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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.

SARS-CoV-2 messenger RNA vaccination in healthy individuals generates immune protection against COVID-19. However, little is known about SARS-CoV-2 mRNA vaccine-induced responses in immunosuppressed patients. We investigated induction of antigen-specific antibody, B cell and T cell responses longitudinally in patients with multiple sclerosis (MS) on anti-CD20 antibody monotherapy (n = 20) compared with healthy controls (n = 10) after BNT162b2 or mRNA-1273 mRNA vaccination. Treatment with anti-CD20 monoclonal antibody (aCD20) significantly reduced spike-specific and receptor-binding domain (RBD)-specific antibody and memory B cell responses in most patients, an effect ameliorated with longer duration from last aCD20 treatment and extent of B cell reconstitution. By contrast, all patients with MS treated with aCD20 generated antigen-specific CD4 and CD8 T cell responses after vaccination. Treatment with aCD20 skewed responses, compromising circulating follicular helper T (T.sub.FH) cell responses and augmenting CD8 T cell induction, while preserving type 1 helper T (T.sub.H1) cell priming. Patients with MS treated with aCD20 lacking anti-RBD IgG had the most severe defect in circulating T.sub.FH responses and more robust CD8 T cell responses. These data define the nature of the SARS-CoV-2 vaccine-induced immune landscape in aCD20-treated patients and provide insights into coordinated mRNA vaccine-induced immune responses in humans. Our findings have implications for clinical decision-making and public health policy for immunosuppressed patients including those treated with aCD20. SARS-CoV-2-specific antibodies and memory B cells are significantly reduced, but CD4.sup.+ and CD8.sup.+ T cells are robustly activated, in patients with multiple sclerosis on anti-CD20 monotherapy versus healthy controls after BNT162b2 or mRNA-1273 mRNA vaccination., Author(s): Sokratis A. Apostolidis [sup.1] [sup.2] [sup.3] , Mihir Kakara [sup.4] [sup.5] , Mark M. Painter [sup.1] [sup.3] [sup.6] , Rishi R. Goel [sup.1] [sup.3] [sup.6] , Divij Mathew [sup.1] [...]

B cell memory after SARS-CoV-2 mRNA vaccination: Clinical trials of mRNA-based vaccines for SARS-CoV-2 have confirmed their ability to provide robust protection against COVID-19. Many studies demonstrate antibody responses to these vaccines, but the timing of B cell memory formation after vaccination is unclear. Goel et al. studied the antibody and B cell memory responses to SARS-CoV-2 mRNA vaccines using a cohort of SARS-CoV-2 naïve and convalescent patients that received the Pfizer or Moderna vaccines. Two shots of the mRNA vaccines were needed to induce peak antibody and memory B cell responses against SARS-CoV-2 in naïve patients, whereas only one shot induced peak responses in convalescent patients. These antibodies could neutralize the more infectious B.1.351 variant. These data provide further evidence of robust, protective immune responses to SARS-CoV-2 after mRNA vaccination. mRNA vaccines for SARS-CoV-2 have been authorized for emergency use. Despite their efficacy in clinical trials, data on mRNA vaccine–induced immune responses are mostly limited to serological analyses. Here, we interrogated antibody and antigen-specific memory B cells over time in 33 SARS-CoV-2 naïve and 11 SARS-CoV-2 recovered subjects. SARS-CoV-2 naïve individuals required both vaccine doses for optimal increases in antibodies, particularly for neutralizing titers against the B.1.351 variant. Memory B cells specific for full-length spike protein and the spike receptor binding domain (RBD) were also efficiently primed by mRNA vaccination and detectable in all SARS-CoV-2 naïve subjects after the second vaccine dose, although the memory B cell response declined slightly with age. In SARS-CoV-2 recovered individuals, antibody and memory B cell responses were significantly boosted after the first vaccine dose; however, there was no increase in circulating antibodies, neutralizing titers, or antigen-specific memory B cells after the second dose. This robust boosting after the first vaccine dose strongly correlated with levels of preexisting memory B cells in recovered individuals, identifying a key role for memory B cells in mounting recall responses to SARS-CoV-2 antigens. Together, our data demonstrated robust serological and cellular priming by mRNA vaccines and revealed distinct responses based on prior SARS-CoV-2 exposure, whereby COVID-19 recovered subjects may only require a single vaccine dose to achieve peak antibody and memory B cell responses. These findings also highlight the utility of defining cellular responses in addition to serologies and may inform SARS-CoV-2 vaccine distribution in a resource-limited setting. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • The first and second mRNA vaccine doses elicit different types of S antibodies • SARS-CoV-2 infections elicit antibodies that bind strongly to common coronaviruses • These common coronavirus-reactive antibodies bind weakly to SARS-CoV-2 • Vaccinations elicit different types of antibodies compared with SARS-CoV-2 infections Anderson et al. show that SARS-CoV-2 infections elicit high levels 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. They find that SARS-CoV-2 vaccination elicits lower levels of original antigenic sin-like antibodies. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Neutralizing antibody titers stabilize ∼6 months after primary vaccination • Memory B cells are stable for >9 months postvaccination and >50% cross-bind Omicron • Omicron-reactive memory B cells are reactivated by a 3rd dose of wild-type vaccine • Low preboost antibody levels correlate with a greater fold increase after boosting Immunization with 2 doses of mRNA vaccine encoding the ancestral SARS-CoV-2 spike protein induces a population of durable memory B cells with broad reactivity to viral variants including Omicron. Boosting with a 3rd dose of ancestral vaccine increases variant-neutralizing antibody levels, highlighting the significance of vaccine-induced B cell memory. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • mRNA vaccines generate antigen-specific T cells in a coordinated immune response • Vaccine-induced T cells resemble the durable memory cells primed by infection • Th1 and cTfh cell responses to the first dose correlate with second-dose responses • SARS-CoV-2-recovered individuals benefit from the first but not the second dose SARS-CoV-2 mRNA vaccines have demonstrated remarkable efficacy, but T cell responses to vaccination have not been well studied. In a longitudinal cohort, Painter et al. show that mRNA vaccines activate SARS-CoV-2-specific T cells that could contribute to durable immunity. The findings highlight the central role of T cells in the two-dose vaccine regimen for individuals not previously infected with SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Summary Long-lived reservoirs of persistent HIV are a major barrier to a cure. CD4+ hematopoietic stem and progenitor cells (HSPCs) have the capacity for lifelong survival, self-renewal, and the generation of daughter cells. Recent evidence shows that they are also susceptible to HIV infection in vitro and in vivo. Whether HSPCs harbor infectious virus or contribute to plasma virus (PV) is unknown. Here, we provide strong evidence that clusters of identical proviruses from HSPCs and their likely progeny often match residual PV. A higher proportion of these sequences match residual PV than proviral genomes from bone marrow and peripheral blood mononuclear cells that are observed only once. Furthermore, an analysis of near-full-length genomes isolated from HSPCs provides evidence that HSPCs harbor functional HIV proviral genomes that often match residual PV. These results support the conclusion that HIV-infected HSPCs form a distinct and functionally significant reservoir of persistent HIV in infected people. Graphical Abstract Highlights • Hematopoietic stem and progenitor cells can serve as long-term reservoirs of HIV • HSPCs harbor both infectious and defective proviral genomes • HSPCs are an important source of residual plasma virus in treated people • Clonally amplified HIV proviruses contribute to residual plasma virus HIV causes an infection that persists even when optimal therapy is used. Zaikos et al. provide evidence that HIV-infected progenitor cells from the bone marrow can amplify virus through normal cellular growth pathways in some treated people. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Human GC responses to SARS-CoV-2 vaccines are restricted to draining lymph nodes • Vaccine-induced GC responses are associated with SARS-CoV-2 neutralizing antibodies • Kidney transplant recipients lack GC responses to SARS-CoV-2 vaccines • Vaccines expand S+RBD− memory B cells in healthy donors and transplant recipients Fine-needle aspiration of lymph nodes in humans reveals that SARS-CoV-2 vaccination induces neutralizing antibody-producing germinal centers, enhanced by repeated vaccination. Conversely, in patients receiving immunosuppressant medication, this process is disrupted, resulting in stunted protective immune responses, highlighting issues about vaccine and booster efficacy in patients with compromised immune systems. [ABSTRACT FROM AUTHOR]

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., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Novel mRNA vaccines for SARS-CoV-2 have been authorized for emergency use. Despite their efficacy in clinical trials, data on mRNA vaccine-induced immune responses are mostly limited to serological analyses. Here, we interrogated antibody and antigen-specific memory B cells over time in 33 SARS-CoV-2 naïve and 11 SARS-CoV-2 recovered subjects. SARS-CoV-2 naïve individuals required both vaccine doses for optimal increases in antibodies, particularly for neutralizing titers against the B.1.351 variant. Memory B cells specific for full-length spike protein and the spike receptor binding domain (RBD) were also efficiently primed by mRNA vaccination and detectable in all SARS-CoV-2 naive subjects after the second vaccine dose, though the memory B cell response declined slightly with age. In SARS-CoV-2 recovered individuals, antibody and memory B cell responses were significantly boosted after the first vaccine dose; however, there was no increase in circulating antibodies, neutralizing titers, or antigen-specific memory B cells after the second dose. This robust boosting after the first vaccine dose strongly correlated with levels of pre-existing memory B cells in recovered individuals, identifying a key role for memory B cells in mounting recall responses to SARS-CoV-2 antigens. Together, our data demonstrated robust serological and cellular priming by mRNA vaccines and revealed distinct responses based on prior SARS-CoV-2 exposure, whereby COVID-19 recovered subjects may only require a single vaccine dose to achieve peak antibody and memory B cell responses. These findings also highlight the utility of defining cellular responses in addition to serologies and may inform SARS-CoV-2 vaccine distribution in a resource-limited setting., (Copyright © 2021, American Association for the Advancement of Science.)

HIV-1 Vpr is necessary for maximal HIV infection and spread in macrophages. Evolutionary conservation of Vpr suggests an important yet poorly understood role for macrophages in HIV pathogenesis. Vpr counteracts a previously unknown macrophage-specific restriction factor that targets and reduces the expression of HIV Env. Here, we report that the macrophage mannose receptor (MR), is a restriction factor targeting Env in primary human monocyte-derived macrophages. Vpr acts synergistically with HIV Nef to target distinct stages of the MR biosynthetic pathway and dramatically reduce MR expression. Silencing MR or deleting mannose residues on Env rescues Env expression in HIV-1-infected macrophages lacking Vpr. However, we also show that disrupting interactions between Env and MR reduces initial infection of macrophages by cell-free virus. Together these results reveal a Vpr-Nef-Env axis that hijacks a host mannose-MR response system to facilitate infection while evading MR's normal role, which is to trap and destroy mannose-expressing pathogens., Competing Interests: JL, MV, MM, DC, MM, BP, ZL, MP, FG, VT, GZ, KC No competing interests declared, (© 2020, Lubow et al.)