Human Immunodeficiency Virus Persistence Research Articles

Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA.

Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be used for absolute quantification without needing a standard curve. Here, we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA. The limit of detection, dynamic ranges, sensitivity, and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat (LTR) and human CD3 gene (for total HIV DNA) and ACH-2 cells (for integrated HIV DNA). Forty-two cases on stable suppressive antiretroviral therapy (ART) were assayed in total HIV DNA and integrated HIV DNA. Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive (CD4 + ) T-cell counts, CD8 + T-cell counts and CD4/CD8 T-cell ratio, respectively. The assay linear dynamic range and lower limit of detection (LLOD) were also assessed. The assay could detect the presence of HIV-1 copies 100% at concentrations of 6.3 copies/reaction, and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction (95% confidence intervals [CI]: 3.6-6.5 copies/reaction) with linearity over a 5-log 10 -unit range in total HIV DNA assay. For the integrated HIV DNA assay, the LLOD was 8.0 copies/reaction (95% CI: 5.8-16.6 copies/reaction) with linearity over a 3-log 10 -unit range. Total HIV DNA in CD4 + T cells was positively associated with integrated HIV DNA ( r = 0.76, P <0.0001). Meanwhile, both total HIV DNA and integrated HIV DNA in CD4 + T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8 + T-cell counts. This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity. It can be readily adapted for measuring HIV DNA with non-B clades, and it could be beneficial for testing in clinical trials.

Viral Persistence in the Gut-Associated Lymphoid Tissue and Barriers to HIV Cure.

More than 40 years after the first reported cases of what then became known as acquired immunodeficiency syndrome (AIDS), tremendous progress has been achieved in transforming the disease from almost universally fatal to a chronic manageable condition. Nonetheless, the efforts to find a preventative vaccine or a cure for the underlying infection with Human Immunodeficiency Virus (HIV) remain largely unsuccessful. Many challenges intrinsic to the virus characteristics and host response need to be overcome for either goal to be achieved. This article will review the obstacles to an effective HIV cure, specifically the steps involved in the generation of HIV latency, focusing on the role of the gut-associated lymphoid tissue, which has received less attention compared with the peripheral blood, despite being the largest repository of lymphoid tissue in the human body, and a large site for HIV persistence.

The hypoxia-regulated ectonucleotidase CD73 is a host determinant of HIV latency

Deciphering the mechanisms underlying viral persistence is critical to achieving a cure for human immunodeficiency virus (HIV) infection. Here, we implement a systems approach to discover molecular signatures of HIV latently infected CD4+ Tcells, identifying the immunosuppressive, adenosine-producing ectonucleotidase CD73 as a key surface marker of latent cells. Hypoxic conditioning, reflecting the lymphoid tissue microenvironment, increases the frequency of CD73+ CD4+ Tcells and promotes HIV latency. Transcriptomic profiles of CD73+ CD4+ Tcells favor viral quiescence, immune evasion, and cell survival. CD73+ CD4+ Tcells are capable of harboring a functional HIV reservoir and reinitiating productive infection exvivo. CD73 or adenosine receptor blockade facilitates latent HIV reactivation invitro, mechanistically linking adenosine signaling to viral quiescence. Finally, tissue imaging of lymph nodes from HIV-infected individuals on antiretroviral therapy reveals spatial association between CD73 expression and HIV persistence invivo. Our findings warrant development of HIV-cure strategies targeting the hypoxia-CD73-adenosine axis.

Regulation of Immune Homeostasis, Inflammation, and HIV Persistence by the Microbiome, Short-Chain Fatty Acids, and Bile Acids.

Despite antiretroviral therapy (ART), people living with human immunodeficiency virus (HIV) (PLWH) continue to experience chronic inflammation and immune dysfunction, which drives the persistence of latent HIV and prevalence of clinical comorbidities. Elucidating the mechanisms that lead to suboptimal immunity is necessary for developing therapeutics that improve the quality of life of PLWH. Although previous studies have found associations between gut dysbiosis and immune dysfunction, the cellular/molecular cascades implicated in the manifestation of aberrant immune responses downstream of microbial perturbations in PLWH are incompletely understood. Recent literature has highlighted that two abundant metabolite families, short-chain fatty acids (SCFAs) and bile acids (BAs), play a crucial role in shaping immunity. These metabolites can be produced and/or modified by bacterial species that make up the gut microbiota and may serve as the causal link between changes to the gut microbiome, chronic inflammation, and immune dysfunction in PLWH. In this review, we discuss our current understanding of the role of the microbiome on HIV acquisition and latent HIV persistence despite ART. Further, we describe cellular/molecular cascades downstream of SCFAs and BAs that drive innate or adaptive immune responses responsible for promoting latent HIV persistence in PLWH. This knowledge can be used to advance HIV cure efforts.

KLRG1 expression on natural killer cells is associated with HIV persistence, and its targeting promotes the reduction of the viral reservoir

Human immunodeficiency virus (HIV) infection induces immunological dysfunction, which limits the elimination of HIV-infected cells during treated infection. Identifying and targeting dysfunctional immune cells might help accelerate the purging of the persistent viral reservoir. Here, we show that chronic HIV infection increases natural killer (NK) cell populations expressing the negative immune regulator KLRG1, both in peripheral blood and lymph nodes. Antiretroviral treatment (ART) does not reestablish these functionally impaired NK populations, and the expression of KLRG1 correlates with active HIV transcription. Targeting KLRG1 with specific antibodies significantly restores the capacity of NK cells to kill HIV-infected cells, reactivates latent HIV present in CD4+ Tcells co-expressing KLRG1, and reduces the intact HIV genomes in samples from ART-treated individuals. Our data support the potential use of immunotherapy against the KLRG1 receptor to impact the viral reservoir during HIV persistence.

Effect of Immune-Modulatory Interventions on Asymptomatic Cytomegalovirus Shedding During Suppressive Antiretroviral Therapy.

Long-term consequences of human immunodeficiency virus (HIV) are likely the result of persistent inflammation and immune dysfunction of which cytomegalovirus (CMV) is a known contributor. We leveraged 2 AIDS Clinical Trials Group clinical trials exploring the effects of immune modulators (ruxolitinib and sirolimus) on inflammation in people with HIV on antiretroviral therapy to determine whether these interventions affected CMV shedding at various mucosal sites. Analyzing 635 mucosal samples collected, we found no significant difference in CMV levels across study arms or time points. Men had more CMV shedding than women. We did confirm an association between higher CMV DNA and immune markers associated with HIV persistence and HIV-associated mortality rates.

Human immunodeficiency virus infection may be a contributing factor to monkeypox infection: Analysis of a 42-case series

To the Editor: An outbreak of monkeypox has emerged, and more than 13,000 cases have already been confirmed worldwide.In our department, we have 42 confirmed cases so far. All of them are cisgender males presenting with lesions in the genital, perianal, or perioral areas (Fig 1). Interestingly, we observe a disproportionate number of individuals living with the human immunodeficiency virus (HIV). Below, we present a retrospective analysis of our confirmed cases with their clinical and epidemiological characteristics (Table I). Differences between groups were analyzed using the Mann–Whitney U test and the t-test for discrete variables according to distribution. Independence between categorical variables was assessed with Fisher's exact test. All tests were performed for a confidence level of 95% in SPSS 22 (IBM Statistics).Table ICharacteristics of the confirmed cases of monkeypox infection in this cohortWith HIV infection (n = 22)Without HIV infection (n = 20)P-valueEpidemiological variables Age (years; mean ± SD)37.7 ± 9.232.5 ± 8.1.059 Cisgender male22 (100%)20 (100%)1.000 MSM20 (90.1%)17 (85%).656 Number of sexual partners over the last 21 d—Mean, [95% Confidence Interval]6.1 [3.0-9.3]3.8 [1.1-6.5] Vaccinated against smallpox5 (22.7%)0.049∗Highlights variables where differences between groups were significant at a 5% level. Prior history of other STI (other than HIV)9 (40.9%)10 (50.0%).757Symptoms Genital lesions16 (72.7%)12 (60.0%).515 Perianal lesions11 (50.0%)11 (55.0%).767 Perioral lesions6 (27.2%)6 (30.0%)1.000 Lesions in other anatomical areas14 (63.6%)6 (30%).037∗Highlights variables where differences between groups were significant at a 5% level. Fever10 (45.5%)12 (60.0%).374 Myalgias/Arthralgias11 (50.0%)12 (60.0%).551 Headache11 (50.0%)10 (50.0%)1.000 Enlarged lymph nodes16 (72.7%)12 (60.0%).515Unless otherwise specified, values are expressed in absolute number (% of the column total) format; HIV, Human Immunodeficiency Virus; MSM, Men who have Sex with Men; SD, Standard Deviation; STI, Sexually Transmissible Infection.∗ Highlights variables where differences between groups were significant at a 5% level. Open table in a new tab Of the 42 patients, 22 (52.4%) had concomitant HIV infection, corresponding to a prevalence of HIV infection more than 100 times higher than that of the general Portuguese population (0.4%).1da Saúde D.-G. Instituto Nacional de Saúde Doutor Ricardo Jorge Infeção VIH e SIDA em Portugal—2020.http://repositorio.insa.pt/handle/10400.18/7243Date: 2020Date accessed: August 15, 2022Google Scholar One individual was diagnosed with acute HIV infection concurrently with monkeypox, while the others were already receiving appropriate antiretroviral treatment.The HIV-infected patients tended to be older, but this did not reach statistical significance. This could explain the differences in smallpox vaccination between the 2 groups.The prevalence of genital, perianal, and perioral lesions was similar overall. All patients had lesions in at least one of these sites, which are typically affected by sexually transmitted infections.A marked difference between the 2 cohorts was in the prevalence of the disseminated form of the disease. Lesions at sites other than the genital, perianal, and perioral areas were found more frequently in patients with HIV infection (63.3% vs 30.0%; P-value 0.037). The frequency of constitutional symptoms (fever, myalgias/arthralgias, and headache) and lymph node enlargement was similar in both groups.These data suggest individuals living with HIV infection are at higher risk of acquiring monkeypox. Although the number of sexual partners in the past 21 days did not differ significantly between groups, we cannot rule out reporting a bias that could affect the interpretation of these results. Similarly, HIV infection could be a surrogate marker for increased sexual risk behavior, which is a recognized risk factor for acquiring monkeypox, rather than a risk factor itself.Additionally, monkeypox may have a more pronounced clinical presentation in HIV-infected persons regardless of prior smallpox vaccination. This may be due in part to the relative immunodeficiency characteristic of HIV infection, even in treated patients.2Klatt N.R. Chomont N. Douek D.C. Deeks S.G. Immune activation and HIV persistence: implications for curative approaches to HIV infection.Immunol Rev. 2013; 254: 326https://doi.org/10.1111/IMR.12065Crossref PubMed Scopus (0) Google Scholar While HIV infection may be a risk factor for monkeypox, monkeypox lesions may also facilitate the transmission of HIV and other sexually transmitted infections.Our data suggest that the current outbreak differs in epidemiology and clinical presentation from a typical endemic monkeypox infection.3Bellinato F. Gisondi P. Girolomoni G. Monkeypox virus infection: what dermatologist needs to know?.J Eur Acad Dermatol Venereol. 2022; 36: e656-e658https://doi.org/10.1111/JDV.18299Crossref Google Scholar,4Beer E.M. Rao V.B. A systematic review of the epidemiology of human monkeypox outbreaks and implications for outbreak strategy.PLoS Negl Trop Dis. 2019; 13e0007791https://doi.org/10.1371/JOURNAL.PNTD.0007791Crossref Google Scholar Monkeypox should be considered in the differential diagnosis of vesicular and ulcerative lesions in the perianal, genital, and perioral areas. Effective communication strategies for vulnerable populations and immunization of at-risk individuals with vaccines already licensed for this disease may improve outcomes.5Monkeypox and smallpox vaccine guidance | monkeypox | poxvirus | CDC.https://www.cdc.gov/poxvirus/monkeypox/clinicians/smallpox-vaccine.htmlDate accessed: June 16, 2022Google Scholar To the Editor: An outbreak of monkeypox has emerged, and more than 13,000 cases have already been confirmed worldwide. In our department, we have 42 confirmed cases so far. All of them are cisgender males presenting with lesions in the genital, perianal, or perioral areas (Fig 1). Interestingly, we observe a disproportionate number of individuals living with the human immunodeficiency virus (HIV). Below, we present a retrospective analysis of our confirmed cases with their clinical and epidemiological characteristics (Table I). Differences between groups were analyzed using the Mann–Whitney U test and the t-test for discrete variables according to distribution. Independence between categorical variables was assessed with Fisher's exact test. All tests were performed for a confidence level of 95% in SPSS 22 (IBM Statistics). Unless otherwise specified, values are expressed in absolute number (% of the column total) format; HIV, Human Immunodeficiency Virus; MSM, Men who have Sex with Men; SD, Standard Deviation; STI, Sexually Transmissible Infection. Of the 42 patients, 22 (52.4%) had concomitant HIV infection, corresponding to a prevalence of HIV infection more than 100 times higher than that of the general Portuguese population (0.4%).1da Saúde D.-G. Instituto Nacional de Saúde Doutor Ricardo Jorge Infeção VIH e SIDA em Portugal—2020.http://repositorio.insa.pt/handle/10400.18/7243Date: 2020Date accessed: August 15, 2022Google Scholar One individual was diagnosed with acute HIV infection concurrently with monkeypox, while the others were already receiving appropriate antiretroviral treatment. The HIV-infected patients tended to be older, but this did not reach statistical significance. This could explain the differences in smallpox vaccination between the 2 groups. The prevalence of genital, perianal, and perioral lesions was similar overall. All patients had lesions in at least one of these sites, which are typically affected by sexually transmitted infections. A marked difference between the 2 cohorts was in the prevalence of the disseminated form of the disease. Lesions at sites other than the genital, perianal, and perioral areas were found more frequently in patients with HIV infection (63.3% vs 30.0%; P-value 0.037). The frequency of constitutional symptoms (fever, myalgias/arthralgias, and headache) and lymph node enlargement was similar in both groups. These data suggest individuals living with HIV infection are at higher risk of acquiring monkeypox. Although the number of sexual partners in the past 21 days did not differ significantly between groups, we cannot rule out reporting a bias that could affect the interpretation of these results. Similarly, HIV infection could be a surrogate marker for increased sexual risk behavior, which is a recognized risk factor for acquiring monkeypox, rather than a risk factor itself. Additionally, monkeypox may have a more pronounced clinical presentation in HIV-infected persons regardless of prior smallpox vaccination. This may be due in part to the relative immunodeficiency characteristic of HIV infection, even in treated patients.2Klatt N.R. Chomont N. Douek D.C. Deeks S.G. Immune activation and HIV persistence: implications for curative approaches to HIV infection.Immunol Rev. 2013; 254: 326https://doi.org/10.1111/IMR.12065Crossref PubMed Scopus (0) Google Scholar While HIV infection may be a risk factor for monkeypox, monkeypox lesions may also facilitate the transmission of HIV and other sexually transmitted infections. Our data suggest that the current outbreak differs in epidemiology and clinical presentation from a typical endemic monkeypox infection.3Bellinato F. Gisondi P. Girolomoni G. Monkeypox virus infection: what dermatologist needs to know?.J Eur Acad Dermatol Venereol. 2022; 36: e656-e658https://doi.org/10.1111/JDV.18299Crossref Google Scholar,4Beer E.M. Rao V.B. A systematic review of the epidemiology of human monkeypox outbreaks and implications for outbreak strategy.PLoS Negl Trop Dis. 2019; 13e0007791https://doi.org/10.1371/JOURNAL.PNTD.0007791Crossref Google Scholar Monkeypox should be considered in the differential diagnosis of vesicular and ulcerative lesions in the perianal, genital, and perioral areas. Effective communication strategies for vulnerable populations and immunization of at-risk individuals with vaccines already licensed for this disease may improve outcomes.5Monkeypox and smallpox vaccine guidance | monkeypox | poxvirus | CDC.https://www.cdc.gov/poxvirus/monkeypox/clinicians/smallpox-vaccine.htmlDate accessed: June 16, 2022Google Scholar None disclosed.

The role of CD101-expressing CD4 T cells in HIV/SIV pathogenesis and persistence.

Despite the advent of effective antiretroviral therapy (ART), human immunodeficiency virus (HIV) continues to pose major challenges, with extensive pathogenesis during acute and chronic infection prior to ART initiation and continued persistence in a reservoir of infected CD4 T cells during long-term ART. CD101 has recently been characterized to play an important role in CD4 Treg potency. Using the simian immunodeficiency virus (SIV) model of HIV infection in rhesus macaques, we characterized the role and kinetics of CD101+ CD4 T cells in longitudinal SIV infection. Phenotypic analyses and single-cell RNAseq profiling revealed that CD101 marked CD4 Tregs with high immunosuppressive potential, distinct from CD101- Tregs, and these cells also were ideal target cells for HIV/SIV infection, with higher expression of CCR5 and α4β7 in the gut mucosa. Notably, during acute SIV infection, CD101+ CD4 T cells were preferentially depleted across all CD4 subsets when compared with their CD101- counterpart, with a pronounced reduction within the Treg compartment, as well as significant depletion in mucosal tissue. Depletion of CD101+ CD4 was associated with increased viral burden in plasma and gut and elevated levels of inflammatory cytokines. While restored during long-term ART, the reconstituted CD101+ CD4 T cells display a phenotypic profile with high expression of inhibitory receptors (including PD-1 and CTLA-4), immunsuppressive cytokine production, and high levels of Ki-67, consistent with potential for homeostatic proliferation. Both the depletion of CD101+ cells and phenotypic profile of these cells found in the SIV model were confirmed in people with HIV on ART. Overall, these data suggest an important role for CD101-expressing CD4 T cells at all stages of HIV/SIV infection and a potential rationale for targeting CD101 to limit HIV pathogenesis and persistence, particularly at mucosal sites.

Antiretroviral Initiation at ≥800 CD4+ Cells/mm3 Associated With Lower Human Immunodeficiency Virus Reservoir Size.

Identifying factors that determine the frequency of latently infected CD4+ T cells on antiretroviral therapy (ART) may inform strategies for human immunodeficiency virus (HIV) cure. We investigated the role of CD4+ count at ART initiation for HIV persistence on ART. Among participants of the Strategic Timing of Antiretroviral Treatment Study, we enrolled people with HIV (PWH) who initiated ART with CD4+ T-cell counts of 500-599, 600-799, or ≥ 800 cells/mm3. After 36-44 months on ART, the levels of total HIV-DNA, cell-associated unspliced HIV-RNA (CA-US HIV-RNA), and two-long terminal repeat HIV-DNA in CD4+ T cells were quantified and plasma HIV-RNA was measured by single-copy assay. We measured T-cell expression of Human Leucocyte Antigen-DR Isotype (HLA-DR), programmed death-1, and phosphorylated signal transducer and activator of transcription-5 (pSTAT5). Virological and immunological measures were compared across CD4+ strata. We enrolled 146 PWH, 36 in the 500-599, 60 in the 600-799, and 50 in the ≥ 800 CD4 strata. After 36-44 months of ART, total HIV-DNA, plasma HIV-RNA, and HLA-DR expression were significantly lower in PWH with CD4+ T-cell count ≥ 800 cells/mm3 at ART initiation compared with 600-799 or 500-599 cells/mm3. The median level of HIV-DNA after 36-44 months of ART was lower by 75% in participants initiating ART with ≥ 800 vs 500-599 cells/mm3 (median [interquartile range]: 16.3 [7.0-117.6] vs 68.4 [13.7-213.1] copies/million cells, respectively). Higher pSTAT5 expression significantly correlated with lower levels of HIV-DNA and CA-US HIV-RNA. Virological measures were significantly lower in females. Initiating ART with a CD4+ count ≥ 800 cells/mm3 compared with 600-799 or 500-599 cells/mm3 was associated with achieving a substantially smaller HIV reservoir on ART.

Sex Differences in Human Immunodeficiency Virus Persistence and Reservoir Size During Aging.

Sex differences in human immunodeficiency virus (HIV) reservoir dynamics remain underexplored. Longitudinal samples from virally suppressed midlife women (n = 59, median age 45 years) and age-matched men (n = 31) were analyzed retrospectively. At each time point, we measured sex hormones (by means of enzyme-linked immunosorbent assay) and cellular HIV DNA and RNA (by means of digital droplet polymerase chain reaction). Number of inducible HIV RNA+ cells, which provides an upper estimate of the replication-competent reservoir, was quantified longitudinally in a different subset of 14 women, across well-defined reproductive stages. Mixed-effects models included normalized reservoir outcomes and sex, time since antiretroviral therapy (ART) initiation, and the sex-by-time interaction as predictors. At ART initiation, women and men had median (interquartile range [IQR]) CD4+ T-cell counts of 204/μL (83-306/μL) versus 238/μL (120-284/μL), respectively; median ages of 45 (42-48) versus 47 (43-51) years; and median follow-up times of 79.2/μL (60.5-121.1/μL) versus 66.2/μL (43.2-80.6/μL) months. We observed a significant decline of total HIV DNA over time in both men and women (P < .01). However, the rates of change differed significantly between the sexes (P < .01), with women having a significantly slower rate of decline than men, more pronounced with age. By contrast, the levels of inducible HIV RNA increased incrementally over time in women during reproductive aging (P < .01). In contrast to men, in whom the HIV reservoir steadily declines with aging, the HIV reservoir in women is more dynamic. Total HIV DNA (including intact and defective genomes) declines more slowly in women than in men, while the inducible HIV RNA+ reservoir, which is highly enriched in replication-competent virus, increases in women after menopause.

Modeling the Effect of Reactive Oxygen Species and CTL Immune Response on HIV Dynamics

Individuals infected by human immunodeficiency virus (HIV) are under oxidative stress due to the imbalance between reactive oxygen species (ROS) production and elimination. This paper presents a mathematical model with the cytotoxic T lymphocytes (CTL) immune response to examine the role of ROS in the dynamics of HIV infection. We classify the equilibria of the model and study the stability of these equilibria. Numerical simulations show that incorporating ROS and CTL immune response into the model leads to very rich dynamics, including bistable phenomena and periodic solutions. Although the current antiretroviral therapy can suppress viral load to the undetectable level, it cannot eradicate the virus. A high level of ROS may be a factor for HIV persistence in patients despite suppressive therapy. These results suggest that oxidative damage and anti-oxidant therapy should be considered in the study of HIV infection and treatment.

The active human immunodeficiency virus reservoir during antiretroviral therapy: emerging players in viral persistence

To discuss the role of CD4+ T cells with active Human immunodeficiency virus (HIV), meaning infected cells with transcriptional and/or translational viral activity during antiretroviral therapy (ART), focusing on new technologies for its detection, potential cell markers for its characterization, and evidences on the contribution of the active HIV reservoir to long-term viral persistence. HIV-infected cells expressing viral ribonucleic acid are systematically detected in subjects on long-term ART. In recent years, powerful new tools have provided significant insights into the nature, quantification, and identification of cells with active HIV, including the identification of new cell markers, and the presence of viral activity in specific cell populations located in different cellular and anatomical compartments. Moreover, studies on viral sequence integrity have identified cell clones with intact viral genomes and active viral transcription that could potentially persist for years. Together, new investigations support the notion that the active reservoir could represent a relevant fraction of long-term infected cells, and therefore, the study of its cell sources and mechanisms of maintenance could represent a significant advance in our understanding of viral persistence and the development of new curative strategies. The presence of HIV-infected cells with viral expression during ART has been traditionally overlooked for years. Based on recent investigations, this active viral reservoir could play an important role in HIV persistence.

Residual Proviral Reservoirs: A High Risk for HIV Persistence and Driving Forces for Viral Rebound after Analytical Treatment Interruption.

Antiretroviral therapy (ART) has dramatically suppressed human immunodeficiency virus (HIV) replication and become undetectable viremia. However, a small number of residual replication-competent HIV proviruses can still persist in a latent state even with lifelong ART, fueling viral rebound in HIV-infected patient subjects after treatment interruption. Therefore, the proviral reservoirs distributed in tissues in the body represent a major obstacle to a cure for HIV infection. Given unavailable HIV vaccine and a failure to eradicate HIV proviral reservoirs by current treatment, it is crucial to develop new therapeutic strategies to eliminate proviral reservoirs for ART-free HIV remission (functional cure), including a sterilizing cure (eradication of HIV reservoirs). This review highlights recent advances in the establishment and persistence of HIV proviral reservoirs, their detection, and potential eradication strategies.

Challenges and Promise of Human Immunodeficiency Virus Remission.

Antiretroviral therapy effectively controls human immunodeficiency virus (HIV) replication but it is unable to fully eradicate the HIV reservoir and treatment must be life-long. Progress toward a strategy for HIV remission will require overcoming key hurdles to fill gaps in our understanding of HIV persistence, but the identification of individuals who have attained sterilizing or functional HIV cure show that such a goal is achievable. In this review, we first outline challenges in targeting the HIV reservoir, including difficulties identifying HIV-infected cells, ongoing work elucidating the complex intracellular environment that contribute to HIV latency, and barriers to reactivating and clearing the HIV reservoir. We then review reported cases of HIV sterilizing cure and explore natural models of HIV remission and the promise that such HIV spontaneous and posttreatment controllers may hold in our search for a broadly-applicable strategy for the millions of patients living with HIV.

CD32+CD4+ memory T cells are enriched for total HIV-1 DNA in tissues from humanized mice

SummaryCD32 has raised conflicting results as a putative marker of the HIV-1 reservoir. We measured CD32 expression in tissues from viremic and virally suppressed humanized mice treated relatively early or late after HIV-1 infection with combined antiretroviral therapy. CD32 was expressed in a small fraction of the memory CD4+ T-cell subsets from different tissues in viremic and aviremic mice, regardless of treatment initiation time. CD32+ memory CD4+ T cells were enriched in cell-associated (CA) HIV-1 DNA but not in CA HIV-1 RNA as compared to the CD32−CD4+ fraction. Using multidimensional reduction analysis, several memory CD4+CD32+ T-cell clusters were identified expressing HLA-DR, TIGIT, or PD-1. Importantly, although tissue-resident CD32+CD4+ memory cells were enriched with translation-competent reservoirs, most of it was detected in memory CD32-CD4+ T cells. Our findings support that CD32 labels highly activated/exhausted memory CD4+ T-cell subsets that contain only a small proportion of the translation-competent reservoir.

Safety and Immunological Evaluation of Interleukin-21 Plus Anti-α4β7 mAb Combination Therapy in Rhesus Macaques.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections compromise gut immunological barriers, inducing high levels of inflammation and a severe depletion of intestinal CD4+ T cells. Expression of α4β7 integrin promotes homing of activated T cells to intestinal sites where they become preferentially infected; blockade of α4β7 with an anti-α4β7 monoclonal antibody (mAb) prior to infection has been reported to reduce gut SIV viremia in rhesus macaques (RMs). Interleukin-21 (IL-21) administration in antiretroviral therapy-treated, SIV-infected RMs reduces gut inflammation and improves gut integrity. We therefore hypothesized that the combination of IL-21 and anti-α4β7 mAb therapies could synergize to reduce inflammation and HIV persistence. We co-administered two intravenous doses of rhesus anti-α4β7 mAb (50 mg/kg) combined with seven weekly subcutaneous infusions of IL-21–IgFc (100 μg/kg) in four healthy, SIV-uninfected RMs to evaluate the safety and immunological profiles of this intervention in blood and gut. Co-administration of IL-21 and anti-α4β7 mAb showed no toxicity at the given dosages as assessed by multiple hematological and chemical parameters and did not alter the bioavailability of the therapeutics or result in the generation of antibodies against the anti-α4β7 mAb or IL-21–IgFc. Upon treatment, the frequency of CD4 memory T cells expressing β7 increased in blood and decreased in gut, consistent with an inhibition of activated CD4 T-cell homing to the gut. Furthermore, the frequency of T cells expressing proliferation and immune activation markers decreased in blood and, more profoundly, in gut. The combined IL-21 plus anti-α4β7 mAb therapy is well-tolerated in SIV-uninfected RMs and reduces the gut homing of α4β7+ CD4 T cells as well as the levels of gut immune activation.

HIV infection suppresses TLR3 activation-mediated antiviral immunity in microglia and macrophages.

Monocytic-lineage cells in the central nervous system (CNS), including microglia and brain resident macrophages, are the key players in the CNS innate immunity against viral infections, including human immunodeficiency virus (HIV). However, these cells also serve as the major targets and reservoirs for HIV in the CNS. To address the question of how HIV can establish persistent infection in the target cells in the CNS, we examined whether HIV has the ability to counteract Toll-like receptor 3 (TLR3) activation-mediated antiviral immunity in microglia and macrophages. We observed that HIV latently infected microglial cells (HC69·5) expressed reduced levels of TLR3 and TLR3 activation-mediated interferons (IFN-α/β and IFN-λ) as compared with the uninfected control cells (C20). In addition, HIV infection of primary human macrophages suppressed the expression of TLR3 and the IFNs. HIV infection also inhibited the expression of the antiviral IFN-stimulated genes (ISGs) and the HIV-restriction miRNAs. Mechanistically, HIV infection inhibited the phosphorylation of IFN regulatory factors (IRF3 and IRF7) and signal transducer and activator of transcription proteins (STAT1 and STAT3) in both HIV latently infected microglia and acutely infected macrophages. These findings provide previously unrecognized and sound mechanisms for HIV infection and persistence in the primary target and reservoir cells in the brain.

Hepatitis C Virus (HCV) Clearance After Treatment With Direct-Acting Antivirals in Human Immunodeficiency Virus (HIV)-HCV Coinfection Modulates Systemic Immune Activation and HIV Transcription on Antiretroviral Therapy.

BackgroundHepatitis C virus (HCV) coinfection among people with human immunodeficiency virus (HIV) might perturb immune function and HIV persistence. We aimed to evaluate the impact of HCV clearance with direct-acting antivirals (DAAs) on immune activation and HIV persistence in HIV/HCV-coinfected individuals on antiretroviral therapy (ART).MethodsIn a prospective observational study, ART-treated participants with HIV/HCV coinfection received sofosbuvir/daclatasvir ± ribavirin (n = 19). Blood samples were collected before DAA therapy, at the end of treatment, and 12 months after DAA termination (12MPT). T- and natural killer (NK)-cell phenotype, soluble plasma factors, cell-associated (CA)-HIV deoxyribonucleic acid (DNA) forms (total, integrated, 2LTR), CA-unspliced (US) and multiple-spliced ribonucleic acid (RNA), and plasma HIV RNA were evaluated.ResultsHepatitis C virus clearance was associated with (1) a downmodulation of activation and exhaustion markers in CD4+, CD8+ T, and NK cells together with (2) decreased plasma levels of Interferon gamma-induced protein 10 (IP10), interleukin-8 (IL-8), soluble (s)CD163 and soluble intercellular adhesion molecule (sICAM). Cell-associated US HIV RNA was significantly higher at 12MPT compared to baseline, with no change in HIV DNA or plasma RNA.ConclusionsElimination of HCV in HIV/HCV-coinfected individuals alters immune function and the transcriptional activity of latently infected cells. This report provides insights into the effects of HCV coinfection in HIV persistence and regards coinfected subjects as a population in which HIV remission might prove to be more challenging.

HIV antibodies level as a marker of HIV persistence: the role of hepatitis C virus coinfection.

Human immunodeficiency virus (HIV) antibodies have been proposed as a measure of the size of the HIV reservoir. The aim of our study is to quantify the anti-HIV antibodies level in a cohort of people living with HIV (PLWH), stratified based on the presence of continuous undetectable HIV viral load and the co-existence of hepatitis C virus infection. A sample of 229 HIV-monoinfected (n = 114) or HIV/HCV-coinfected [either with resolved HCV infection (n = 75) or active HCV coinfection (n = 40)] patients, followed up a median of 34 (IQR 20-44) months, was studied. Anti-HIV index was obtained as the 1:800 dilution of HIV antibodies. CD4+ T cell count, time with undetectable HIV viral load, annual increase of CD4+ T cell count, anti-HCV therapy, and diagnosis of cirrhosis were analyzed. Patients with a continued suppressed HIV viral load had significant lower anti-HIV index compared with those with virologic failure during the follow-up. Significant higher CD4+ T cell increase was observed in those with a lower anti-HIV index. HIV-monoinfected patients showed an anti-HIV index significantly lower than patients with HCV coinfection. Resolved HCV infection after interferon-based therapy, but not with direct acting antivirals, was associated with a lower anti-HIV index. HIV/HCV-coinfected patients showed higher HIV antibodies level when compared with HIV-monoinfected individuals. A decrease in anti-HIV index in HIV/HCV-coinfected patients was detected when a sustained virological HCV response was obtained after interferon-based therapy, in possible relation with the direct or indirect effect of interferon on PLWH CD4 T cells.

An analytical pipeline for identifying and mapping the integration sites of HIV and other retroviruses

BackgroundAll retroviruses, including human immunodeficiency virus (HIV), must integrate a DNA copy of their genomes into the genome of the infected host cell to replicate. Although integrated retroviral DNA, known as a provirus, can be found at many sites in the host genome, integration is not random. The adaption of linker-mediated PCR (LM-PCR) protocols for high-throughput integration site mapping, using randomly-sheared genomic DNA and Illumina paired-end sequencing, has dramatically increased the number of mapped integration sites. Analysis of samples from human donors has shown that there is clonal expansion of HIV infected cells and that clonal expansion makes an important contribution to HIV persistence. However, analysis of HIV integration sites in samples taken from patients requires extensive PCR amplification and high-throughput sequencing, which makes the methodology prone to certain specific artifacts.ResultsTo address the problems with artifacts, we use a comprehensive approach involving experimental procedures linked to a bioinformatics analysis pipeline. Using this combined approach, we are able to reduce the number of PCR/sequencing artifacts that arise and identify the ones that remain. Our streamlined workflow combines random cleavage of the DNA in the samples, end repair, and linker ligation in a single step. We provide guidance on primer and linker design that reduces some of the common artifacts. We also discuss how to identify and remove some of the common artifacts, including the products of PCR mispriming and PCR recombination, that have appeared in some published studies. Our improved bioinformatics pipeline rapidly parses the sequencing data and identifies bona fide integration sites in clonally expanded cells, producing an Excel-formatted report that can be used for additional data processing.ConclusionsWe provide a detailed protocol that reduces the prevalence of artifacts that arise in the analysis of retroviral integration site data generated from in vivo samples and a bioinformatics pipeline that is able to remove the artifacts that remain.