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.

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.

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.

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.

A 77 Amino Acid Region in the N-Terminal Half of the HSV-1 E3 Ubiquitin Ligase ICP0 Contributes to Counteracting an Established Type 1 Interferon Response.

Herpes simplex virus 1 (HSV-1) is a human pathogen capable of establishing lifelong latent infections that can reactivate under stress conditions. A viral immediate early protein that plays important roles in the HSV-1 lytic and latent infections is the viral E3 ubiquitin ligase, ICP0. ICP0 transactivates all temporal classes of HSV-1 genes and facilitates viral gene expression. ICP0 also impairs the antiviral effects of interferon (IFN)-ß, a component of host innate defenses known to limit viral replication. To begin to understand how ICP0 allows HSV-1 to disarm the IFN-ß response, we performed genetic analyses using a series of ICP0 truncation mutants in the absence and presence of IFN-ß in cell culture. We observed that IFN-ß pretreatment of cells significantly impaired the replication of the ICP0 truncation mutants, n212 and n312, which code for the first 211 and 311 amino acids of ICP0, respectively; this effect of IFN-ß correlated with decreased HSV-1 early and late gene expression. This increased sensitivity to IFN-ß was not as apparent with the ICP0 mutant, n389. Our mapping studies indicate that loss of 77 amino acids from residues 312 to 388 in the N-terminal half of ICP0 resulted in a virus that was significantly more sensitive to cells pre-exposed to IFN-ß. This 77 amino acid region contains a phospho-SUMO-interacting motif or -SIM, which we propose participates in ICP0's ability to counteract the antiviral response established by IFN-ß. IMPORTANCE Interferons (IFNs) are secreted cellular factors that are induced by viral infection and limit replication. HSV-1 is largely refractory to the antiviral effects of type 1 IFNs, which are synthesized shortly after viral infection, in part through the activities of the viral regulatory protein, ICP0. To understand how ICP0 impedes the antiviral effects of type 1 IFNs, we used a series of HSV-1 ICP0 mutants and examined their viral replication and gene expression levels in cells stimulated with IFN-ß (a type 1 IFN). Our mapping data identifies a discrete 77 amino acid region in the N-terminal half of ICP0 that facilitates HSV-1 resistance to IFN-ß. This region of ICP0 is modified by phosphorylation and binds to the posttranslational modification SUMO, suggesting that HSV, and potentially other viruses, may counteract type 1 IFN signaling by altering SUMO and/or SUMO modified cellular proteins.

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.

Differences in inducibility of the latent HIV reservoir in perinatal and adult infection.

The HIV latent reservoir in resting memory CD4+ T cells precludes cure. Therapeutics to reactivate and eliminate this reservoir are in clinical trials in adults, but not yet in pediatric populations. We determined, ex vivo, the inducibility of the latent reservoir in perinatal infection as compared with adult infections using the Tat/rev induced limiting dilution assay (TILDA), in which a single round (12 hours) of CD4+ T cell stimulation with PMA/ionomycin maximally activates T cells and leads to proviral expression with multiply spliced HIV RNA production. Markers of immune activation and exhaustion were measured to assess interactions with inducibility. Although rates of T cell activation with PMA/ionomycin were similar, the latent reservoir in perinatal infection was slower to reactivate and of lower magnitude compared with adult infection, independent of proviral load. An enhanced TILDA with the addition of phytohemagglutin and a duration of 18 hours augmented proviral expression in perinatal but not adult infection. The baseline HLA-DR+CD4+ T cell level was significantly lower in perinatal compared with adult infections, but not correlated with induced reservoir size. These data support the hypothesis that there are differences in kinetics of latency reversal and baseline immune activation in perinatal compared with adult infections, with implications for latency reversal strategies toward reservoir clearance and remission.

The Latent Human Immunodeficiency Virus (HIV) Reservoir Resides Primarily in CD32-CD4+ T Cells in Perinatally HIV-Infected Adolescents With Long-Term Virologic Suppression.

Background: High-level expression of the Fcγ receptor, CD32hi, on CD4+ T cells was associated with enhanced human immunodeficiency virus (HIV) infection of the latent reservoir in a study of adults receiving antiretroviral therapy. We tested the hypothesis that CD32 was the preferential marker of the latent HIV reservoir in virally suppressed, perinatally HIV-infected adolescents. Methods: The frequency of CD32hiCD4+ T cells was determined by flow cytometry (N = 5) and the inducible HIV reservoir in both CD32hi and CD32-CD4+ T cells was quantified (N = 4) with a quantitative viral outgrowth assay. Viral outgrowth was measured by the standard p24 enzyme-linked immunosorbent assay and an ultrasensitive p24 assay (Simoa; Quanterix) with lower limits of quantitation. Results: We found a 59.55-fold enrichment in the absolute number of infectious cells in the CD32- population compared with CD32hi cells. Exponential HIV replication occurred exclusively in CD32-CD4+ T cells (mean change, 17.46 pg/mL; P = .04). Induced provirus in CD32hiCD4+ T cells replicated to substantially lower levels, which did not increase significantly over time (mean change, 0.026 pg/mL; P = .23) and were detected only with the Simoa assay. Conclusions: Our data suggests that the latent HIV reservoir resides mainly in CD32-CD4+ T cells in virally suppressed, perinatally HIV-infected adolescents, which has implications for reservoir elimination strategies.

Reactivation of Latent Epstein-Barr Virus: A Comparison after Exposure to Gamma, Proton, Carbon, and Iron Radiation.

Among the many stressors astronauts are exposed to during spaceflight, cosmic radiation may lead to various serious health effects. Specifically, space radiation may contribute to decreased immunity, which has been documented in astronauts during short- and long-duration missions, as evidenced by several changes in cellular immunity and plasma cytokine levels. Reactivation of latent herpes viruses, either directly from radiation of latently infected cells and/or from perturbation of the immune system, may result in disease in astronauts. Epstein‒Barr virus (EBV) is one of the eight human herpes viruses known to infect more than 90% of human adults and persists for the life of the host without normally causing adverse effects. Reactivation of several latent viruses in astronauts is well documented, although the mechanism of reactivation is not well understood. We studied the effect of four different types of radiation, (1) 137Cs gamma rays, (2) 150-MeV protons, (3) 600 MeV/n carbon ions, and (4) 600 MeV/n iron ions on the activation of lytic gene transcription and of reactivation of EBV in a latently infected cell line (Akata) at doses of 0.1, 0.5, 1.0, and 2.0 Gy. The data showed that for all doses used in this study, lytic gene transcription was induced and median viral loads were significantly higher for all types of radiation than in corresponding control samples, with the increases detected as early as four days post-exposure and generally tapering off at later time points. The viability and size of EBV-infected Akata cells were highly variable and exhibited approximately the same trend in time for all radiation types at 0.1, 0.5, 1.0, and 2.0 Gy. This work shows that reactivation of viruses can occur due to the effect of different types of radiation on latently infected cells in the absence of changes or cytokines produced in the immune system. In general, gamma rays are more effective than protons, carbon ions, and iron ions in inducing latent virus reactivation, though these high-energy particles did induce more sustained and later reactivation of EBV lytic gene transcription. These findings also challenge the common relative biological effectiveness concept that is often used in radiobiology for other end points.

Depletion of the Insulator Protein CTCF Results in Herpes Simplex Virus 1 Reactivation In Vivo.

Herpes simplex virus 1 (HSV-1) establishes a lifelong latent infection in host peripheral neurons, including the neurons of the trigeminal ganglia (TG). HSV-1 can reactivate from neurons to cause recurrent infection. During latency, the insulator protein CTCF occupies DNA binding sites on the HSV-1 genome, and these sites have been previously characterized as functional enhancer-blocking insulators. Previously, CTCF was found to be dissociated from wild-type virus postreactivation but not in mutants that do not reactivate, indicating that CTCF eviction may also be an important component of reactivation. To further elucidate the role of CTCF in reactivation of HSV-1, we used recombinant adeno-associated virus (rAAV) vectors to deliver a small interfering RNA targeting CTCF to peripheral neurons latent with HSV-1 in rabbit TG. Our data show that CTCF depletion resulted in long-term and persistent shedding of infectious virus in the cornea and increased ICP0 expression in the ganglia, indicating that CTCF depletion facilitates HSV-1 reactivation.IMPORTANCE Increasing evidence has shown that the insulator protein CTCF regulates gene expression of DNA viruses, including the gammaherpesviruses. While CTCF occupation and insulator function control gene expression in DNA viruses, CTCF eviction has been correlated to increased lytic gene expression and the dissolution of transcriptional domains. Our previous data have shown that in the alphaherpesvirus HSV-1, CTCF was found to be dissociated from the HSV-1 genome postreactivation, further indicating a global role for CTCF eviction in the transition from latency to reactivation in HSV-1 genomes. Using an rAAV8, we targeted HSV-1-infected peripheral neurons for CTCF depletion to show that CTCF depletion precedes the shedding of infectious virus and increased lytic gene expression in vivo, providing the first evidence that CTCF depletion facilitates HSV-1 reactivation.

Alternative Sample Types for HIV-1 Antiretroviral Drug Resistance Testing.

Monitoring human immunodeficiency virus type 1 (HIV-1) drug resistance is critical for assessing ART effectiveness and treatment outcomes for HIV-1-infected individuals, including children, worldwide. Traditionally, testing for HIV-1 drug resistance has primarily been performed on plasma samples, and with commercially available, clinically validated assays that are costly and difficult to access. While plasma is the preferred sample for HIV-1 drug resistance genotyping, plasma analysis requires sophisticated laboratory equipment, personnel, space, and stringent storage conditions for maintenance of sample integrity and transport. With the limitations in feasibility and affordability of providing these ideal conditions for plasma genotyping in resource-constrained settings, the field has gained substantial experience with the dried blood spot (DBS) technique as an alternative. Moreover, DBS analysis can be used to comprehensively monitor the spread of the epidemic with applications to more-sensitive and quantitative technologies to assess HIV-1 globally.

Inhibition of H3K27me3-specific histone demethylases JMJD3 and UTX blocks reactivation of herpes simplex virus 1 in trigeminal ganglion neurons.

Herpes simplex virus 1 (HSV-1) genomes are associated with the repressive heterochromatic marks H3K9me2/me3 and H3K27me3 during latency. Previous studies have demonstrated that inhibitors of H3K9me2/me3 histone demethylases reduce the ability of HSV-1 to reactivate from latency. Here we demonstrate that GSK-J4, a specific inhibitor of the H3K27me3 histone demethylases UTX and JMJD3, inhibits HSV-1 reactivation from sensory neurons in vitro. These results indicate that removal of the H3K27me3 mark plays a key role in HSV-1 reactivation.

Role of polycomb proteins in regulating HSV-1 latency.

Herpes simplex virus (HSV) establishes a latent infection within sensory neurons of humans. Latency is characterized by the transcriptional repression of lytic genes by the condensation of lytic gene regions into heterochromatin. Recent data suggest that facultative heterochromatin predominates, and that cellular Polycomb proteins are involved in the establishment and maintenance of transcriptional repression during latency. This review summarizes these data and discusses the implication of viral and cellular factors in regulating heterochromatin composition.

Dynamic dosing assay relating real-time respiration responses of Staphylococcus aureus biofilms to changing microchemical conditions.

Bacterial biofilms are a metabolically heterogeneous community of bacteria distributed in an extracellular matrix comprised primarily of hydrated polysaccharides. Effective inhibitory concentrations measured under planktonic conditions are not applicable to biofilms, and inhibition concentrations measured for biofilms vary widely. Here, we introduce a novel microfluidic approach for screening respiration inhibition of bacteria in a biofilm array morphology. The device geometry and operating conditions allow antimicrobial concentration and flux to vary systematically and predictably with space and time. One experiment can screen biofilm respiratory responses to many different antimicrobial concentrations and dosing rates in parallel. To validate the assay, onset of respiration inhibition following NaN3 exposure is determined optically using an O2-sensing thin film. Onset of respiration inhibition obeys a clear and reproducible pattern based on time for diffusive transport of the respiration inhibitor to each biofilm in the array. This approach can be used for high-throughput screening of antimicrobial effectiveness as a function of microbial characteristics, antimicrobial properties, or antimicrobial dosing rates. The approach may also be useful in better understanding acquired antimicrobial resistance or for screening antimicrobial combinations.

Window on a microworld: simple microfluidic systems for studying microbial transport in porous media.

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 microm. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.