The coronavirus disease 2019 (COVID-19) pandemic has demonstrated a clear need for high-throughput, multiplexed and sensitive assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses and their emerging variants. Here, we present a cost-effective virus and variant detection platform, called microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN), which combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel to test for up to 21 viruses, including SARS-CoV-2, other coronaviruses and both influenza strains, and demonstrated its diagnostic-grade performance on 525 patient specimens in an academic setting and 166 specimens in a clinical setting. We further developed an mCARMEN panel to enable the identification of 6 SARS-CoV-2 variant lineages, including Delta and Omicron, and evaluated it on 2,088 patient specimens with near-perfect concordance to sequencing-based variant classification. Lastly, we implemented a combined Cas13 and Cas12 approach that enables quantitative measurement of SARS-CoV-2 and influenza A viral copies in samples. The mCARMEN platform enables high-throughput surveillance of multiple viruses and variants simultaneously, enabling rapid detection of SARS-CoV-2 variants.

ABSTRACT The rational design of HIV-1 immunogens to trigger the development of broadly neutralizing antibodies (bNAbs) requires understanding the viral evolutionary pathways influencing this process. An acute HIV-1-infected individual exhibiting >50% plasma neutralization breadth developed neutralizing antibody specificities against the CD4-binding site (CD4bs) and V1V2 regions of Env gp120. Comparison of pseudoviruses derived from early and late autologous env sequences demonstrated the development of >2 log resistance to VRC13 but not to other CD4bs-specific bNAbs. Mapping studies indicated that the V3 and CD4-binding loops of Env gp120 contributed significantly to developing resistance to the autologous neutralizing response and that the CD4-binding loop (CD4BL) specifically was responsible for the developing resistance to VRC13. Tracking viral evolution during the development of this cross-neutralizing CD4bs response identified amino acid substitutions arising at only 4 of 11 known VRC13 contact sites (K282, T283, K421, and V471). However, each of these mutations was external to the V3 and CD4BL regions conferring resistance to VRC13 and was transient in nature. Rather, complete resistance to VRC13 was achieved through the cooperative expression of a cluster of single amino acid changes within and immediately adjacent to the CD4BL, including a T359I substitution, exchange of a potential N -linked glycosylation (PNLG) site to residue S362 from N363, and a P369L substitution. Collectively, our data characterize complex HIV-1 env evolution in an individual developing resistance to a VRC13-like neutralizing antibody response and identify novel VRC13-associated escape mutations that may be important to inducing VRC13-like bNAbs for lineage-based immunogens. IMPORTANCE The pursuit of eliciting broadly neutralizing antibodies (bNAbs) through vaccination and their use as therapeutics remains a significant focus in the effort to eradicate HIV-1. Key to our understanding of this approach is a more extensive understanding of bNAb contact sites and susceptible escape mutations in HIV-1 envelope ( env ). We identified a broad neutralizer exhibiting VRC13-like responses, a non-germline restricted class of CD4-binding site antibody distinct from the well-studied VRC01-class. Through longitudinal envelope sequencing and Env-pseudotyped neutralization assays, we characterized a complex escape pathway requiring the cooperative evolution of four amino acid changes to confer complete resistance to VRC13. This suggests that VRC13-class bNAbs may be refractory to rapid escape and attractive for therapeutic applications. Furthermore, the identification of longitudinal viral changes concomitant with the development of neutralization breadth may help identify the viral intermediates needed for the maturation of VRC13-like responses and the design of lineage-based immunogens.

Purpose:To evaluate the impact of TearCare (TC) treatment on clinical, quality of life, and functional visual outcome metrics in patients with dry eye disease (DED) and meibomian gland disease (MGD). Methods:This is a prospective, single-center clinical trial. Adults with MGD and a DED diagnosis and tear break-up time (TBUT) <10 seconds were included. All subjects had at least 20/40 vision and no surgery or new treatment for DED within 60 days prior to enrollment. All patients had one baseline visit prior to undergoing TC and one follow-up visit 1 month after TC. At each visit, the meibomian gland secretion score (MGSS), TBUT, and corneal fluorescein staining (KFL) were assessed. DED symptoms were evaluated using the Ocular Surface Disease Index (OSDI) questionnaire, Visual Function Questionnaire 25 (VFQ-25), and the Fatigue Severity Scale. Reading speed was determined through the International Reading Speed Texts (IReST), Minnesota Low Vision Reading Test (MNREAD), and Wilkins Rate of Reading Test (WRRT). Results:Thirty-two subjects were included. The average age was 55.9 years. Sixteen (52%) participants had a clinically significant improvement in reading speed after treatment with TC, defined as >10 words per minute increase in their IReST score. Improvement on the IReST and the MNREAD reached statistical significance (p = 0.012 and p = 0.028, respectively). OSDI scores significantly decreased and VFQ-25 scores significantly increased after TC treatment (p < 0.001). All of the clinical exam parameters showed statistically significant improvements after treatment (p < 0.001). Conclusion:TC is an effective treatment both clinically and with respect to visual function. Patients who had TC exhibited improvements in quality of life and improved reading speed after a single treatment. This treatment should be frequently considered and utilized to reduce the disease burden of DED related to MGD.

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.

Human immunodeficiency virus 1 (HIV-1) reservoir cells persist lifelong despite antiretroviral treatment1,2 but may be vulnerable to host immune responses that could be exploited in strategies to cure HIV-1. Here we used a single-cell, next-generation sequencing approach for the direct ex vivo phenotypic profiling of individual HIV-1infected memory CD4+ T cells from peripheral blood and lymph nodes of people living with HIV-1 and receiving antiretroviral treatment for approximately 10 years. We demonstrate that in peripheral blood, cells harbouring genome-intact proviruses and large clones of virally infected cells frequently express ensemble signatures of surface markers conferring increased resistance to immune-mediated killing by cytotoxic T and natural killer cells, paired with elevated levels of expression of immune checkpoint markers likely to limit proviral gene transcription; this phenotypic profile might reduce HIV-1 reservoir cell exposure to and killing by cellular host immune responses. Viral reservoir cells harbouring intact HIV-1 from lymph nodes exhibited a phenotypic signature primarily characterized by upregulation of surface markers promoting cell survival, including CD44, CD28, CD127 and the IL-21 receptor. Together, these results suggest compartmentalized phenotypic signatures of immune selection in HIV-1 reservoir cells, implying that only small subsets of infected cells with optimal adaptation to their anatomical immune microenvironment are able to survive during long-term antiretroviral treatment. The identification of phenotypic markers distinguishing viral reservoir cells may inform future approaches for strategies to cure and eradicate HIV-1.

CXCR5 (CD185 or Burkitt lymphoma receptor 1) is a receptor activated by the chemokine CXCL13. This G-protein coupled receptor (GPCR) is expressed on T-follicular helper cells (Tfh), B cells, and CD4+ T cells in secondary lymphoid organs such as the spleen, lymph nodes, and Peyer's patches. CXCR5 is involved in Tfh cell migration to B cell follicles, where they interact with B cells to initiate the humoral immune response. Aberrant activation of the CXCR5-CXCL13 signalling axis contributes to autoimmune conditions, including multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis. The CXCL13:CXCR5 axis is also involved in angioimmunoblastic T-cell lymphoma (AITL). Cancer and normal cells within the tumor microenvironment secrete CXCL13 to contribute to the proliferation and metastasis of these lymphomas. Antagonists of CXCR5 would be potential drug molecules to reduce the severity of cancers and autoimmune diseases. Protein engineering can make CXCL13 an effective antagonist by modifying the N-terminal sequence, known to interact with a cavity among transmembrane helices in CXCR5 and induce signaling. About 168,000 variant CXCL13 sequences were expressed by a phage display library, reduced to 98 potential CXCR5 antagonists after assays to identify inhibitors by bioinformatics, and experimentally tested for antagonism. The most potent antagonist will be further validated for its ability to prevent cell migration and cancer cell survival by fusing to IgG and using in CAR T therapy.

AbstractBackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection is poorly understood, partly because few studies have systematically applied genomic analysis to distinguish reinfection from persistent RNA detection related to initial infection. We aimed to evaluate the characteristics of SARS-CoV-2 reinfection and persistent RNA detection using independent genomic, clinical, and laboratory assessments.MethodsAll individuals at a large academic medical center who underwent a SARS-CoV-2 nucleic acid amplification test (NAAT) ≥45 days after an initial positive test, with both tests between 14 March and 30 December 2020, were analyzed for potential reinfection. Inclusion criteria required having ≥2 positive NAATs collected ≥45 days apart with a cycle threshold (Ct) value <35 at repeat testing. For each included subject, likelihood of reinfection was assessed by viral genomic analysis of all available specimens with a Ct value <35, structured Ct trajectory criteria, and case-by-case review by infectious diseases physicians.ResultsAmong 1569 individuals with repeat SARS-CoV-2 testing ≥45 days after an initial positive NAAT, 65 (4%) met cohort inclusion criteria. Viral genomic analysis characterized mutations present and was successful for 14/65 (22%) subjects. Six subjects had genomically supported reinfection, and 8 subjects had genomically supported persistent RNA detection. Compared to viral genomic analysis, clinical and laboratory assessments correctly distinguished reinfection from persistent RNA detection in 12/14 (86%) subjects but missed 2/6 (33%) genomically supported reinfections.ConclusionsDespite good overall concordance with viral genomic analysis, clinical and Ct value-based assessments failed to identify 33% of genomically supported reinfections. Scaling-up genomic analysis for clinical use would improve detection of SARS-CoV-2 reinfections.

There are emerging reports of false-positive HIV nucleic acid testing (NAT) in patients who have received chimeric antigen receptor (CAR) T-cell therapies. We present a case of a 66-year-old-woman with primary-refractory stage IIIA double-hit high-grade B-cell lymphoma, in whom we detected false-positive HIV-1 NAT results after receipt of a third-generation self-inactivating investigational lentivirus-based CAR T-cell therapy. We reviewed the current state of the science on HIV-1 NAT and found that all reported false-positive cases have occurred in the setting of lentivirus-based CAR T-cell therapy and testing with FDA-approved platforms targeting the 5’LTR genomic region. Herein, we offer recommendations for HIV diagnostic testing in patients undergoing this mode of therapy. Clinicians managing this patient population should be aware of cross-reactivity between these therapeutic agents and commonly used HIV-1 NAT assays.

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.

Rationale: The leading cause of death in coronavirus disease 2019 (COVID-19) is severe pneumonia, with many patients developing acute respiratory distress syndrome (ARDS) and diffuse alveolar damage (DAD). Whether DAD in fatal COVID-19 is distinct from other causes of DAD remains unknown. Objective: To compare lung parenchymal and vascular alterations between patients with fatal COVID-19 pneumonia and other DAD-causing etiologies using a multidimensional approach. Methods: This autopsy cohort consisted of consecutive patients with COVID-19 pneumonia (n = 20) and with respiratory failure and histologic DAD (n = 21; non-COVID-19 viral and nonviral etiologies). Premortem chest computed tomography (CT) scans were evaluated for vascular changes. Postmortem lung tissues were compared using histopathological and computational analyses. Machine-learning-derived morphometric analysis of the microvasculature was performed, with a random forest classifier quantifying vascular congestion (CVasc) in different microscopic compartments. Respiratory mechanics and gas-exchange parameters were evaluated longitudinally in patients with ARDS. Measurements and Main Results: In premortem CT, patients with COVID-19 showed more dilated vasculature when all lung segments were evaluated (P = 0.001) compared with controls with DAD. Histopathology revealed vasculopathic changes, including hemangiomatosis-like changes (P = 0.043), thromboemboli (P = 0.0038), pulmonary infarcts (P = 0.047), and perivascular inflammation (P < 0.001). Generalized estimating equations revealed significant regional differences in the lung microarchitecture among all DAD-causing entities. COVID-19 showed a larger overall CVasc range (P = 0.002). Alveolar-septal congestion was associated with a significantly shorter time to death from symptom onset (P = 0.03), length of hospital stay (P = 0.02), and increased ventilatory ratio [an estimate for pulmonary dead space fraction (Vd); p = 0.043] in all cases of ARDS. Conclusions: Severe COVID-19 pneumonia is characterized by significant vasculopathy and aberrant alveolar-septal congestion. Our findings also highlight the role that vascular alterations may play in Vd and clinical outcomes in ARDS in general.

Evaluate SARS-CoV-2 RNA and inflammatory cytokines and chemokines in the CSF of patients with acute COVID-19 and neurologic symptoms, and to compare these to controls and patients with known neurotropic pathogens.

Letters29 March 2022Implementation of a SARS-CoV-2 Genotyping Panel for Prompt Omicron Variant Identification: A Pragmatic Tool for Clinical LaboratoriesFREEEliezer Zachary Nussbaum, MD, Vamsi Thiriveedhi, MS, Rockib Uddin, BS, Ha Eun Cho, BS, Seamus Carroll, BA, Eric S. Rosenberg, MD, Jacob E. Lemieux, MD, DPhil, Sarah E. Turbett, MDEliezer Zachary Nussbaum, MDDivision of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts (E.Z.N., R.U., J.E.L.)Search for more papers by this author, Vamsi Thiriveedhi, MSDepartment of Pathology, Massachusetts General Hospital, Boston, Massachusetts (V.T., H.E.C., S.C.)Search for more papers by this author, Rockib Uddin, BSDivision of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts (E.Z.N., R.U., J.E.L.)Search for more papers by this author, Ha Eun Cho, BSDepartment of Pathology, Massachusetts General Hospital, Boston, Massachusetts (V.T., H.E.C., S.C.)Search for more papers by this author, Seamus Carroll, BADepartment of Pathology, Massachusetts General Hospital, Boston, Massachusetts (V.T., H.E.C., S.C.)Search for more papers by this author, Eric S. Rosenberg, MDDivision of Infectious Diseases and Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts (E.S.R., S.E.T.)Search for more papers by this author, Jacob E. Lemieux, MD, DPhilDivision of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts (E.Z.N., R.U., J.E.L.)Search for more papers by this author, Sarah E. Turbett, MDDivision of Infectious Diseases and Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts (E.S.R., S.E.T.)Search for more papers by this authorAuthor, Article and Disclosure Informationhttps://doi.org/10.7326/M22-0023 SectionsSupplemental MaterialAboutVisual Abstract ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinkedInRedditEmail Background: The emergence of the SARS-CoV-2 Omicron variant has important clinical and therapeutic implications. Certain SARS-CoV-2 monoclonal antibody therapies are ineffective or have reduced efficacy against this variant (1). Whole-genome sequencing (WGS) is the gold standard for variant identification, but it requires costly equipment and can be labor intensive, and the results are generally not available to inform real-time therapeutic decisions. An assay that quickly differentiates Omicron from other circulating SARS-CoV-2 strains may help with clinical decision making.Objective: To determine if a real-time nucleic acid amplification-based SARS-CoV-2 mutational panel can accurately identify SARS-CoV-2 variants, including Omicron.Methods and Findings: Many Omicron sequences include the ΔH69/V70 deletion mutation, resulting in the inability of certain SARS-CoV-2 nucleic acid amplification tests (NAATs) to detect the spike gene target (S gene target failure [SGTF]), while still detecting SARS-CoV-2 RNA (2, 3). With the initial reports of Omicron, we screened SARS-CoV-2 NAAT–positive specimens tested within the Mass General Brigham health care system for SGTF using the TaqPath COVID-19 Combo Kit polymerase chain reaction assay (Thermo Fisher Scientific). We subsequently analyzed SGTF specimens for specific mutation target sequences found in Omicron and Delta variants using a polymerase chain reaction–based SARS-CoV-2 mutational panel (TaqMan SARS-CoV-2 Mutational Panel, Thermo Fisher Scientific) that had previously been validated for the detection of select mutations (ΔH69/V70, L452R, E484K, and N501Y) using well-characterized frozen archived clinical respiratory samples positive for Alpha, Beta, Gamma, and Delta variants (Supplement). To enhance Omicron detection, we incorporated 2 additional primer sequences (P681H and K417N) into the mutational panel and analyzed samples according to the following algorithm: Samples were analyzed using at least 3 mutation targets (L452R, K417N, and P681H); if the results of these target sequences resulted in an undetermined variant determination but a K417N mutation was identified, suggesting potential Omicron, the 3 mutation targets were repeated and 3 additional mutation targets were added (ΔH69/V70, L484K, and N501Y) to confirm the identification of Omicron. Variant determinations were made on the basis of the mutational profiles outlined in Table 1. To validate performance of this algorithm, we compared the results from the mutational panel to 119 clinical respiratory tract samples confirmed to be Omicron (n = 69) or Delta (n = 50) by WGS during this time frame (Supplement).Table 1. SARS-CoV-2 Mutational Panel Mutation Targets and Expected Mutation Patterns for Omicron and Delta Variants and Mutation Panel Variant Determinations for 1328 SGTF SARS-CoV-2–Positive Respiratory Tract SpecimensFor the WGS-confirmed Omicron cases, we performed chart review of these persons, recording select clinical information (Table 2). This study was approved by the Mass General Brigham Institutional Review Board for the protection of human subjects under protocol 2019P003305.Table 2. Clinical Characteristics of Patients With Omicron InfectionFrom 1 December to 30 December 2021, we screened 2399 SARS-CoV-2 NAAT-positive specimens for SGTF. Of those, 1328 (55%) were positive for SGTF, with amplification of the ORF1ab and N gene targets. We identified 1260 of 1328 (95%) as Omicron and 14 of 1328 (1%) as Delta variants on the basis of mutation patterns listed in Table 1. In 54 of 1328 (4%) cases, a variant determination could not be made on the basis of the mutation profile; mean cycle threshold for these samples was 31.1 (SD, 4.9). Of the 69 samples that were confirmed to be Omicron by WGS, all were correctly identified by the mutational panel. Of the 50 samples confirmed to be Delta by WGS, 47 of 50 (94%) were correctly identified by the mutational panel; for the remaining 3 WGS-confirmed Delta samples, a variant determination could not be made on the basis of the mutation profile (Supplement). Sensitivity and specificity of the mutational panel for Omicron detection was 100%. Sensitivity and specificity for Delta detection was 94% and 100%, respectively.Pertinent clinical characteristics of the 69 persons with WGS-confirmed Omicron infection are summarized in Table 2. Of note, 12 of 69 (17%) patients were referred for casirivimab–imdevimab or bamlanivimab–etesevimab therapy, with confirmed receipt in 8 patients (12%).Discussion: Our results indicate that a real-time NAAT-based SARS-CoV-2 mutational panel accurately identifies mutations associated with Omicron, leading to correct identification of this variant among samples with SGTF. Although testing for SGTF was a useful screening tool for Omicron detection, the presence of SGTF alone did not predict identification of this variant in all cases and may be a less reliable surrogate for Omicron detection when prevalence is lower, underscoring the importance of more specific methods for real-time variant identification.These results have important practical implications. Compared with the higher cost and slower turnaround time of WGS, the mutational panel provided less expensive, reliable results within the same day of testing using commercially available assays and instruments available in many clinical laboratories. Because casirivimab–imdevimab and bamlanivimab–etesevimab are ineffective against Omicron, these results would allow clinicians to make real-time determinations about appropriate allocation of monoclonal antibody therapy. Indeed, 17% of patients with confirmed Omicron infection were referred for either casirivimab-imdevimab or bamlanivimab-etesevimab, from which they were unlikely to benefit. Although sotrovimab has demonstrated retained activity against Omicron (1), supply of this therapy is limited, highlighting the importance of prompt Omicron identification to ensure optimal allocation.Our study has some limitations. First, although we identified 1262 samples as Omicron using the mutational panel, we were only able to do confirmatory testing with WGS for 69 samples during this time period. Second, our study evaluated the accuracy of only the SARS-CoV-2 mutational panel among samples with SGTF; the ability of this panel to identify Omicron variants without using SGTF as a surrogate marker for ΔH69/V70 deletions (for example, BA.2) was not assessed. Third, the mutational panel evaluated in this study contains mutation targets that may have reduced performance for Omicron (ΔH69/V70 and P681H) because of nonspecific amplification of independent mutations similar to the mutation targets; we account for this potential limitation by including multiple mutation targets in the panel and using overall mutational patterns when making a variant determination. Finally, the mutational panel in this study does not include newer mutational sequences specifically designed for Omicron detection (G339D and Q493R); these targets should be evaluated for potential use in future mutational panels.In conclusion, we show that a NAAT-based SARS-CoV-2 genotyping panel is an accurate and practical tool for real-time identification of Omicron; clinical use of these assays should be considered to help inform therapeutic decisions, particularly when effective therapy is in short supply.References1. Hoffmann M , Krüger N , Schulz S , et al. The Omicron variant is highly resistant against antibody-mediated neutralization: implications for control of the COVID-19 pandemic. Cell. 2022;185:447-456.e11. [PMID: 35026151] doi:10.1016/j.cell.2021.12.032 CrossrefMedlineGoogle Scholar2. World Health Organization. Enhancing response to Omicron SARS-CoV-2 variant: technical brief and priority actions for member states. Accessed at www.who.int/publications/m/item/enhancing-readiness-for-omicron-(b.1.1.529)-technical-brief-and-priority-actions-for-member-states on 23 December 2021. Google Scholar3. Kidd M , Richter A , Best A , et al. S-variant SARS-CoV-2 lineage B1.1.7 is associated with significantly higher viral load in samples tested by TaqPath polymerase chain reaction. J Infect Dis. 2021;223:1666-1670. [PMID: 33580259] doi:10.1093/infdis/jiab082 CrossrefMedlineGoogle Scholar Comments0 CommentsSign In to Submit A Comment Author, Article and Disclosure InformationAffiliations: Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts (E.Z.N., R.U., J.E.L.)Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts (V.T., H.E.C., S.C.)Division of Infectious Diseases and Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts (E.S.R., S.E.T.)* Drs. Lemieux and Turbett contributed equally to this work.Financial Support: Partially funded by the Centers for Disease Control and Prevention Broad Agency Announcements 75D30120C09610 and 75D30120C09605.Disclosures: Disclosures can be viewed at www.acponline.org/authors/icmje/ConflictOfInterestForms.do?msNum=M22-0023.Reproducible Research Statement:Study validation protocol: See the Supplement. Statistical code and genotyping data set: Available on request from Dr. Turbett (e-mail, Turbett.[email protected]HARVARD.EDU). The code for genome assembly is available at https://dockstore.org/organizations/BroadInstitute/collections/pgs. All genomes have been submitted to NCBI GenBank; accession numbers are included in the supplement.Corresponding Author: Sarah E. Turbett, MD, Division of Infectious Diseases, Departments of Medicine and Pathology, Massachusetts General Hospital, Gray-Bigelow 5-526, 55 Fruit Street, Boston, MA 02114; e-mail, Turbett.[email protected]HARVARD.EDU.This article was published at Annals.org on 29 March 2022. PreviousarticleNextarticle Advertisement FiguresReferencesRelatedDetails Metrics LatestKeywordsNucleic acidsRNAInfectious diseasesCOVID-19MutationGenotypingClinical laboratories CopyrightCopyright © 2022 by American College of Physicians. All Rights Reserved.Loading ...

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.