Low unspliced cell-associated HIV RNA in early treated adolescents living with HIV on long suppressive ART.

Introduction: Initiation of antiretroviral treatment (ART) in patients early after HIV-infection and long-term suppression leads to low or undetectable levels of HIV RNA and cell-associated (CA) HIV DNA and RNA. Both CA-DNA and CA-RNA, overestimate the size of the HIV reservoir but CA-RNA as well as p24/cell-free viral RNA can be indicators of residual viral replication. This study describes HIV RNA amounts and levels of cytokines/soluble markers in 40 well-suppressed adolescents who initiated ART early in life and investigated which viral markers may be informative as endpoints in cure clinical trials within this population. Methods: Forty adolescents perinatally infected with HIV on suppressive ART for >5 years were enrolled in the CARMA study. HIV DNA and total or unspliced CA-RNA in PBMCs were analyzed by qPCR/RT-qPCR and dPCR/RT-dPCR. Cell-free HIV was determined using an ultrasensitive viral load (US-VL) assay. Plasma markers and p24 were analyzed by digital ELISA and correlations between total and unspliced HIV RNA and clinical markers, including age at ART, Western Blot score, levels of cytokines/inflammation markers or HIV CA-DNA, were tested. Results: CA-RNA was detected in two thirds of the participants and was comparable in RT-qPCR and RT-dPCR. Adolescents with undetectable CA-RNA showed significantly lower HIV DNA compared to individuals with detectable CA-RNA. Undetectable unspliced CA-RNA was positively associated with age at ART initiation and Western Blot score. We found that a higher concentration of TNF-α was predictive of higher CA-DNA and CA-RNA. Other clinical characteristics like US-VL, time to suppression, or percent CD4+ T-lymphocytes were not predictive of the CA-RNA in this cross-sectional study. Conclusions: Low CA-DNA after long-term suppressive ART is associated with lower CA-RNA, in concordance with other reports. Patients with low CA-RNA levels in combination with low CA-DNA and low Western Blot scores should be further investigated to characterize candidates for treatment interruption trials. Unspliced CA-RNA warrants further investigation as a marker that can be prioritized in paediatric clinical trials where the sample volume can be a significant limitation.

Emergency myelopoiesis distinguishes multisystem inflammatory syndrome in children from pediatric severe COVID-19.

BACKGROUND: Multisystem inflammatory syndrome in children (MIS-C) is a hyperinflammatory condition caused by recent SARS-CoV-2 infection, but the underlying immunological mechanisms driving this distinct syndrome are unknown. METHODS: We utilized high dimensional flow cytometry, cell-free (cf) DNA, and cytokine and chemokine profiling to identify mechanisms of critical illness distinguishing MIS-C from severe acute COVID-19 (SAC). RESULTS: Compared to SAC, MIS-C patients demonstrated profound innate immune cell death and features of emergency myelopoiesis (EM), an understudied phenomenon observed in severe inflammation. EM signatures were characterized by fewer mature myeloid cells in the periphery and decreased expression of HLA-DR and CD86 on antigen presenting cells. IL-27, a cytokine known to drive hematopoietic stem cells towards EM, was increased in MIS-C, and correlated with immature cell signatures in MIS-C. Upon recovery, EM signatures decreased, and IL-27 plasma levels returned to normal levels. Despite profound lymphopenia, we report a lack of cfDNA released by adaptive immune cells and increased CCR7 expression on T cells indicative of egress out of peripheral blood. CONCLUSIONS: Immune cell signatures of EM combined with elevated innate immune cell-derived cfDNA levels distinguish MIS-C from SAC in children and provide mechanistic insight into dysregulated immunity contributing towards MIS-C, offering potential diagnostic and therapeutic targets.

Age at ART initiation and proviral reservoir size in perinatal HIV-1 infection: considerations for ART-free remission.

PURPOSE OF REVIEW: Achieving ART-free remission without the need for lifelong antiretroviral treatment (ART) is a new objective in HIV-1 therapeutics. This review comprehensively examines the literature to evaluate whether the age at ART initiation in children with perinatal HIV-1 influences the size and decay of the HIV-1 reservoir. The insights gathered from this review serve to inform the field on the unique dynamics of HIV-1 reservoir size in perinatal HIV-1 infection as a function of age at ART initiation, as well as inform biomarker profiling and timing of ART-free remission strategies for children living with HIV-1 globally. RECENT FINDINGS: Recent studies demonstrate that initiating very early effective ART in neonates is feasible and limits HIV-1 reservoir size. The clinical relevance of limiting the HIV-1 reservoir size in perinatal infection was recently demonstrated in the Tatelo Study, which investigated a treatment switch from ART to two broadly neutralizing antibodies (bNAbs) in very early treated children. Low proviral reservoir size was associated with sustained virologic control for 24 weeks on bNAbs. SUMMARY: Immediate and early ART initiation for neonates and infants with perinatal HIV-1 is essential to restricting HIV-1 reservoir size that may enable ART-free remission.

HIV-1 reservoir size after neonatal antiretroviral therapy and the potential to evaluate antiretroviral-therapy-free remission (IMPAACT P1115): a phase 1/2 proof-of-concept study.

BACKGROUND: Infants born with HIV-1 require lifelong antiretroviral therapy (ART). We aimed to assess whether very early ART in neonates might restrict HIV-1 reservoirs, an important step towards ART-free remission. METHODS: IMPAACT P1115 is an ongoing, phase 1/2, proof-of-concept study in which infants were enrolled at 30 research clinics in 11 countries (Brazil, Haiti, Kenya, Malawi, South Africa, Tanzania, Thailand, Uganda, the USA, Zambia, and Zimbabwe) into two cohorts. Infants at least 34 weeks' gestational age at high risk for in-utero HIV-1 with either untreated maternal HIV-1 (cohort 1) or who were receiving pre-emptive triple antiretroviral prophylaxis outside of the study (maternal ART permissible; cohort 2) were included. All infants initiated treatment within 48 h of life. Cohort 1 initiated three-drug nevirapine-based ART, and cohort 2 initiated three-drug nevirapine-based prophylaxis then three-drug nevirapine-based ART following HIV diagnosis by age 10 days. We added twice-daily coformulated oral ritonavir 75 mg/m2 and lopinavir 300 mg/m2 from 14 days of life and 42 weeks postmenstrual age. We discontinued nevirapine 12 weeks after two consecutive plasma HIV-1 RNA levels below limit of detection. We tracked virological suppression, safety outcomes, and meeting a predetermined biomarker profile at age 2 years (undetectable RNA since week 48, HIV-1 antibody-negative, HIV-1 DNA not detected, and normal CD4 count and CD4 percentage) to assess qualification for analytical treatment interruption. This study is registered with ClinicalTrials.gov, NCT02140255. FINDINGS: Between Jan 23, 2015, and Dec 14, 2017, 440 infants were included in cohort 1 and 20 were included in cohort 2. 54 of these infants (34 from cohort 1 and 20 from cohort 2) had confirmed in-utero HIV-1 and were enrolled to receive study ART. 33 (61%) of 54 infants were female and 21 (39%) were male. The estimated probability of maintaining undetectable plasma RNA through to 2 years was 33% (95% CI 17-49) in cohort 1 and 57% (28-78) in cohort 2. Among infants maintaining protocol-defined virological control criteria through to study week 108, seven of 11 (64%, 95% CI 31-89) in cohort 1 and five of seven (71%, 29-96) in cohort 2 had no detected HIV-1 DNA. Ten of 12 (83%, 52-100) in cohort 1 and all seven (100%, 59-100) in cohort 2 tested HIV-1 antibody-negative at week 108. Among 54 infants initiated on very early ART, ten (19%; six in cohort 1 and four in cohort 2) met all criteria for possible analytical treatment interruption. Reversible grade 3 or 4 adverse events occurred in 15 (44%) of 34 infants in cohort 1 and seven (35%) of 20 infants in cohort 2. INTERPRETATION: Very early ART for in-utero HIV-1 can achieve sustained virological suppression in association with biomarkers indicating restricted HIV-1 reservoirs by age 2 years, which might enable potential ART-free remission. FUNDING: National Institute of Allergy and Infectious Diseases, the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and the National Institute of Mental Health.

Current HIV/SIV Reservoir Assays for Preclinical and Clinical Applications: Recommendations from the Experts 2022 NIAID Workshop Summary.

Since the first HIV-cured person was reported in 2009, a strong interest in developing highly sensitive HIV and SIV reservoir assays has emerged. In particular, the question arose about the comparative value of state-of-the-art assays to measure and characterize the HIV reservoir, and how these assays can be applied to accurately detect changes in the reservoir during efforts to develop a cure for HIV infection. Second, it is important to consider the impact on the outcome of clinical trials if these relatively new HIV reservoir assays are incorporated into clinical trial endpoints and/or used for clinical decision-making. To understand the advantages and limitations and the regulatory implications of HIV reservoir assays, the National Institute of Allergy and Infectious Diseases (NIAID) sponsored and convened a meeting on September 16, 2022, to discuss the state of knowledge concerning these questions and best practices for selecting HIV reservoir assays for a particular research question or clinical trial protocol.

Immune activation and exhaustion marker expression on T-cell subsets in ART-treated adolescents and young adults with perinatal HIV-1 infection as correlates of viral persistence.

HIV-1 infection in memory CD4+ T cells forms a latent reservoir that is a barrier to cure. Identification of immune biomarkers that correlate with HIV-1 reservoir size may aid with evaluating efficacy of HIV-1 eradication strategies, towards ART-free remission and cure. In adults living with non-perinatal HIV-1, the immune exhaustion marker PD-1 on central memory CD4+ T cells (Tcm) correlates with measures of HIV-1 reservoir size. Immune correlates of HIV-1 are less defined in adolescents and young adults with perinatal HIV-1. With multi-parameter flow cytometry, we examined immune activation (CD69, CD25, HLA-DR), and exhaustion (PD-1, TIGIT, TIM-3 and LAG-3) markers on CD4+ T cell subsets (naïve (Tn), central memory (Tcm), and the combination (Ttem) of transitional (Ttm) and effector memory (Tem) cells, in 10 adolescents and young adults living with perinatal HIV-1 (median age 15.9 years; median duration of virologic suppression 7.0 years), in whom HIV-1 reservoir size was measured with the Intact Proviral HIV-1 DNA Assay (IPDA) and an enhanced Tat/Rev limiting dilution assay (TILDA). RNA-seq was also performed on the unstimulated CD4+ T cells. The median total HIV-1 DNA concentration in memory CD4+ T cells was 211.90 copies per million CD4+ T cells. In the 7 participants with subtype B HIV-1 infection, the median intact proviral DNA load was 7.96 copies per million CD4+ T cells. Levels of HLA-DR and TIGIT on the Ttem were correlated with total HIV-1 DNA (r=0.76, p=0.015) and (r=0.72, p=0.023), respectively, but not with intact proviral load or induced reservoir size. HIV-1 DNA load was also positively correlated with transcriptional clusters associated with HLA-DR expression by RNA-seq. In contrast, PD-1 expression on Tcm was inversely correlated with total HIV-1 DNA (r=-0.67, p=0.039). Reservoir size by IPDA and TILDA were correlated (r=0.81, p=0.036). Thus, in this cohort of youths with long-standing treated perinatal infection, HLA-DR and TIGIT on Ttem were the key correlates of HIV-1 infected cell frequencies by total HIV-1 DNA, and not PD-1. Total HIV-1 DNA was negatively correlated with PD-1 expressing Tcm. These differences in longstanding perinatal HIV-1 infection compared with adult infection requires further study in larger cohorts.

Extended Analysis of HIV Infection in Cisgender Men and Transgender Women Who Have Sex with Men Receiving Injectable Cabotegravir for HIV Prevention: HPTN 083.

HPTN 083 demonstrated that injectable cabotegravir (CAB) was superior to oral tenofovir disoproxil fumarate-emtricitabine (TDF-FTC) for HIV prevention in cisgender men and transgender women who have sex with men. We previously analyzed 58 infections in the blinded phase of HPTN 083 (16 in the CAB arm and 42 in the TDF-FTC arm). This report describes 52 additional infections that occurred up to 1 year after study unblinding (18 in the CAB arm and 34 in the TDF-FTC arm). Retrospective testing included HIV testing, viral load testing, quantification of study drug concentrations, and drug resistance testing. The new CAB arm infections included 7 with CAB administration within 6 months of the first HIV-positive visit (2 with on-time injections, 3 with ≥1 delayed injection, and 2 who restarted CAB) and 11 with no recent CAB administration. Three cases had integrase strand transfer inhibitor (INSTI) resistance (2 with on-time injections and 1 who restarted CAB). Among 34 CAB infections analyzed to date, diagnosis delays and INSTI resistance were significantly more common in infections with CAB administration within 6 months of the first HIV-positive visit. This report further characterizes HIV infections in persons receiving CAB preexposure prophylaxis and helps define the impact of CAB on the detection of infection and the emergence of INSTI resistance.

How CD4+ T Cells Transcriptional Profile Is Affected by Culture Conditions: Towards the Design of Optimal In Vitro HIV Reactivation Assays.

Most of the current assays directed at the investigation of HIV reactivation are based on cultures of infected cells such as Peripheral Blood Mononuclear Cells (PBMCs) or isolated CD4+ T cells, stimulated in vitro with different activator molecules. The culture media in these in vitro tests lack many age- and donor-specific immunomodulatory components normally found within the autologous plasma. This triggered our interest in understanding the impact that different matrices and cell types have on T cell transcriptional profiles following in vitro culture and stimulation. METHODS: Unstimulated or stimulated CD4+ T cells of three young adults with perinatal HIV-infection were isolated from PBMCs before or after culture in RPMI medium or autologous plasma. Transcriptomes were sequenced using Oxford Nanopore technologies. RESULTS: Transcriptional profiles revealed the activation of similar pathways upon stimulation in both media with a higher magnitude of TCR cascade activation in CD4+ lymphocytes cultured in RPMI. CONCLUSIONS: These results suggest that for studies aiming at quantifying the magnitude of biological mechanisms under T cell activation, the autologous plasma could better approximate the in vivo environment. Conversely, if the study aims at defining qualitative aspects, then RPMI culture could provide more evident results.

HIV-1 remission and possible cure in a woman after haplo-cord blood transplant.

Previously, two men were cured of HIV-1 through CCR5Δ32 homozygous (CCR5Δ32/Δ32) allogeneic adult stem cell transplant. We report the first remission and possible HIV-1 cure in a mixed-race woman who received a CCR5Δ32/Δ32 haplo-cord transplant (cord blood cells combined with haploidentical stem cells from an adult) to treat acute myeloid leukemia (AML). Peripheral blood chimerism was 100% CCR5Δ32/Δ32 cord blood by week 14 post-transplant and persisted through 4.8 years of follow-up. Immune reconstitution was associated with (1) loss of detectable replication-competent HIV-1 reservoirs, (2) loss of HIV-1-specific immune responses, (3) in vitro resistance to X4 and R5 laboratory variants, including pre-transplant autologous latent reservoir isolates, and (4) 18 months of HIV-1 control with aviremia, off antiretroviral therapy, starting at 37 months post-transplant. CCR5Δ32/Δ32 haplo-cord transplant achieved remission and a possible HIV-1 cure for a person of diverse ancestry, living with HIV-1, who required a stem cell transplant for acute leukemia.

Markers of intestinal immune activation and inflammation are not associated with preterm birth among women with low level HIV viremia.

BACKGROUND: Maternal markers of intestinal immune activation may be used to predict preterm birth (PTB) in pregnant women living with HIV. METHODS: This study used de-identified samples from the International Maternal Pediatric Adolescent AIDS Clinical Trials Group (IMPAACT) Protocol P1025 study. Singleton pregnancies with ≥3 ml plasma available and HIV viral load ≤400 copies/ml within 4 weeks of specimen collection were included. Frequency matching of PTB cases and term birth controls was performed on basis of maternal race, number of available plasma specimens, and timing of plasma sample collection in a 1:1 ratio. Plasma progesterone, 25-hydroxy vitamin D, soluble CD14, intestinal fatty acid binding protein (I-FABP), Lipopolysaccharide (LPS)-binding protein, and inflammatory cytokines (IL-1B, IFN-gamma, IL-6, TNF-alpha) were measured. Generalized mixed linear regression modeling was used to examine the association between PTB and biomarkers, adjusting for covariates and confounders. Data analyses were performed using SAS 9.4 (Cary, NC). RESULTS: We included 104 PTB compared to 104 controls. Third trimester log2 IL-1B was lower among PTB versus term birth controls by univariate analysis (-1.50 ± 2.26 vs. -.24 ± 2.69, p = .01) though this association was no longer significant by regression modeling. In an uncontrolled, exploratory sub-analysis, subjects with prior PTB had increased odds of PTB with higher I-FABP [aOR 2.72, 95% CI 1.18-6.24] and lower IFN-gamma [aOR .23, 95% CI .12-.41] after adjustment for covariates and confounders. CONCLUSIONS: Intestinal immune activation measured by soluble CD14 or intestinal fatty acid binding protein was not associated with preterm birth among pregnant women with low-level HIV viremia.

Willingness of Racially Diverse Young Adults Living with HIV to Participate in HIV Cure Research: A Cross-Sectional Survey in the United States.

Nearly half of new HIV cases in the United States are among youth. Little is known about the willingness of young adults living with HIV (YLWH) to participate in HIV cure-related research. In 2021, we recruited 271 YLWH aged 18-29 for an online survey. We asked questions about willingness to participate in HIV cure research, perceived risks and benefits, acceptable trade-offs, and perceptions on analytical treatment interruptions. We conducted descriptive analyses to summarize data and bivariate analyses to explore correlations by demographics. Most respondents (mean age = 26) identified as men (86%) and Black Americans (69%). YLWH expressed high willingness to consider participating in cell- and gene-based approaches (75%) and immune-based approaches (71%). Approximately 45% would be willing to let their viral load become detectable for a period of time during an HIV cure study, 27% would not be willing, and 28% did not know. The social risk most likely to deter participation was the possibility of transmitting HIV to sex partners while off HIV medications (65% of respondents would be deterred a great deal or a lot). Compared to the 25-29 age group (n = 192), the 18-24 age group (n = 79) was more likely to indicate that having to disclose HIV status would matter a great deal in considering participation in HIV cure research (38% vs. 21%, p = .003). Inclusion and engagement of YLWH are critical for advancing novel HIV curative agents. Our article concludes with possible considerations for engaging YLWH in HIV cure research. Physical, clinical, and social risks will need to be kept to a minimum, and research teams will need to proactively mitigate the possibility of transmitting HIV to sex partners while off HIV medications.

Infants Receiving Very Early Antiretroviral Therapy Have High CD4 Counts in the First Year of Life.

We followed 54 infants with in utero HIV after initiating very early antiretroviral treatment. At weeks 24 and 48, ≥80% had CD4 ≥1500 cells/mm3 and CD4% ≥25%. Routine Pneumocystis jirovecii pneumonia prophylaxis in the first year of life may not be necessary for all very early treated infants. CLINICAL TRIALS REGISTRATION: NCT02140255.

Advances in Pediatric HIV-1 Cure Therapies and Reservoir Assays.

Significant advances in the field of HIV-1 therapeutics to achieve antiretroviral treatment (ART)-free remission and cure for persons living with HIV-1 are being made with the advent of broadly neutralizing antibodies and very early ART in perinatal infection. The need for HIV-1 remission and cure arises due to the inability of ART to eradicate the major reservoir for HIV-1 in resting memory CD4+ T cells (the latent reservoir), and the strict adherence to lifelong treatment. To measure the efficacy of these cure interventions on reservoir size and to dissect reservoir dynamics, assays that are sensitive and specific to intact proviruses are critical. In this review, we provided a broad overview of some of the key interventions underway to purge the reservoir in adults living with HIV-1 and ones under study in pediatric populations to reduce and control the latent reservoir, primarily focusing on very early treatment in combination with broadly neutralizing antibodies. We also summarized assays currently in use to measure HIV-1 reservoirs and their feasibility and considerations for studies in children.

Assessing the Variability of Cell-Associated HIV DNA Quantification through a Multicenter Collaborative Study.

Reliable and accurate quantification of cell-associated HIV DNA (CA HIV DNA) is critical for early infant diagnosis, clinical management of patients under therapy, and to inform new therapeutics efficacy. The present study assessed the variability of CA HIV DNA quantification obtained from various assays and the value of using reference materials to help harmonize the measurements. Using a common set of reagents, our multicenter collaborative study highlights significant variability of CA HIV DNA quantification and lower limit of quantification across assays. The quantification of CA HIV DNA from a panel of infected PBMCs can be harmonized through cross-subtype normalization but assay calibration with the commonly used 8E5 cell line failed to reduce quantification variability between assays, demonstrating the requirement to thoroughly evaluate reference material candidates to help improve the comparability of CA HIV DNA diagnostic assay performance. IMPORTANCE Despite a global effort, HIV remains a major public health burden with an estimated 1.5 million new infections occurring in 2020. HIV DNA is an important viral marker, and its monitoring plays a critical role in the fight against HIV: supporting diagnosis in infants and underpinning clinical management of patients under therapy. Our study demonstrates that HIV DNA measurement of the same samples can vary significantly from one laboratory to another, due to heterogeneity in the assay, protocol, and reagents used. We show that when carefully selected, reference materials can reduce measurement variability and harmonize HIV DNA quantification across laboratories, which will help contribute to improved diagnosis and clinical management of patients living with HIV.

Characterization of Human Immunodeficiency Virus (HIV) Infections in Women Who Received Injectable Cabotegravir or Tenofovir Disoproxil Fumarate/Emtricitabine for HIV Prevention: HPTN 084.

BACKGROUND: HIV Prevention Trials Network 084 demonstrated that long-acting injectable cabotegravir (CAB) was superior to daily oral tenofovir (TFV) disoproxil fumarate (TDF)/emtricitabine (FTC) for preventing human immunodeficiency virus (HIV) infection in sub-Saharan African women. This report describes HIV infections that occurred in the trial before unblinding. METHODS: Testing was performed using HIV diagnostic assays, viral load testing, a single-copy RNA assay, and HIV genotyping. Plasma CAB, plasma TFV, and intraerythrocytic TFV-diphosphate concentrations were determined by liquid chromatography-tandem mass spectrometry. RESULTS: Forty HIV infections were identified (CAB arm, 1 baseline infection, 3 incident infections; TDF/FTC arm, 36 incident infections). The incident infections in the CAB arm included 2 with no recent drug exposure and no CAB injections and 1 with delayed injections; in 35 of 36 cases in the TDF/FTC arm, drug concentrations indicated low or no adherence. None of the cases had CAB resistance. Nine women in the TDF/FTC arm had nonnucleoside reverse-transcriptase inhibitor resistance; 1 had the nucleoside reverse-transcriptase inhibitor resistance mutation, M184V. CONCLUSIONS: Almost all incident HIV infections occurred in the setting of unquantifiable or low drug concentrations. CAB resistance was not detected. Transmitted nonnucleoside reverse-transcriptase inhibitor resistance was common; 1 woman may have acquired nucleoside reverse-transcriptase inhibitor resistance from study drug exposure.

Experience and Outcomes of Breastfed Infants of Women Living With HIV in the United States: Findings From a Single-Center Breastfeeding Support Initiative.

We assessed breastfeeding outcomes for a cohort of infants born to women living with HIV (WLHIV) at an urban health care center in the United States. Ten infants were exclusively breastfed for a mean duration of 4.4 (1.0-8.6) months. All had negative HIV RNA PCRs at a median age of 16 months.

Distinct Cytokine and Chemokine Dysregulation in Hospitalized Children With Acute Coronavirus Disease 2019 and Multisystem Inflammatory Syndrome With Similar Levels of Nasopharyngeal Severe Acute Respiratory Syndrome Coronavirus 2 Shedding.

BACKGROUND: Multisystem inflammatory syndrome in children (MIS-C) is a severe clinical phenotype of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that remains poorly understood. METHODS: Hospitalized children <18 years of age with suspected coronavirus disease 2019 (COVID-19) (N = 53) were recruited into a prospective cohort study; 32 had confirmed COVID-19, with 16 meeting the US Centers for Disease Control criteria for MIS-C. Differences in nasopharyngeal viral ribonucleic acid (RNA) levels, SARS-CoV-2 seropositivity, and cytokine/chemokine profiles were examined, including after adjustments for age and sex. RESULTS: The median ages for those with and without MIS-C were 8.7 years (interquartile range [IQR], 5.5-13.9) and 2.2 years (IQR, 1.1-10.5), respectively (P = .18), and nasopharyngeal levels of SARS-CoV-2 RNA did not differ significantly between the 2 groups (median 63 848.25 copies/mL versus 307.1 copies/mL, P = .66); 75% of those with MIS-C were antibody positive compared with 44% without (P = .026). Levels of 14 of 37 cytokines/chemokines (interleukin [IL]-1RA, IL-2RA, IL-6, IL-8, tumor necrosis factor-α, IL-10, IL-15, IL-18, monocyte chemoattractant protein [MCP]-1, IP-10, macrophage-inflammatory protein [MIP]-1α, MCP-2, MIP-1ß, eotaxin) were significantly higher in children with MIS-C compared to those without, irrespective of age or sex (false discovery rate <0.05; P < .05). CONCLUSIONS: The distinct pattern of heightened cytokine/chemokine dysregulation observed with MIS-C, compared with acute COVID-19, occurs across the pediatric age spectrum and with similar levels of nasopharyngeal SARS-CoV-2 RNA.

Insights from Clonal Expansion and HIV Persistence in Perinatal Infections.

The latent HIV reservoir forms early in the course of infection and is maintained for life despite effective antiretroviral treatment (ART), including early treatment. Perinatal HIV infection presents a unique opportunity to limit seeding of the reservoir through early ART. However, a greater understanding of the persistence of the integrated proviruses is needed for targeting the residual proviruses that form barriers to cure. A study was performed by Bale and Katusiime et al. (M. J. Bale, M. G. Katusiime, D. Wells, X. Wu, et al., mBio 12:e00568-21, 2021, https://doi.org/10.1128/mBio.00568-21) using in-depth integration site analysis in 11 children before ART and after up to nine years of ART. They have identified early development of long-lived proviruses, although the replication competence is unknown. A small fraction of cells bearing integrated proviruses clonally expand early during infection and persist. Integration in the oncogenes STAT5B and BACH2 were also found; these findings confirm the early development of clonal proliferation in perinatal HIV infection despite early effective ART, with a propensity for oncogenes.

Clinical validation of a quantitative HIV-1 DNA droplet digital PCR assay: Applications for detecting occult HIV-1 infection and monitoring cell-associated HIV-1 dynamics across different subtypes in HIV-1 prevention and cure trials.

BACKGROUND: In HIV-1-exposed infants, nucleic acid testing (NAT) is required to diagnose infection since passively transferred maternal antibodies preclude antibody testing. The sensitivity of clinical NAT assays is lowered with infant antiretroviral prophylaxis and, with empiric very early antiretroviral treatment of high-risk infants, thereby impacting early infant diagnosis. Similarly, adult HIV-1 infections acquired under pre-exposure prophylaxis may occur at low levels, with undetectable plasma viremia and indeterminate antibody tests, for which HIV-1 DNA testing maybe a useful adjunct. Cell-associated HIV-1 DNA concentrations are also used to monitor HIV-1 persistence in viral reservoirs with relevance to HIV-1 cure therapeutics, particularly in perinatal infections. OBJECTIVES: We clinically validated an HIV-1 DNA quantitative assay using droplet digital PCR (ddPCR), across different HIV-1 subtypes. STUDY DESIGN: The analytical sensitivity and specificity of an HIV-1 DNA ddPCR assay was determined using serial dilutions of a plasmid containing HIV-1 LTR-gag spiked into peripheral blood mononuclear cells (PBMCs), with MOLT-4 cells or PBMCs infected with different HIV-1 subtypes (A, B and C), and U1 cells spiked into PBMCs. Inter- and intra-run variability were used to determine assay precision. RESULTS: The HIV-1 LTR-gag ddPCR assay was reliable and reproducible, and exhibited high analytical specificity with sensitivity to near single copy level, across multiple HIV-1 subtypes, and a limit of detection of 4.09 copies/million PBMCs. CONCLUSIONS: This assay has applications for detecting occult HIV-1-infection in the setting of combination and long-acting regimens used for HIV-1 prevention, across different HIV-1 subtypes, in infants and adults, and in HIV-1 cure interventions.