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Sustained, drug-free control of HIV-1 replication is naturally achieved in less than 0.5% of infected individuals (here termed ‘elite controllers’), despite the presence of a replication-competent viral reservoir1. Inducing such an ability to spontaneously maintain undetectable plasma viraemia is a major objective of HIV-1 cure research, but the characteristics of proviral reservoirs in elite controllers remain to be determined. Here, using next-generation sequencing of near-full-length single HIV-1 genomes and corresponding chromosomal integration sites, we show that the proviral reservoirs of elite controllers frequently consist of oligoclonal to near-monoclonal clusters of intact proviral sequences. In contrast to individuals treated with long-term antiretroviral therapy, intact proviral sequences from elite controllers were integrated at highly distinct sites in the human genome and were preferentially located in centromeric satellite DNA or in Kruppel-associated box domain-containing zinc finger genes on chromosome 19, both of which are associated with heterochromatin features. Moreover, the integration sites of intact proviral sequences from elite controllers showed an increased distance to transcriptional start sites and accessible chromatin of the host genome and were enriched in repressive chromatin marks. These data suggest that a distinct configuration of the proviral reservoir represents a structural correlate of natural viral control, and that the quality, rather than the quantity, of viral reservoirs can be an important distinguishing feature for a functional cure of HIV-1 infection. Moreover, in one elite controller, we were unable to detect intact proviral sequences despite analysing more than 1.5 billion peripheral blood mononuclear cells, which raises the possibility that a sterilizing cure of HIV-1 infection, which has previously been observed only following allogeneic haematopoietic stem cell transplantation2,3, may be feasible in rare instances. In individuals who have achieved natural control of HIV-1 without drug treatment, intact proviral sequences are integrated into genomic regions that are not permissive to active viral transcription, indicating deep latency of the virus.

Chromosomal integration of genome-intact HIV-1 sequences into the host genome creates a reservoir of virally infected cells that persists throughout life, necessitating indefinite antiretroviral suppression therapy. During effective antiviral treatment, the majority of these proviruses remain transcriptionally silent, but mechanisms responsible for viral latency are insufficiently clear. Here, we used matched integration site and proviral sequencing (MIP-Seq), an experimental approach involving multiple displacement amplification of individual proviral species, followed by near-full-length HIV-1 next-generation sequencing and corresponding chromosomal integration site analysis to selectively map the chromosomal positions of intact and defective proviruses in 3 HIV-1–infected individuals undergoing long-term antiretroviral therapy. Simultaneously, chromatin accessibility and gene expression in autologous CD4(+) T cells were analyzed by assays for transposase-accessible chromatin using sequencing (ATAC-Seq) and RNA-Seq. We observed that in comparison to proviruses with defective sequences, intact HIV-1 proviruses were enriched for non-genic chromosomal positions and more frequently showed an opposite orientation relative to host genes. In addition, intact HIV-1 proviruses were preferentially integrated in either relative proximity to or increased distance from active transcriptional start sites and to accessible chromatin regions. These studies strongly suggest selection of intact proviruses with features of deeper viral latency during prolonged antiretroviral therapy, and may be informative for targeting the genome-intact viral reservoir.

Summary Humoral responses in COVID-19 disease are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined postmortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers, a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+ TFH cell differentiation together with an increase in T-bet+ TH1 cells and aberrant extra-follicular TNF-α accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific “disease-related” B cell populations. These data identify defective Bcl-6+ TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections and suggest that achieving herd immunity through natural infection may be difficult., Highlights 1. Acute phase SARS-CoV-2-specific T cells display an activated cytotoxic phenotype 2. Broad and polyfunctional SARS-CoV-2-specific T cell responses in convalescent phase 3. Detection of SARS-CoV-2-specific T cell responses also in seronegative individuals, Buggert and colleagues provide a phenotypic and functional map of SARS-CoV-2-specific T cells across the full spectrum of exposure, infection, and COVID-19 severity. They observe that SARS-CoV-2-specific T cells generate a broad, robust and functionally replete response in convalescent individuals, that may provide protection from recurrent episodes of severe COVID-19.

Humoral responses in COVID-19 disease are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined postmortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers, a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+TFH cell differentiation together with an increase in T-bet+TH1 cells and aberrant extra-follicular TNF-a accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections and suggest that achieving herd immunity through natural infection may be difficult. Funding: This work was supported by NIH U19 AI110495 to SP, NIH R01 AI146779 to AGS, NIH R01AI137057 and DP2DA042422 to DL, BMH was supported by NIGMS T32 GM007753, TMC was supported by T32 AI007245. Funding for these studies from the Massachusetts Consortium of Pathogen Readiness, the Mark and Lisa Schwartz Foundation and Enid Schwartz is also acknowledged. Conflict of Interest: None. Ethical Approval: This study was performed with the approval of the Institutional Review Boards at the Massachusetts General Hospital and the Brigham and Women's Hospital.

Summary: Although dendritic cells are among the human cell population best equipped for cell-intrinsic antiviral immune defense, they seem highly susceptible to infection with the Zika virus (ZIKV). Using highly purified myeloid dendritic cells isolated from individuals with naturally acquired acute infection, we here show that ZIKV induces profound perturbations of transcriptional signatures relative to healthy donors. Interestingly, we noted a remarkable downregulation of antiviral interferon-stimulated genes and innate immune sensors, suggesting that ZIKV can actively suppress interferon-dependent immune responses. In contrast, several host factors known to support ZIKV infection were strongly upregulated during natural ZIKV infection; these transcripts included AXL, the main entry receptor for ZIKV; SOCS3, a negative regulator of ISG expression; and IDO-1, a recognized inducer of regulatory T cell responses. Thus, during in vivo infection, ZIKV can transform the transcriptome of dendritic cells in favor of the virus to render these cells highly conducive to ZIKV infection. : Sun et al. find distinct transcriptional signatures in myeloid dendritic cells isolated from individuals with naturally acquired Zika Virus infection. These data indicate that Zika virus can reprogram dendritic cells to increase their susceptibility to further ZIKV infection. Keywords: Zika virus, flavivirus, interferon stimulated genes, dendritic cells, acute infection, RNA-seq, SOCS3, IDO-1, AXL

HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells.

Neonatal HIV-1 infection is associated with rapidly progressive and frequently fatal immune deficiency if left untreated. Immediate institution of antiretroviral therapy (ART), ideally within hours after birth, may restrict irreversible damage to the developing neonatal immune system and possibly provide opportunities for facilitating drug-free viral control during subsequent treatment interruptions. However, the virological and immunological effects of ART initiation within hours after delivery have not been systematically investigated. We examined a unique cohort of neonates with HIV-1 infection from Botswana who started ART shortly after birth and were followed longitudinally for about 2 years in comparison to control infants started on treatment during the first year after birth. We demonstrate multiple clear benefits of rapid antiretroviral initiation, including an extremely small reservoir of intact proviral sequences, a reduction in abnormal T cell immune activation, a more polyfunctional HIV-1–specific T cell response, and an innate immune profile that displays distinct features of improved antiviral activity and is associated with intact proviral reservoir size. Together, these data offer rare insight into the evolutionary dynamics of viral reservoir establishment in neonates and provide strong empirical evidence supporting the immediate initiation of ART for neonates with HIV-1 infection.

Little is known about the genotypic make-up of HIV-1 DNA genomes during the earliest stages of HIV-1 infection. Here, we use near-full-length, single genome next-generation sequencing to longitudinally genotype and quantify subtype C HIV-1 DNA in four women identified during acute HIV-1 infection in Durban, South Africa, through twice-weekly screening of high-risk participants. In contrast to chronically HIV-1-infected patients, we found that at the earliest phases of infection in these four participants, the majority of viral DNA genomes are intact, lack APOBEC-3G/F-associated hypermutations, have limited genome truncations, and over one year show little indication of cytotoxic T cell-driven immune selections. Viral sequence divergence during acute infection is predominantly fueled by single-base substitutions and is limited by treatment initiation during the earliest stages of disease. Our observations provide rare longitudinal insights of HIV-1 DNA sequence profiles during the first year of infection to inform future HIV cure research., The dynamics of HIV-1 DNA sequences early after HIV-1 transmission remains poorly characterized. Here, the authors perform a longitudinal evaluation of HIV-1 DNA sequences in subtype C-infected individuals during acute infection, providing a landscape of the nature and evolution of the very early viral genome.

Background Notwithstanding 1 documented case of HIV-1 cure following allogeneic stem cell transplantation (allo-SCT), several subsequent cases of allo-SCT in HIV-1 positive individuals have failed to cure HIV-1 infection. The aim of our study was to describe changes in the HIV reservoir in a single chronically HIV-infected patient on suppressive antiretroviral therapy who underwent allo-SCT for treatment of acute lymphoblastic leukemia. Methods and findings We prospectively collected peripheral blood mononuclear cells (PBMCs) by leukapheresis from a 55-year-old man with chronic HIV infection before and after allo-SCT to measure the size of the HIV-1 reservoir and characterize viral phylogeny and phenotypic changes in immune cells. At day 784 post-transplant, when HIV-1 was undetectable by multiple measures—including PCR measurements of both total and integrated HIV-1 DNA, replication-competent virus measurement by large cell input quantitative viral outgrowth assay, and in situ hybridization of colon tissue—the patient consented to an analytic treatment interruption (ATI) with frequent clinical monitoring. He remained aviremic off antiretroviral therapy until ATI day 288, when a low-level virus rebound of 60 HIV-1 copies/ml occurred, which increased to 1,640 HIV-1 copies/ml 5 days later, prompting reinitiation of ART. Rebounding plasma HIV-1 sequences were phylogenetically distinct from proviral HIV-1 DNA detected in circulating PBMCs before transplantation. The main limitations of this study are the insensitivity of reservoir measurements, and the fact that it describes a single case. Conclusions allo-SCT led to a significant reduction in the size of the HIV-1 reservoir and a >9-month-long ART-free remission from HIV-1 replication. Phylogenetic analyses suggest that the origin of rebound virus was distinct from the viruses identified pre-transplant in the PBMCs., Andrew Badley and colleagues study the viral reservoir in an individual with HIV infection after allogeneic stem cell transplantation., Author summary Why was this study done? Currently there is no cure for HIV infection. The only previously documented case of HIV cure occurred in the setting of stem cell transplantation for acute myeloid leukemia. However, other similar cases have not resulted in HIV cure. This observational study was done to further describe in detail the effects of allogeneic stem cell transplantation on residual HIV in a patient being treated for acute lymphoblastic leukemia. What did the researchers do and find? We prospectively collected blood and tissue samples from before and after stem cell transplantation and measured the HIV reservoir size using multiple complementary techniques. We found that the size of the HIV reservoir decreased substantially after transplantation to levels at or below the limit of detection of most assays. We observed a prolonged remission from HIV rebound after antiretroviral treatment interruption in the post-transplant period. The genetic sequence of the rebounding virus in the blood clustered closely with sequences from blood prior to treatment interruption. What do these findings mean? These findings affirm that allogeneic stem cell transplantation can profoundly decrease the size of the HIV reservoir. However, current technologies for measuring reservoir size in blood are insufficiently sensitive to predict HIV cure. Until new biomarkers of HIV cure are developed, the decision to discontinue antiretroviral therapy after allogeneic stem cell transplantation to assess a possible cure should be undertaken cautiously.

Therapeutic strategies aimed at achieving antiretroviral therapy (ART)-free HIV remission in infected individuals are under active investigation. Considering the vast majority of HIV-infected individuals experience plasma viral rebound upon cessation of therapy, clinical trials evaluating the efficacy of curative strategies would likely require inclusion of ART interruption. However, it is unclear what impact short-term analytical treatment interruption (ATI) and subsequent reinitiation of ART have on immunologic and virologic parameters of HIV-infected individuals. Here, we show a significant increase of HIV burden in the CD4+ T cells of infected individuals during ATI that was correlated with the level of plasma viral rebound. However, the size of the HIV reservoirs as well as immune parameters, including markers of exhaustion and activation, returned to pre-ATI levels 6–12 months after the study participants resumed ART. Of note, the proportions of near full-length, genome-intact and structurally defective HIV proviral DNA sequences were similar prior to ATI and following reinitiation of ART. In addition, there was no evidence of emergence of antiretroviral drug resistance mutations within intact HIV proviral DNA sequences following reinitiation of ART. These data demonstrate that short-term ATI does not necessarily lead to expansion of the persistent HIV reservoir nor irreparable damages to the immune system in the peripheral blood, warranting the inclusion of ATI in future clinical trials evaluating curative strategies., Author summary While we have made considerable advancements in the treatment of HIV, most infected individuals require life-long treatment to suppress plasma viremia, underscoring the need for the development of additional therapeutic strategies that would allow durable virologic remission in the absence of antiretroviral therapy (ART). While a definitive cure has not yet been identified, the field is moving in a promising direction, and with continued efforts we may arrive at a clinically acceptable alternative to ART. Clinical validation of new treatment options likely requires patients to stop therapy while monitoring for viral rebound, but the effect of treatment interruption and its precise impact on immunologic and virologic parameters in HIV-infected individuals has not been fully delineated. In this work, we measured a significant increase of HIV burden in the CD4+ T cells of infected individuals who underwent ATI with subsequent plasma viral rebound. However, the size of the HIV reservoirs as well as immune parameters returned to pre-ATI levels 6–12 months after the participants resumed ART. These data suggest ATI does not lead to expansion of the persistent HIV reservoir nor irreversible damages to the immune system in the peripheral blood.

Background With the expansion of HIV treatment programs in sub-Saharan Africa, there are increased cases of HIV drug resistance. In Botswana where the national HIV treatment program has been in place since 2002, patients with HIV strains resistant to the core antiretroviral classes are a reality. There is need to investigate how some of the less frequently used antiretroviral classes such as enfuvirtide (T-20) and its derivatives would fair in this population. Methods A total of 164 samples from 129 patients initiating combination antiretroviral therapy (cART) and 35 patients failing NRTI – and NNRTI-based cART in studies conducted in Botswana were available for analysis. Viral RNA was isolated from plasma and RT-PCR targeting HIV-1 gp41 was run and the product sequenced. Sequences were edited using Sequencher and alignments were made using Clustal-X. A search on the Los Alamos HIV database yielded 106 gp41 sequences from unique Botswana patients and these were included in the analysis. The IAS-USA, 2015 Resistance Mutations update report was used to define the T-20 drug resistance mutations. Results A total of 154 samples were successfully sequenced, 126 from treatment naive patients and 28 from virologic failure patients. Additionally, 106 gp41 sequences from previous studies conducted in Botswana were included in the analysis. No major T-20 was detected in any of the 260 sequences. The N42S mutation which is associated with T-20 hypersensitivity was found in (87.3%) and this is consistent with published data from HIV-1C studies. The I69V mutation (95.6%) was the most common detected HR1 polymorphism. The most common HR2 polymorphism detected was I135L (98.4%) followed by E151A (92.3%). Conclusions These results provide invaluable data on gp41 diversity in Botswana and show that there is no background resistance to T-20 or its derivatives. T-20 would be an alternative drug for patients failing cART in Botswana.