B cells were stable through 9 months post-vaccination with no evidence of decline over time, and ∼40-50% of RBD-specific memory B cells were capable of simultaneously recognizing the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells induced by the first 2 doses of mRNA vaccine were boosted significantly by a 3rd dose and the magnitude of this boosting was similar to memory B cells specific for other variants. Pre-3 rd dose memory B cell frequencies correlated with the increase in neutralizing antibody titers after the 3 rd dose. In contrast, pre-3 rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit reactivation of immunological memory and constrain further antibody boosting by mRNA vaccines. These data provide a deeper understanding of how the quantity and quality of antibody and memory B cell responses change over time and number of antigen exposures. These data also provide insight into potential immune dynamics following recall responses to additional vaccine doses or post-vaccination infections.