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Messenger RNA (mRNA) vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective at inducing protective immunity. However, weak antibody responses are seen in some individuals, and cellular correlates of immunity remain poorly defined, especially for B cells. Here we used unbiased approaches to longitudinally dissect primary antibody, plasmablast, and memory B cell (MBC) responses to the two-dose mRNA-1273 vaccine in SARS-CoV-2–naive adults. Coordinated immunoglobulin A (IgA) and IgG antibody responses were preceded by bursts of spike-specific plasmablasts after both doses but earlier and more intensely after dose 2. While antibody and B cell cellular responses were generally robust, they also varied within the cohort and decreased over time after a dose-2 peak. Both antigen-nonspecific postvaccination plasmablast frequency after dose 1 and their spike-specific counterparts early after dose 2 correlated with subsequent antibody levels. This correlation between early plasmablasts and antibodies remained for titers measured at 6 months after vaccination. Several distinct antigen-specific MBC populations emerged postvaccination with varying kinetics, including two MBC populations that correlated with 2- and 6-month antibody titers. Both were IgG-expressing MBCs: one less mature, appearing as a correlate after the first dose, while the other MBC correlate showed a more mature and resting phenotype, emerging as a correlate later after dose 2. This latter MBC was also a major contributor to the sustained spike-specific MBC response observed at month 6. Thus, these plasmablasts and MBCs that emerged after both the first and second doses with distinct kinetics are potential determinants of the magnitude and durability of antibodies in response to mRNA-based vaccination.

Significance HIV-1 infection requires lifelong treatment with antiretroviral therapy (ART) due to viral rebound of a latent reservoir of intact, transcriptionally silent provirus found to persist in the genome of CD4+ T cells. One major challenge to understanding the nature of the latent reservoir is accurately characterizing the measuring of the size of the reservoir. Herein, we use quadruplex polymerase chain reaction (Q4PCR) to assess the dynamics of the latent reservoir in HIV+ individuals who have been on long-term ART for up to 10 y. Our results show that Q4PCR can be used to accurately measure the latent reservoir, while providing the added benefit of assessing the genetic diversity of the reservoir to better understand changes to clonal dynamics over time., HIV-1 infection produces a long-lived reservoir of latently infected CD4+ T cells that represents the major barrier to HIV-1 cure. The reservoir contains both intact and defective proviruses, but only the proviruses that are intact can reinitiate infection upon cessation of antiretroviral therapy (ART). Here we combine four-color quantitative PCR and next-generation sequencing (Q4PCR) to distinguish intact and defective proviruses and measure reservoir content longitudinally in 12 infected individuals. Q4PCR differs from other PCR-based methods in that the amplified proviruses are sequence verified as intact or defective. Samples were collected systematically over the course of up to 10 y beginning shortly after the initiation of ART. The size of the defective reservoir was relatively stable with minimal decay during the 10-y observation period. In contrast, the intact proviral reservoir decayed with an estimated half-life of 4.9 y. Nevertheless, both intact and defective proviral reservoirs are dynamic. As a result, the fraction of intact proviruses found in expanded clones of CD4+ T cells increases over time with a concomitant decrease in overall reservoir complexity. Thus, reservoir decay measurements by Q4PCR are quantitatively similar to viral outgrowth assay (VOA) and intact proviral DNA PCR assay (IPDA) with the addition of sequence information that distinguishes intact and defective proviruses and informs reservoir dynamics. The data are consistent with the notion that intact and defective proviruses are under distinct selective pressure, and that the intact proviral reservoir is progressively enriched in expanded clones of CD4+ T cells resulting in diminishing complexity over time.

Several highly potent and broadly neutralizing monoclonal antibodies against HIV have recently been isolated from B cells of infected individuals. However, the effects of these antibodies on the persistent viral reservoirs in HIV-infected individuals receiving antiretroviral therapy (ART) are unknown. We show that several HIV-specific monoclonal antibodies--in particular, PGT121, VRC01, and VRC03--potently inhibited entry into CD4(+) T cells of HIV isolated from the latent viral reservoir of infected individuals whose plasma viremia was well controlled by ART. In addition, we demonstrate that HIV replication in autologous CD4(+) T cells derived from infected individuals receiving ART was profoundly suppressed by three aforementioned and other HIV-specific monoclonal antibodies. These findings have implications for passive immunotherapy as an approach toward controlling plasma viral rebound in patients whose ART is withdrawn.

Highly active antiretroviral therapy (HAART) decreases plasma viremia below the limits of detection in the majority of HIV-infected individuals, thus serving to slow disease progression. However, HAART targets only actively replicating virus and is unable to eliminate latently infected, resting CD4 + T cells. Such infected cells are potentially capable of reinitiating virus replication upon cessation of HAART, thus leading to viral rebound. Agents that would eliminate these reservoirs, when used in combination with HAART, could thus provide a strategy for the eradication of HIV. Prostratin is a preclinical candidate that induces HIV expression from latently infected CD4 + T cells, potentially leading to their elimination through a virus-induced cytopathic effect or host anti-HIV immunity. Here, we report the synthesis of a series of designed prostratin analogs and report in vitro and ex vivo studies of their activity relevant to induction of HIV expression. Members of this series are up to 100-fold more potent than the preclinical lead (prostratin) in binding to cell-free PKC, and in inducing HIV expression in a latently infected cell line and prostratin-like modulation of cell surface receptor expression in primary cells from HIV-negative donors. Significantly, selected members were also tested for HIV induction in resting CD4 + T cells isolated from infected individuals receiving HAART and were found to exhibit potent induction activity. These more potent agents and by extension related tunable analogs now accessible through the studies described herein should facilitate research and preclinical advancement of this strategy for HIV/AIDS eradication.

We and others have reported that the vast majority of virus-producing CD4 + T cells during the acute infection of rhesus macaques with simian immunodeficiency virus (SIV) or CXCR4 (X4)-using simian/human immunodeficiency viruses (SHIVs) exhibited a nonactivated phenotype. These findings have been extended to show that resting CD4 + T lymphocytes collected from SIV- or X4-SHIV-infected animals during the first 10 days of infection continue to release virus ex vivo. Furthermore, we observed high frequencies of integrated viral DNA (up to 5.1 × 10 4 DNA copies per 10 5 cells) in circulating resting CD4 + T cells during the first 10 days of the infection. Integration of SIV DNA was detected only in memory CD4 + T cells and SHIVs preferentially integrated into resting naïve CD4 + T cells. Taken together, these results show that during the acute infection large numbers of resting CD4 + T cells carry integrated nonhuman primate lentiviral DNA and are the major source of progeny virions irrespective of coreceptor usage. Prompt and sustained interventions are therefore required to block the rapid systemic dissemination of virus and prevent an otherwise fatal clinical outcome.

HIV envelope binds to and signals through its primary cellular receptor, CD4, and through a coreceptor, either CC chemokine receptor 5 (CCR5) or CXC chemokine receptor 4 (CXCR4). Here, we evaluate the response of peripheral blood mononuclear cells to a panel of genetically diverse R5 and X4 envelope proteins. Modulation of gene expression was evaluated by using oligonucleotide microarrays. Activation of transcription factors was evaluated by using an array of oligonucleotides encoding transcription factor binding sites. Responses were strongly influenced by coreceptor specificity. Treatment of cells from CCR5Δ32 homozygous donors with glycoprotein (gp)120 derived from an R5 virus demonstrated that the majority of responses elicited by R5 envelopes required engagement of CCR5. R5 envelopes, to a greater extent than X4 envelopes, induced the expression of genes belonging to mitogen-activated protein kinase signal transduction pathways and genes regulating the cell cycle. A number of genes induced by R5, but not X4, envelopes were also up-regulated in the resting CD4+T cell population of HIV-infected individuals. These results suggest that R5 envelope facilitates replication of HIV in the pool of resting CD4+T cells. Additionally, signaling by R5 gp120 may facilitate the transmission of R5 viruses by inducing a permissive environment for HIV replication.

Progression of HIV disease is associated with the appearance of numerous B cell defects. We describe herein a population of immature/transitional B cells that is overly represented in the peripheral blood of individuals with advancing HIV disease. These B cells, identified by the expression of CD10, were unresponsive by proliferation to B cell receptor triggering and possessed a phenotype and an Ig diversity profile that confirmed their immature/transitional stage of differentiation. Consistent with an immature status, their lack of proliferation to B cell receptor triggering was reversed with CD40 ligand, but not B cell activation factor. Finally, levels of CD10 expression on B cells were directly correlated with serum levels of IL-7, suggesting that increased levels of IL-7 modulate human B cell maturation either directly or indirectly by means of a homeostatic effect on lymphopenia. Taken together, these data offer insight into human B cell development as well as B cell dysfunction in advanced HIV disease that may be linked to IL-7-dependent homeostatic events.

CD8+T cells are critical for effective host defenses against viral infections. Studies addressing HIV-induced immune responses in infected individuals have suggested that CD8+T cells play an important role in controlling viral replication. However, despite an abundance of HIV-specific CD8+T cells, HIV is not contained in many untreated patients. Active HIV replication is associated with numerous immunologic changes, the most notable and consistent of which is an increase in CD8+T cells expressing CD38. Previous studies have demonstrated that the expression of CD38 on CD8+T cells is associated with poor prognostic outcome in infected individuals with detectable plasma viremia; however, the relationship between the expression of CD38 and the frequency of HIV-specific CD8+T cells is unclear. We demonstrate a correlation between levels of HIV-specific CD8+T cells and levels of CD8+T cells expressing CD38 in untreated patients. The distribution of HIV-specific CD8+T cells was heavily skewed toward CD38+CD8+T cells in patients with a high percentage of CD38+CD8+T cells. Spontaneous/Fas-mediated apoptosis in CD38+CD8+T cells was significantly higher in patients with high percentages of CD38+CD8+T cells. Our data suggest that a substantial proportion of the HIV-specific CD8+T cells present in CD38+CD8+T cells in patients with active viral replication arise by HIV-driven aberrant immune activation and may not manifest effective cytolytic activity against infected targets due to a high degree of susceptibility to apoptosis, thus providing an explanation of why HIV is not successfully contained by CD8+T cells in such individuals.

HIV infection induces a wide array of B cell dysfunctions. We have characterized the effect of plasma viremia on the responsiveness of B cells to CD4 + T cell help in HIV-infected patients. In HIV-negative donors, B cell proliferation correlated with CD154 expression on activated CD4 + T cells and with the availability of IL-2, whereas in HIV-infected viremic patients, reduced B cell proliferation was observed despite normal CD154 expression on activated CD4 + T cells. Reduced triggering of B cells by activated CD4 + T cells was clearly observed in HIV-infected viremic patients compared with aviremic patients with comparable CD4 + T cell counts, and a dramatic improvement in B cell function was observed in patients whose plasma viremia was controlled by effective antiretroviral therapy. The degree of B cell dysfunction in viremic patients correlated strongly with the inability of B cells to express CD25 in response to activated CD4 + T cells, resulting in an inability to mount a normal proliferative response to IL-2. Similar defects in responsiveness to IL-2 were observed in the B cells of HIV-infected viremic patients in the context of B cell receptor stimulation. These data provide new insight into the mechanisms associated with ineffective humoral responses in HIV disease.

Although continuous highly active antiretroviral therapy (HAART) is effective for many HIV-infected patients, it can be toxic and prohibitive in cost. By decreasing the total amount of time patients receive medications, intermittent HAART could reduce toxicity and cost. Therefore, we initiated a pilot study in which 10 HIV-infected individuals receiving effective therapy that resulted in levels of HIV RNA + T cell counts >300 cells per mm 3 of whole blood received repeated cycles of 7 days on HAART followed by 7 days off of HAART. Patients maintained suppression of plasma viremia for 32–68 weeks. There was no significant increase in HIV proviral DNA or replication-competent HIV in peripheral CD4 + T cells or HIV RNA in peripheral blood or lymph node mononuclear cells. There was no significant change in CD4 + T cell counts, no significant increase in CD4 + or CD8 + T cells expressing activation markers or producing IFN-γ in response to HIV, no increase in CD4 + T cell proliferation to p24 antigen, and no evidence for the development of resistance to HAART medications. There was a significant decrease in serum cholesterol and triglyceride levels. Thus, in this proof-of-concept study, short-cycle intermittent HAART maintained suppression of plasma viremia as well as HIV replication in reservoir sites while preserving CD4 + T cell counts. In addition, there was a decrease in serum cholesterol and triglyceride levels. Intermittent therapy may be an important strategy to reduce cost and toxicity for HIV-infected individuals.

CD8+T cell-mediated antiviral activity against HIV has been described consistently in infected individuals; however, the role of this activity in controlling replication of HIV in the latently infected, resting CD4+T cell reservoir is unclear. By using anex vivosystem, we show that replication of HIV in this viral reservoir is effectively suppressed in coculture by autologous CD8+T cells in long-term nonprogressors (LTNPs) and in patients whose viremia was controlled by highly active antiretroviral therapy (HAART), but not in therapy-naive patients who had substantial levels of plasma viremia. This antiviral activity was largely independent of cytotoxic CD8+T lymphocytes (CTL). When the role of soluble CD8+T cell-derived factors was examined, we found that CC-chemokines played a major role in inhibition of viral replication in the latent viral reservoir in some LTNPs and patients receiving HAART, but not in chronically infected patients who were not receiving antiretroviral therapy. Potent antiviral activity, independent of CC-chemokines, was found mainly in patients in whom HAART was initiated shortly after the acute phase of HIV infection. These results indicate that CD8+T cells provide potent suppressive activity against HIV replication in the latent viral reservoir via direct cellular contact in patients who are naturally LTNPs or in those who are treated with HAART. Furthermore, the profound antiviral activity exerted by non-CC-chemokine soluble factors in infected patients who began HAART early in HIV infection suggests that preservation of this HIV-suppressive mechanism by early initiation of therapy may play an important role in the containment of viral replication in infected patients following interruption of therapy.

Identifying the immunologic and virologic consequences of discontinuing antiretroviral therapy in HIV-infected patients is of major importance in developing long-term treatment strategies for patients with HIV-1 infection. We designed a trial to characterize these parameters after interruption of highly active antiretroviral therapy (HAART) in patients who had maintained prolonged viral suppression on antiretroviral drugs. Eighteen patients with CD4 + T cell counts ≥ 350 cells/μl and viral load below the limits of detection for ≥1 year while on HAART were enrolled prospectively in a trial in which HAART was discontinued. Twelve of these patients had received prior IL-2 therapy and had low frequencies of resting, latently infected CD4 cells. Viral load relapse to >50 copies/ml occurred in all 18 patients independent of prior IL-2 treatment, beginning most commonly during weeks 2–3 after cessation of HAART. The mean relapse rate constant was 0.45 (0.20 log 10 copies) day −1 , which was very similar to the mean viral clearance rate constant after drug resumption of 0.35 (0.15 log 10 copies) day −1 ( P = 0.28). One patient experienced a relapse delay to week 7. All patients except one experienced a relapse burden to >5,000 RNA copies/ml. Ex vivo labeling with BrdUrd showed that CD4 and CD8 cell turnover increased after withdrawal of HAART and correlated with viral load whereas lymphocyte turnover decreased after reinitiation of drug treatment. Virologic relapse occurs rapidly in patients who discontinue suppressive drug therapy, even in patients with a markedly diminished pool of resting, latently infected CD4 + T cells.

Although highly active antiretroviral therapy (HAART) in the form of triple combinations of drugs including protease inhibitors can reduce the plasma viral load of some HIV-1-infected individuals to undetectable levels, it is unclear what the effects of these regimens are on latently infected CD4+T cells and what role these cells play in the persistence of HIV-1 infection in individuals receiving such treatment. The present study demonstrates that highly purified CD4+T cells from 13 of 13 patients receiving HAART with an average treatment time of 10 months and with undetectable (in vitro. Phenotypic analysis of HIV-1 produced by activation of latently infected CD4+T cells revealed the presence in some patients of syncytium-inducing virus. In addition, the presence of unintegrated HIV-1 DNA in infected resting CD4+T cells from patients receiving HAART, even those with undetectable plasma viremia, suggests persistent active virus replicationin vivo.