Parallel analysis of transcription, integration, and sequence of single HIV-1 proviruses.

HIV-1-infected cells that persist despite antiretroviral therapy (ART) are frequently considered "transcriptionally silent," but active viral gene expression may occur in some cells, challenging the concept of viral latency. Applying an assay for profiling the transcriptional activity and the chromosomal locations of individual proviruses, we describe a global genomic and epigenetic map of transcriptionally active and silent proviral species and evaluate their longitudinal evolution in persons receiving suppressive ART. Using genome-wide epigenetic reference data, we show that proviral transcriptional activity is associated with activating epigenetic chromatin features in linear proximity of integration sites and in their inter- and intrachromosomal contact regions. Transcriptionally active proviruses were actively selected against during prolonged ART; however, this pattern was violated by large clones of virally infected cells that may outcompete negative selection forces through elevated intrinsic proliferative activity. Our results suggest that transcriptionally active proviruses are dynamically evolving under selection pressure by host factors.

Loss of Bcl-6-Expressing T Follicular Helper Cells and Germinal Centers in COVID-19.

Humoral responses in coronavirus disease 2019 (COVID-19) are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined post mortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers and 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.

Evaluating the Impact of Residency Research Productivity on Craniofacial Surgeons' Career Trajectory.

OBJECTIVE: To elucidate trends in publication productivity over the past 50 years and how they correlate with the stage of training as well as the competitiveness of residency and fellowship programs. The authors hypothesize that research qualifications play an important role in obtaining a competitively ranked craniofacial surgery fellowship and that over the past 50 years, there has been an upward shift in the research qualifications held by applicants at the time of application. DESIGN: A cross-sectional study was performed involving all 121 academic craniofacial surgeons in the United States for whom sex, years since fellowship, current institution rank, current academic position, and years since graduation were available. MAIN OUTCOME MEASURES: The relationship between research qualifications, as determined by the overall number of publications, number of first authorship publications, H-index scores, and years since fellowship completion, was examined. The study also analyzed the placement of surgeons at programs ranked in the top 25 versus the lower 25. RESULTS: As the number of decades since beginning craniofacial fellowship decreased, the average number of first authorship publications and average H-index increased. Those who attended higher-ranking residency and fellowship programs had a higher average number of publications, number of first authorship publications, and H-index. CONCLUSIONS: Over the past 5 decades, research productivity at all stages of medical training has surged, potentially fueled by the trend toward pass/fail grading in medical school evaluations and standardized exams and the need to differentiate oneself in an increasingly competitive field.

A Possible Sterilizing Cure of HIV-1 Infection Without Stem Cell Transplantation.

BACKGROUND: A sterilizing cure of HIV-1 infection has been reported in 2 persons living with HIV-1 who underwent allogeneic hematopoietic stem cell transplantations from donors who were homozygous for the CCR5Δ32 gene polymorphism. However, this has been considered elusive during natural infection. OBJECTIVE: To evaluate persistent HIV-1 reservoir cells in an elite controller with undetectable HIV-1 viremia for more than 8 years in the absence of antiretroviral therapy. DESIGN: Detailed investigation of virologic and immunologic characteristics. SETTING: Tertiary care centers in Buenos Aires, Argentina, and Boston, Massachusetts. PATIENT: A patient with HIV-1 infection and durable drug-free suppression of HIV-1 replication. MEASUREMENTS: Analysis of genome-intact and replication-competent HIV-1 using near-full-length individual proviral sequencing and viral outgrowth assays, respectively; analysis of HIV-1 plasma RNA by ultrasensitive HIV-1 viral load testing. RESULTS: No genome-intact HIV-1 proviruses were detected in analysis of a total of 1.188 billion peripheral blood mononuclear cells and 503 million mononuclear cells from placental tissues. Seven defective proviruses, some of them derived from clonally expanded cells, were detected. A viral outgrowth assay failed to retrieve replication-competent HIV-1 from 150 million resting CD4+ T cells. No HIV-1 RNA was detected in 4.5 mL of plasma. LIMITATIONS: Absence of evidence for intact HIV-1 proviruses in large numbers of cells is not evidence of absence of intact HIV-1 proviruses. A sterilizing cure of HIV-1 can never be empirically proved. CONCLUSION: Genome-intact and replication-competent HIV-1 were not detected in an elite controller despite analysis of massive numbers of cells from blood and tissues, suggesting that this patient may have naturally achieved a sterilizing cure of HIV-1 infection. These observations raise the possibility that a sterilizing cure may be an extremely rare but possible outcome of HIV-1 infection. PRIMARY FUNDING SOURCE: National Institutes of Health and Bill & Melinda Gates Foundation.

Pathology after Chlamydia trachomatis infection is driven by nonprotective immune cells that are distinct from protective populations.

Infection with Chlamydia trachomatis drives severe mucosal immunopathology; however, the immune responses that are required for mediating pathology vs. protection are not well understood. Here, we employed a mouse model to identify immune responses required for C. trachomatis-induced upper genital tract pathology and to determine whether these responses are also required for bacterial clearance. In mice as in humans, immunopathology was characterized by extravasation of leukocytes into the upper genital tract that occluded luminal spaces in the uterus and ovaries. Flow cytometry identified these cells as neutrophils at early time points and CD4+ and CD8+ T cells at later time points. To determine what draws these cells to C. trachomatis-infected tissue, we measured the expression of 700 inflammation-related genes in the upper genital tract and found an up-regulation of many chemokines, including a node of interaction between CXCL9/10/11 and their common receptor CXCR3. Either depleting neutrophils or reducing T-cell numbers by CXCR3 blockade was sufficient to significantly ameliorate immunopathology but had no effect on bacterial burden, demonstrating that these responses are necessary for mucosal pathology but dispensable for C. trachomatis clearance. Therapies that specifically target these host responses may therefore prove useful in ameliorating C. trachomatis-induced pathology without exacerbating infection or transmission.

Gynecologic Care for Sexual and Gender Minority Patients.

Sexual and gender minority (SGM) people, including lesbian, gay, bisexual, transgender, and queer individuals, are a diverse population with a wide spectrum of gynecologic needs. Institutionalized cisheteronormativity, stigmatization, lack of provider training, and fear of discrimination contribute to health disparities in this patient population. In this article, we review key topics in the gynecologic care of SGM patients and provide strategies to enable gynecologists to provide SGM people with equitable and inclusive full spectrum reproductive health care.

Progressive transformation of the HIV-1 reservoir cell profile over two decades of antiviral therapy.

HIV-1 establishes a life-long reservoir of virally infected cells which cannot be eliminated by antiretroviral therapy (ART). Here, we demonstrate a markedly altered viral reservoir profile of long-term ART-treated individuals, characterized by large clones of intact proviruses preferentially integrated in heterochromatin locations, most prominently in centromeric satellite/micro-satellite DNA. Longitudinal evaluations suggested that this specific reservoir configuration results from selection processes that promote the persistence of intact proviruses in repressive chromatin positions, while proviruses in permissive chromosomal locations are more likely to be eliminated. A bias toward chromosomal integration sites in heterochromatin locations was also observed for intact proviruses in study participants who maintained viral control after discontinuation of antiretroviral therapy. Together, these results raise the possibility that antiviral selection mechanisms during long-term ART may induce an HIV-1 reservoir structure with features of deep latency and, possibly, more limited abilities to drive rebound viremia upon treatment interruptions.

Immunogenicity and Protective Capacity of a Virus-like Particle Vaccine against Chlamydia trachomatis Type 3 Secretion System Tip Protein, CT584.

An effective vaccine against Chlamydia trachomatis is urgently needed as infection rates continue to rise and C. trachomatis causes reproductive morbidity. An obligate intracellular pathogen, C. trachomatis employs a type 3 secretion system (T3SS) for host cell entry. The tip of the injectosome is composed of the protein CT584, which represents a potential target for neutralization with vaccine-induced antibody. Here, we investigate the immunogenicity and efficacy of a vaccine made of CT584 epitopes coupled to a bacteriophage virus-like particle (VLP), a novel platform for Chlamydia vaccines modeled on the success of HPV vaccines. Female mice were immunized intramuscularly, challenged transcervically with C. trachomatis, and assessed for systemic and local antibody responses and bacterial burden in the upper genital tract. Immunization resulted in a 3-log increase in epitope-specific IgG in serum and uterine homogenates and in the detection of epitope-specific IgG in uterine lavage at low levels. By contrast, sera from women infected with C. trachomatis and virgin controls had similarly low titers to CT584 epitopes, suggesting these epitopes are not systemically immunogenic during natural infection but can be rendered immunogenic by the VLP platform. C. trachomatis burden in the upper genital tract of mice varied after active immunization, yet passive protection was achieved when immune sera were pre-incubated with C. trachomatis prior to inoculation into the genital tract. These data demonstrate the potential for antibody against the T3SS to contribute to protection against C. trachomatis and the value of VLPs as a novel platform for C. trachomatis vaccines.

Signatures of immune selection in intact and defective proviruses distinguish HIV-1 elite controllers.

Increasing evidence suggests that durable drug-free control of HIV-1 replication is enabled by effective cellular immune responses that may induce an attenuated viral reservoir configuration with a weaker ability to drive viral rebound. Here, we comprehensively tracked effects of antiviral immune responses on intact and defective proviral sequences from elite controllers (ECs), analyzing both classical escape mutations and HIV-1 chromosomal integration sites as biomarkers of antiviral immune selection pressure. We observed that, within ECs, defective proviruses were commonly located in permissive genic euchromatin positions, which represented an apparent contrast to autologous intact proviruses that were frequently located in heterochromatin regions; this suggests differential immune selection pressure on intact versus defective proviruses in ECs. In comparison to individuals receiving antiretroviral therapy, intact and defective proviruses from ECs showed reduced frequencies of escape mutations in cytotoxic T cell epitopes and antibody contact regions, possibly due to the small and poorly inducible reservoir that may be insufficient to drive effective viral escape in ECs. About 15% of ECs harbored nef deletions in intact proviruses, consistent with increased viral vulnerability to host immunity in the setting of nef dysfunction. Together, these results suggest a distinct signature of immune footprints in proviral sequences from ECs.

The Loss of Bcl-6 Expressing T Follicular Helper Cells and the Absence of Germinal Centers in 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.