Summary Latent replication-competent HIV-1 persists in individuals on long-term antiretroviral therapy (ART). We developed the Full-Length Individual Proviral Sequencing (FLIPS) assay to determine the distribution of latent replication-competent HIV-1 within memory CD4+ T cell subsets in six individuals on long-term ART. FLIPS is an efficient, high-throughput assay that amplifies and sequences near full-length (~9 kb) HIV-1 proviral genomes and determines potential replication-competency through genetic characterization. FLIPS provides a genome-scale perspective which addresses the limitations of other methods that also genetically characterize the latent reservoir. Using FLIPS, we identified 5% proviruses as intact and potentially replication-competent. Intact proviruses were unequally distributed between T cell subsets, with effector memory cells containing the largest proportion of genetically intact HIV-1 proviruses. We identified multiple identical intact proviruses suggesting a role for cellular proliferation in the maintenance of the latent HIV-1 reservoir.

Inflammation in HIV infection is predictive of non-AIDS morbidity and death1, higher set point plasma virus load2 and virus acquisition3; thus, therapeutic agents are in development to reduce its causes and consequences. However, inflammation may simultaneously confer both detrimental and beneficial effects. This dichotomy is particularly applicable to type I interferons (IFN-I) which, while contributing to innate control of infection4–10, also provide target cells for the virus during acute infection, impair CD4 T-cell recovery, and are associated with disease progression6,7,11–19. Here we manipulated IFN-I signalling in rhesus macaques (Macaca mulatta) during simian immunodeficiency virus (SIV) transmission and acute infection with two complementary in vivo interventions. We show that blockade of the IFN-I receptor caused reduced antiviral gene expression, increased SIV reservoir size and accelerated CD4 T-cell depletion with progression to AIDS despite decreased T-cell activation. In contrast, IFN-α2a administration initially upregulated expression of antiviral genes and prevented systemic infection. However, continued IFN-α2a treatment induced IFN-I desensitization and decreased antiviral gene expression, enabling infection with increased SIV reservoir size and accelerated CD4 T-cell loss. Thus, the timing of IFN-induced innate responses in acute SIV infection profoundly affects overall disease course and outweighs the detrimental consequences of increased immune activation. Yet, the clinical consequences of manipulation of IFN signalling are difficult to predict in vivo and therapeutic interventions in human studies should be approached with caution.

Passive immunization with HIV-1-neutralizing monoclonal antibodies (mAbs) is being considered for prevention and treatment of HIV-1 infection. As therapeutic agents, mAbs could be used to suppress active virus replication, maintain suppression induced by antiretroviral therapy (ART), and/or decrease the size of the persistent virus reservoir. We assessed the impact of VRC01, a potent human mAb targeting the HIV-1 CD4 binding site, on ART-treated and untreated HIV-1-infected subjects. Among six ART-treated individuals with undetectable plasma viremia, two infusions of VRC01 did not reduce the peripheral blood cell-associated virus reservoir measured 4 weeks after the second infusion. In contrast, six of eight ART-untreated, viremic subjects infused with a single dose of VRC01 experienced a 1.1 to 1.8 log10 reduction in plasma viremia. The two subjects with minimal responses to VRC01 were found to have predominantly VRC01-resistant virus before treatment. Notably, two subjects with plasma virus load <1000 copies/ml demonstrated virus suppression to undetectable levels for over 20 days until VRC01 levels declined. Among the remaining four subjects with baseline virus loads between 3000 and 30,000 copies, viremia was only partially suppressed by mAb infusion, and we observed strong selection pressure for the outgrowth of less neutralization-sensitive viruses. In summary, a single infusion of mAb VRC01 significantly decreased plasma viremia and preferentially suppressed neutralization-sensitive virus strains. These data demonstrate the virological effect of this neutralizing antibody and highlight the need for combination strategies to maintain virus suppression.

Coronavirus disease 2019 (COVID-19) is known to cause multi-organ dysfunction 1 – 3 during acute infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with some patients experiencing prolonged symptoms, termed post-acute sequelae of SARS-CoV-2 (refs. 4 , 5 ). However, the burden of infection outside the respiratory tract and time to viral clearance are not well characterized, particularly in the brain 3 , 6 – 14 . Here we carried out complete autopsies on 44 patients who died with COVID-19, with extensive sampling of the central nervous system in 11 of these patients, to map and quantify the distribution, replication and cell-type specificity of SARS-CoV-2 across the human body, including the brain, from acute infection to more than seven months following symptom onset. We show that SARS-CoV-2 is widely distributed, predominantly among patients who died with severe COVID-19, and that virus replication is present in multiple respiratory and non-respiratory tissues, including the brain, early in infection. Further, we detected persistent SARS-CoV-2 RNA in multiple anatomic sites, including throughout the brain, as late as 230 days following symptom onset in one case. Despite extensive distribution of SARS-CoV-2 RNA throughout the body, we observed little evidence of inflammation or direct viral cytopathology outside the respiratory tract. Our data indicate that in some patients SARS-CoV-2 can cause systemic infection and persist in the body for months.

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The interaction between follicular T helper cells (TFH) and B cells in the lymph nodes and spleen has a major impact on the development of antigen-specific B cell responses during infection or vaccination. Recent studies described a functional equivalent of these cells among circulating CD4 T cells, referred to as peripheral TFH cells. Here, we characterize the phenotype and in vitro B cell helper activity of peripheral TFH populations, as well as the effect of HIV infection on these populations. In co-culture experiments we confirmed CXCR5+ cells from HIV-uninfected donors provide help to B cells and more specifically, we identified a CCR7highCXCR5highCCR6highPD-1high CD4 T cell population that secretes IL-21 and enhances isotype-switched immunoglobulin production. This population is significantly decreased in treatment-naïve, HIV-infected individuals and can be recovered after anti-retroviral therapy. We found impaired immunoglobulin production in co-cultures from HIV-infected individuals and found no correlation between the frequency of peripheral TFH cells and memory B cells, or with neutralization activity in untreated HIV infection in our cohort. Furthermore, we found that within the peripheral TFH population, the expression level of TFH-associated genes more closely resembles a memory, non-TFH population, as opposed to a TFH population. Overall, our data identify a heterogeneous population of circulating CD4 T cells that provides in vitro help to B cells, and challenges the origin of these cells as memory TFH cells.

Cytolytic CD8 T cells play a crucial role in the control and elimination of virus-infected cells and are a major focus of HIV cure efforts. However, it has been shown that HIV-specific CD8 T cells are infrequently found within germinal centers (GCs), a predominant site of active and latent HIV infection. We demonstrate that HIV infection induces marked changes in the phenotype, frequency, and localization of CD8 T cells within the lymph node (LN). Significantly increased frequencies of CD8 T cells in the B cell follicles and GCs were found in LNs from treated and untreated HIV-infected individuals. This profile was associated with persistent local immune activation but did not appear to be directly related to local viral replication. Follicular CD8 (fCD8) T cells, despite compromised cytokine polyfunctionality, showed good cytolytic potential characterized by high ex vivo expression of granzyme B and perforin. We used an anti-HIV/anti-CD3 bispecific antibody in a redirected killing assay and found that fCD8 T cells had better killing activity than did non-fCD8 T cells. Our results indicate that CD8 T cells with potent cytolytic activity are recruited to GCs during HIV infection and, if appropriately redirected to kill HIV-infected cells, could be an effective component of an HIV cure strategy.

Memory T cells maintained a relatively constant HIV-1 DNA integrant pool that was genetically stable during long-term effective ART. These integrants appear to be maintained by cellular proliferation and longevity of infected cells, rather than by ongoing viral replication.