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Objectives Elevated levels of interleukin-6 (IL-6), D-dimer, and C-reactive protein (hsCRP) are associated with increased incidence of comorbid disease and mortality among people living with HIV (PLWH). Prior studies suggest a genetic basis for these biomarker elevations in the general population. The study objectives are to identify the genetic basis for these biomarkers among PLWH. Methods Baseline levels of hsCRP, D-dimer and IL-6, and single nucleotide polymorphisms (SNPs) were determined for 7,768 participants in three HIV treatment trials. Single variant analysis was performed for each biomarker on samples from each of three ethnic groups (African [AFR], Admixed American [AMR], European [EUR]) within each trial including covariates relevant to biomarker levels. For each ethnic group, the results were pooled across trials, then further pooled across ethnicities. Results The transethnic analysis identified three, two and one known loci associated with hsCRP, D-dimer and IL-6 levels, respectively, and two novel loci, FGB and GCNT1, associated with D-dimer levels. Lead SNPs exhibited similar effects across ethnicities. Additionally, three novel, ethnic-specific loci were identified: CATSPERG associated with D-dimer in AFR and PROX1-AS1 and TRAPPC9 associated with IL-6 in AFR and AMR, respectively. Conclusions Eleven loci associated with three biomarker levels were identified in PLWH from the three studies including six loci known in the general population and five novel loci associated with D-dimer and IL-6 levels. These findings support the hypothesis that host genetics may partially contribute to chronic inflammation in PLWH and help to identify potential targets for intervention of serious non-AIDS complications.

Recently, a genome-wide association study using plasma HIV RNA from antiretroviral therapy-naive patients reported that 14 naturally occurring nonsynonymous single-nucleotide polymorphisms (SNPs) in HIV derived from antiretrovirus drug-naive patients were associated with virus load (VL). Those SNPs were detected in reverse transcriptase, RNase H, integrase, envelope, and Nef. However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP and examined their replicative abilities. An HIV variant containing a Met-to-Ile change at codon 50 in integrase [HIV(IN:M50I)] was found as an impaired virus. Despite the mutation being in integrase, the virus release was significantly suppressed (P

OBJECTIVES: The Strategic Timing of AntiRetroviral Treatment (START) and Strategies for Management of Antiretroviral Therapy (SMART) trials demonstrated that ART can partly reverse clinically defined immune dysfunction induced by HIV replication. As control of HIV replication is influenced by the HLA region, we explored whether HLA alleles independently influence the risk of clinical events in HIV+ individuals. DESIGN: Cohort study. METHODS: In START and SMART participants, associations between imputed HLA alleles and AIDS, infection-related cancer, herpes virus-related AIDS events, chronic inflammation-related conditions and bacterial pneumonia were assessed. Cox regression was used to estimate hazard ratios (HRs) for the risk of events among allele carriers versus non-carriers. Models were adjusted for sex, age, geography, race, time-updated CD4+ T-cell counts and HIV viral load (VL) and stratified by treatment group within trials. HLA class I and II alleles were analyzed separately. The Benjamini-Hochberg procedure was used to limit the false discovery rate to

BACKGROUND: HIV-infected patients with poor virologic control and multidrug-resistant virus have limited therapeutic options. The current study was undertaken to evaluate the safety, immunologic effects, and antiviral activity of peripheral lymphocytes transferred from an elite controller, whose immune system is able to control viral replication without antiretroviral medications, to an HLA-B*2705–matched progressor. METHODS: Approximately 22 billion cells were collected from an elite controller by lymphapheresis and infused within 6 hours into a recipient with a preinfusion CD4(+) T cell count of 10 cells/μL (1%) and HIV plasma viral load of 114,993 copies/mL. RESULTS: Donor cells were cleared from the recipient’s peripheral blood by day 8. A transient decrease in viral load to 58,421 (day 3) was followed by a rebound to 702,972 (day 6) before returning to baseline values by day 8. The decreased viral load was temporally associated with peak levels of donor T cells, including CD8(+) T cells that had high levels of expression of Ki67, perforin, and granzyme B. Notably, recipient CD8(+) T cells also showed increased expression of these markers, especially in HIV-specific tetramer–positive cells. CONCLUSION: These results suggest that the adoptive transfer of lymphocytes from an HIV-infected elite controller to an HIV-infected patient with progressive disease may be able to perturb the immune system of the recipient in both positive and negative ways. TRIAL REGISTRATION: ClinicalTrials.gov NCT00559416. FUNDING: Intramural Research Programs of the US NIH Clinical Center and the National Institute of Allergy and Infectious Diseases (NIAID); the National Cancer Institute.

The programed death-1 (PD1)/programed death-ligand 1 (PD-L1) pathway plays a critical role in regulating immune responses against pathogens by controlling the balance between immunity and limiting tissue damage. In chronic viral infections such as HIV/SIV, hepatitis B virus, and hepatitis C virus, failure to completely eliminate virus leads to a sustained inflammatory/activated environment accompanied by the accumulation of T cells with diminished in-vitro effector function, ‘exhausted T cells’ [1–3]. In HIV/SIV infections, chronic T-cell immune activation is reflected by increased expression of a variety of immunomodulatory receptors including, PD1, CTLA-4, LAG3, CD244/2B4, CD160, and others [4–11]. In-vitro and in-vivo studies of HIV/SIV infection have shown that blockade of PD1 is associated with enhanced virus-specific responses and improved viral control [12–15]. These observations support the hypothesis that PD1/PD-L1 interactions contribute to the functional dysregulation, exhaustion, and ineffective viral control [8,16–18]. This evidence suggests that the PD1/PD-L1 pathway may be a potential target for immune intervention in patients with HIV infection. PD1 is expressed upon activation by T, B, and natural killer (NK) cells, dendritic cells, and activated monocytes, although its function in the latter is not well defined [19]. PD1 interacts with two ligands, PD-L1 and PD-L2. PD-L1 is mainly expressed by hematopoietic cells, such as T and B cells, dendritic cells, macrophages, and some epithelial cells (lung and vascular endothelium) [19–22]. In contrast, PD-L2 expression is more restricted and is inducible in dendritic cells, macrophages, bone marrow–derived mast cells, and some B cells [19,23]. PD1 interaction with its ligands regulates T cell receptor signaling by distinct mechanisms leading to diminished effector function, including cytokine secretion, proliferation, cytotoxicity, motility, and cell survival [24–27]. In addition, engagement of PD1 with its ligands induces reverse signaling on PD-L1/PD-L2 expressing cells [19,28,29]. Lymphoid organs are the primary targets of HIV/SIV infection [30–33]. In HIV/SIV-infected lymph nodes, the germinal center (GC) T follicular helper CD4+ T cells (CD4+ Tfh) exhibit higher expression of PD1 compared with extra follicular CD4+ and CD8+ T cells. PD-L1 expression has been observed inside and outside germinal centers [34,35]. To obtain further insights as to the potential role of PD-L1 blockade in the treatment of HIV infection, we analyzed the expression of PD1 and PD-L1 in human lymph node from 23 patients infected with HIV. In addition, we have evaluated the safety and the effect on SIV viral load of in-vivo PD1/PD-L1 blockade using a fully human anti-PD-L1 mAb (MSB0010718C, Avelumab, EMD-Serono, Inc., Rockland, Massachusetts, USA) in rhesus (Rh) macaques chronically infected with SIVmac239, that had previously received IL-15 [36].

Ionizing irradiation is used routinely to induce myeloablation and immunosuppression. However, it has not been possible to evaluate the extent of ablation without invasive biopsy. For lymphoid recovery, peripheral blood (PB) lymphocytes (PBLs) have been used for analysis, but they represent

Background Although rates of severe HIV-associated neurocognitive disorders have declined in the post-antiretroviral treatment (ART) era, subtle deficits persist, possibly exacerbated by treatment non-adherence. The actual effects of ART interruption/initiation on brain glucose metabolism as a reflection of viral replication and neuroinflammation remain unclear. Our study investigates how treatment initiation and interruption alter brain glucose metabolism in SIV-infected macaques, using 18F-FDG PET in correlation with plasma and CSF viral loads (VL) and cytokine levels. Methods SIV-infected macaques (n = 7) underwent ART initiation only, ART interruption only, or both. Five uninfected animals served as controls. 18F-FDG PET imaging was performed at baseline and 1, 3, and 6 months after treatment modification. Mean and maximum standardized uptake values (SUV) for the whole-brain and subregions were calculated. Plasma and CSF VL and cytokine levels were measured. Paired t tests evaluated acute changes in whole-brain SUV from baseline to 1 month, while mixed-effect linear regression models evaluated changes over multiple timepoints and correlated SUV values with disease markers. Results ART interruption was associated with increased SUVmean and SUVmax acutely, after 1 month (SUVmean 95% CI [0.044–0.786 g/ml], p = 0.037; SUVmax 95% CI [0.122–3.167 g/ml], p = 0.041). The correlation between SUV and time, however, was not significant when evaluated across all timepoints. Increased SUVmean and SUVmax correlated with decreased CD4+ and CD8+ T-cell counts and increased plasma VL. SUVmax was positively associated with increases in CSF VL, and there were borderline positive associations between SUVmax and IL-2, and between SUVmean and IL-15. The treatment initiation group showed no associations between imaging and disease biomarkers despite viral suppression, reduced cytokine levels, and increased CD4+ and CD8+ T-cell counts. Conclusions ART interruption is associated with increased brain glucose metabolism within 1 month of treatment cessation, which, in concert with increased levels of pro-inflammatory cytokines in the CSF, may reflect neuroinflammation in the setting of viral rebound. Although we cannot assert neurologic damage in association with cerebral hypermetabolism, it is a concerning outcome of ART non-adherence. Treatment initiation, meanwhile, did not result in significant changes in brain metabolism. HIV-induced neuroinflammation may require a longer period to abate than our follow-up period allowed. Electronic supplementary material The online version of this article (10.1186/s12974-018-1244-z) contains supplementary material, which is available to authorized users.

Background: Despite successfully suppressed viremia by treatment, patients with high levels of biomarkers of coagulation/inflammation are at an increased risk of developing non-AIDS defining serious illnesses such as cardiovascular diseases. Thus, there is a relationship between persistent immune activation and coagulation/inflammation, although the mechanisms are poorly understood. Platelets play an important role in this process. Although interactions between platelets and elements of the innate immune system, such as monocytes, are well described, little is known about the interaction between platelets and the adaptive immune system. Design: We investigated the interaction of a component of the coagulation system, platelets, and the adaptive immune system T cells. Methods: Healthy controls and combination antiretroviral therapy (cART)-treated HIV-infected patients with viral loads of less than 40 copies/ml for more than 15 months were analysed for platelet–T-cell conjugate formation. Results: Platelets can form conjugates with T cells and were preferentially seen in CD4+ and CD8+ T-cell subsets with more differentiated phenotypes [memory, memory/effector and terminal effector memory (TEM)]. Compared with healthy controls, these conjugates in patients with HIV infection were more frequent, more often composed of activated platelets (CD42b+CD62P+), and were significantly associated with the D-dimer serum levels. Conclusion: These data support a model in which platelet–T-cell conjugates may play a critical role in the fast recruitment of antigen-experienced T cells to the place of injury. This mechanism can contribute in maintaining a state of coagulation/inflammation observed in these patients contributing to the pathology of the disease.

Persistent immune activation in chronic HIV infection leads to exhaustion/activation of virus-specific CD8 T cells accompanied by expression of checkpoint receptors such as PD1. Here, we hypothesized that blockade of the PD1/PD-L1 pathway in combination with IL-15 (known to induce proliferation of CD8 T cells) may have a higher impact in improving HIV-specific responses. We assessed the in vitro effects of a checkpoint inhibitor, anti-PD-L1 (Avelumab), in combination with rhIL15 in HIV-specific CD8 T cell proliferation and cytokine secretion. Cell Trace Violet (CTV) labeled PBMCs from HIV infected patients (HIV+, n= 15) and healthy controls (HC, n= 7) were stimulated with HIVGag and CEF-peptides in the following conditions: rhIL-15, anti-PD-L1, and combination rhIL- 15/anti-PD-L1. After 5 days of culture, cells were re-stimulated and analyzed by flow cytometry. We found that rhIL-15 promoted higher antigen-independent proliferation (CTVlow) and cytokine secretion IFNg+ (p=0.009) and TNFa+ (p=0.01) in CD8 T cells from HIV+ patients than HC. In addition, rhIL-15 induced an increased trend in HIVGag-specific CD8 T cells (CTVlowIFNg+) but did not reach significance (p=0.1250). In HC, rhIL15 increased CEF-specific CD8 T cell CTVlowIFNg+ (p=0.03) and TNFa+ (p=0.03). Anti-PD-L1 alone did not show any effect in CD8 T cells from HIV+ patients and HC. In contrast, the combined treatment increased the CTVlowIFNg+ HIVGag-specific CD8 T cells in HIV+ patients (p=0.039) and CTVlowIFNg+ CEF-specific CD8 T cells in HC (p= 0.031). Overall, there was a synergistic effect in HIV-specific CD8 T cell function with the combined treatment. Further studies should assess the potential use of this combined treatment in the context of HIV infection.

The PD1/PD-L1 pathway contributes to the pathogenesis of HIV/SIV infection. Expression of PD1 by HIV/SIV-specific CD8 T cells can limit antiviral responses and expression of checkpoint receptors including PD1 by HIV infected cells can contribute to maintenance of viral reservoirs. In addition, reports have shown that IL-15 induces the expansion of effector memory CD8 T and NK cells. In this study, we hypothesized that expansion of memory CD8 T cells in the context of PD1/PD-L1 blockade may lead to enhanced anti-SIV responses. We design a study in which we evaluated the effect of rhIL-15 administered in cycles of ten days by continuous infusion in combination with weekly administration of anti-PD-L1 (Avelumab) over 24 weeks. After seven doses of anti-PD-L1 and completion of two cycles of rhIL15, cART therapy was interrupted and anti-PD-L1 administration continued to complete 24 weeks treatment. We found that administration of rhIL-15 in combination with anti-PD-L1 treatment was well tolerated. rhIL-15 infusions were associated with transient increases in proliferation of NK cells and effector memory CD8 T cells. rhIL-15/anti-PD-L1 treatment led to a sustained expression of CXCR3+ with low to undetectable expression of PD1on CD8 T cells, suggesting potential of increased tissue trafficking. Multidimensional single cell analysis showed that CXCR3+PD1−/low CD8 T cells secrete cytokine in response to viral antigens. Despite these clear biological effects, no impact was observed on rebound plasma viremia after cART discontinuation and during anti-PD-L1 administration. Futures studies should characterize the CXCR3+PD1−/low CD8 T cell subset expanded by this treatment to understand the potential impact of this subset in viral control.

Interleukin 27 (IL-27) is an immunomodulatory cytokine with important roles in both the innate and adaptive immune systems. In the last five years, the addition of exogenous IL-27 to primary cell cultures has been demonstrated to decrease HIV-1 replication in a number of cell types including peripheral blood mononuclear cells (PBMCs), CD4+ T cells, macrophages and dendritic cells. These in-vitro findings suggest that IL-27 may have therapeutic value in the setting of HIV-1 infection. In this review, we describe the current knowledge of the biology of IL-27, its effects primarily on HIV-1 replication but also in other viral infections and explore its potential role as a therapeutic cytokine for the treatment of patients with HIV-1 infection.

IL-27 promotes the differentiation of monocytes to HIV-resistant macrophages by down-regulating host factor SPTBN1., The susceptibility of macrophages to HIV-1 infection is modulated during monocyte differentiation. IL-27 is an anti-HIV cytokine that also modulates monocyte activation. In this study, we present new evidence that IL-27 promotes monocyte differentiation into macrophages that are nonpermissive for HIV-1 infection. Although IL-27 treatment does not affect expression of macrophage differentiation markers or macrophage biological functions, it confers HIV resistance by down-regulating spectrin β nonerythrocyte 1 (SPTBN1), a required host factor for HIV-1 infection. IL-27 down-regulates SPTBN1 through a TAK-1–mediated MAPK signaling pathway. Knockdown of SPTBN1 strongly inhibits HIV-1 infection of macrophages; conversely, overexpression of SPTBN1 markedly increases HIV susceptibility of IL-27–treated macrophages. Moreover, we demonstrate that SPTBN1 associates with HIV-1 gag proteins. Collectively, our results underscore the ability of IL-27 to protect macrophages from HIV-1 infection by down-regulating SPTBN1, thus indicating that SPTBN1 is an important host target to reduce HIV-1 replication in one major element of the viral reservoir.

Background A high incidence of nontraumatic osteonecrosis has been reported in HIV-infected patients. We investigated the levels of D-dimer and C-reactive protein (CRP) in a cohort of HIV-infected adults with and without osteonecrosis of the femoral head. Methods Forty-three HIV-infected patients with osteonecrosis of the femoral head and a comparison group of 50 HIV-infected patients with negative MRI of the hips and for whom serial plasma samples were available were included. D-dimer and CRP levels were measured prior to and at the time of diagnosis for osteonecrosis patients, at the time of negative MRI of the hips for controls, and at least 6 months later for both groups. Results Biomarker levels were elevated at the time of diagnosis in the osteonecrosis cohort compared with controls. Median D-dimer value was 0.32 μg/ml in the osteonecrosis group compared with less than 0.22 μg/ml in the control group (P = 0.016). For CRP, the corresponding values were 2.52 mg/l and 1.23 mg/l (P = 0.003). Postdiagnosis, D-dimer and CRP levels were also elevated in the osteonecrosis patients compared with controls. Linear regression demonstrated a rise in D-dimer levels from prediagnosis to diagnosis in the osteonecrosis patients whereas CRP levels did not change significantly over time. Conclusion Compared to controls, patients who developed osteonecrosis had elevated levels of D-dimer and CRP at diagnosis. D-dimer levels increased whereas CRP levels did not change significantly from prediagnosis to diagnosis. These data suggest that patients with higher levels of inflammation are at an increased risk of osteonecrosis.

Background: Selecting the optimal combination of HIV drugs for an individual in resourcelimited settings is challenging because of the limited availability of drugs and genotyping. Objective: The evaluation as a potential treatment support tool of computational models that predict response to therapy without a genotype, using cases from the Phidisa cohort in South Africa. Methods: Cases from Phidisa of treatment change following failure were identified that had the following data available: baseline CD4 count and viral load, details of failing and previous antiretroviral drugs, drugs in new regimen and time to follow-up. The HIV Resistance Response Database Initiative’s (RDI’s) models used these data to predict the probability of a viral load < 50 copies/mL at follow-up. The models were also used to identify effective alternative combinations of three locally available drugs. Results: The models achieved accuracy (area under the receiver–operator characteristic curve) of 0.72 when predicting response to therapy, which is less accurate than for an independent global test set (0.80) but at least comparable to that of genotyping with rules-based interpretation. The models were able to identify alternative locally available three-drug regimens that were predicted to be effective in 69% of all cases and 62% of those whose new treatment failed in the clinic. Conclusion: The predictive accuracy of the models for these South African patients together with the results of previous studies suggest that the RDI’s models have the potential to optimise treatment selection and reduce virological failure in different patient populations, without the use of a genotype. Keywords: HIV therapy; mathematical modelling; treatment; genotype

Disruption of vascular integrity by trauma and other tissue insults leads to inflammation and activation of the coagulation cascade. The serine protease thrombin links these 2 processes. The proinflammatory function of thrombin is mediated by activation of protease-activated receptor 1 (PAR-1). We found that peripheral blood effector memory CD4(+) and CD8(+) T lymphocytes expressed PAR-1 and that expression was increased in CD8(+) T cells from human immunodeficiency virus (HIV)-infected patients. Thrombin enhanced cytokine secretion in CD8(+) T cells from healthy controls and HIV-infected patients. In addition, thrombin induced chemokinesis, but not chemotaxis, of CD8(+) T cells, which led to structural changes, including cell polarization and formation of a structure rich in F-actin and phosphorylated ezrin-radexin-moesin proteins. These findings suggest that thrombin mediates cross-talk between the coagulation system and the adaptive immune system at sites of vascular injury through increased T-cell motility and production of proinflammatory cytokines.

HIV-induced immune activation leads to expansion of a subset of human CD8 + T cells expressing HLA-DR antigens. Expansion of CD8 + HLA-DR + T cells can be also observed in non-HIV settings including several autoimmune diseases and aging. Although these cells are felt to represent “immune exhaustion” and/or to be anergic, their precise role in host defense has remained unclear. Here, we report that this subset of cells exhibits a restricted repertoire, shows evidence of multiple rounds of division, but lacks markers of recent TCR engagement. Detailed cell cycle analysis revealed that compared with their CD8 + HLA-DR − counterpart, the CD8 + HLA-DR + T-cell pool contained an increased fraction of cells in S-phase with elevated levels of the G2/M regulators: cyclin A2, CDC25C, Cdc2 (CDK1), indicating that these cells are not truly anergic but rather experiencing proliferation in vivo. Together, these data support a hypothesis that antigen stimulation leads to the initial expansion of a CD8 + pool of cells in vivo that undergo further expansion independent of ongoing TCR engagement. No qualitative differences were noted between CD8 + HLA-DR + cells from HIV + and HIV − donors, indicating that the generation of CD8 + HLA-DR + T cells is a part of normal immune regulation that is exaggerated in the setting of HIV-1 infection.

The notion of a “suppressive” process that is active during inflammation was first introduced in the late 1960s [1, 2]. Research in this field faced a major setback in the early 1980s when DNAbased studies revealed that the genetic locus that was thought to control suppressor T-cell activity in mice (the I-J gene within the major histocompatibility complex region) did not exist [3]. Research in the field was revitalized by Sakaguchi et al [4] in 1995, when it was demonstrated that the adoptive transfer of CD4 T cells depleted of interleukin 2 (IL-2) receptor α-chain–positive (CD25) cells led to a spectrum of autoimmune diseases in immunocompromised mice and that cotransfer of the CD25 cells prevented these diseases. In 2003, the forkhead box protein P3 (FoxP3) was identified as a master control gene for the development and in vivo suppressor activity of these CD4CD25 T cells that have become commonly referred to as regulatory T cells or “Tregs” [5, 6]. A substantial number of reports have been generated in both human and murine systems on the characterization of Tregs and their role in the regulation of the immune response in health and disease. Today, Tregs are widely recognized as a subset of CD4CD25FoxP3 T cells capable of suppressing the activation, proliferation, and function of a wide variety of immune effector cells, including CD4 and CD8 T cells, natural killer (NK) and NKT cells, B cells, and antigen-presenting cells, such as dendritic cells and macrophages [7, 8]. There are at least 2 different types of Tregs present in humans: natural Tregs and induced (or adaptive) Tregs. Both Treg populations are characterized by high levels of expression of CD25 on the cell surface and intracellular expression of FoxP3 [6]. Although natural Tregs arise during the normal process of T-cell development in the thymus and survive in the periphery [9], induced or adaptive Tregs can be converted from extrathymic naive CD4 T cells in vivo by antigen stimulation in mice [10] or in vitro in mouse or human cells by culturing CD4FoxP3 T cells with different cytokines, including transforming growth factor β, IL-2, or interleukin 15 [11–13]. Intermittent in vivo administration of IL2 can lead to an expansion of Treg-like cells (CD4CD45ROCD25 T cells) in patients with human immunodeficiency virus type 1 (HIV-1) infection [14] or hepatitis C virus infection [15]. Although no clinical benefit was seen in a phase III study of IL-2 in patients with HIV-1 infection [16], patients with hepatitis C virus–induced vasculitis had remission of their disease. The immune systems of patients with HIV-1 infection are characterized by inadequate control of HIV-1 replication, progressive loss of CD4 T cells, and chronic generalized immune activation [17–19]. There is increasing evidence that clinical progression of HIV-1 infection is critically linked to the negative consequences of immune activation and that the level of immune activation is directly correlated to the plasma levels of HIV-1. [20]. The importance of immune activation in HIV-1 infection has been reinforced by the strong associations between baseline levels of CD4 and CD8 T cells that express CD38 and HLA-DR [21–23], plasma levels of interleukin 6, D-dimer or soluble CD14, and all-cause mortality [24, 25]. Given their potent suppressive capability, the role of Tregs in HIV-1 infection and, in particular, their impact on the host response to HIV-1 and immune activation have received much attention in recent years. In the context of HIV-1 infection, Tregs can be postulated to be beneficial by dampening excessive immune activation (the good) (Figure 1) or to be harmful by suppressing HIV-1–specific immune responses Received and accepted 7 February 2012; electronically published 28 March 2012. Correspondence: H. Clifford Lane, MD, Clinical and Molecular Retrovirology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (clane@niaid.nih.gov). The Journal of Infectious Diseases 2012;205:1479–82 Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2012. DOI: 10.1093/infdis/jis238

We previously showed that HIV infection leads to expansion of a rapidly proliferating pool (s1) of CD4 and CD8 T lymphocytes. In the current study, we used in vivo labeling with bromodeoxyuridine to characterize the kinetics of naive, memory, and activated (HLA-DR+/CD38+) subpopulations of CD4 and CD8 T lymphocytes, and to examine the relationship between kinetic parameters and baseline CD4 counts, HIV viral load, potential markers of microbial translocation, and cytokine levels. Activated cells showed the highest proliferation rates, followed by effector and central memory cells, with naive cells showing the lowest rates, for both CD4 and CD8 T cells. HIV viral load correlated with s1 of CD4 and CD8 effector memory cells, as well as CD8 naive cells, whereas CD4 cell counts correlated inversely with naive CD4 s1. Endotoxin levels showed a weak negative association with CD4 but not CD8 s1. INF-γ and TNF-α were associated with s1 for CD4 and CD8 cells, respectively. Thus, HIV is the primary driving force behind the activation and proliferation of most subsets of both CD4 and CD8 T lymphocytes, whereas naive CD4 cell proliferation likely represents a homeostatic response. Microbial translocation does not appear to play an important role in this proliferation.

Cytosolic foreign DNA is detected by pattern recognition receptors and mainly induces type I IFN production. We found that transfection of different types of DNA into various untreated cells induces type III IFN (IFN-λ1) rather than type I IFN, indicating the presence of uncharacterized DNA sensor(s). A pull-down assay using cytosolic proteins identified that Ku70 and Ku80 are the DNA-binding proteins. The knockdown studies and the reporter assay revealed that Ku70 is a novel DNA sensor inducing the IFN-lambda1 activation. The functional analysis of IFNL1 promoter revealed that positive-regulatory domain I and IFN-stimulated response element sites are predominantly involved in the DNA-mediated IFNL1 activation. A pull-down assay using nuclear proteins demonstrated that the IFN-λ1 induction is associated with the activation of IFN regulatory factor-1 and -7. Thus, to our knowledge, we show for the first time that Ku70 mediates type III IFN induction by DNA.

Immune activation plays an important role in the pathogenesis of HIV disease. Although the causes are not fully understood, the forces that lead to immune dysfunction differ for CD4 and CD8 T cells. In this study, we report that the molecular pathways that drive immune activation during chronic HIV infection are influenced by differences in the homeostatic regulation of the CD4 and CD8 T cell pools. Proliferation of CD4 T cells is controlled more tightly by CD4 T cell numbers than is CD8 T cell proliferation. This difference reflects the importance of maintaining a polyclonal CD4 T cell pool in host surveillance. Both pools of T cells were found to be driven by viral load and its associated state of inflammation. In the setting of HIV-induced lymphopenia, naive CD4 T cells were recruited mainly into the proliferating pool in response to CD4 T cell depletion, whereas naive CD8 T cell proliferation was driven mainly by levels of HIV RNA. RNA analysis revealed increased expression of genes associated with type I IFN and common γ chain cytokine signaling in CD4 T cell subsets and only type I IFN-associated genes in CD8 T cell subsets. In vitro studies demonstrated enhanced STAT1 phosphorylation in response to IFN-α and increased expression of the IFNAR1 transcripts in naive and memory CD4 T cells compared with that observed in CD8 T cells. CD4 T cell subsets also showed enhanced STAT1 phosphorylation in response to exogenous IL-7.

A randomized, controlled clinical trial was started in the pre-HAART era to compare the efficacy of zidovudine (AZT) and interferon-alpha (IFN-alpha) either alone or in combination to reduce HIV viremia, maintain CD4(+) cell count, and decrease time to AIDS progression and death. The purpose of the current study was to compare the effects of AZT and IFN on HIV load using modern technology. One hundred and eighty patients with CD4(+) counts above 500 cells/mm(3) were randomized to receive AZT alone, IFN-alpha alone, or AZT and IFN-alpha in combination. CD4(+) cell count and HIV load at baseline and at the last follow-up visit were compared, and time to AIDS or death was calculated by treatment group. At a mean follow-up of 45 weeks, the mean change in log HIV RNA was -0.06 for the AZT alone group, -0.47 for the AZT plus IFN-alpha group (P = 0.01 versus AZT group), and -0.35 for the IFN-alpha alone group (P = 0.02 versus AZT group). There was no significant difference among groups in change in total CD4(+) count or in time to AIDS or death. Since treatment with IFN-alpha produces significant decreases in HIV load, additional studies of IFN-alpha as part of a combination regimen are warranted.

IL-27, a member of the IL-12 cytokine family composed of IL-12, IL-23 and IL-35, has previously been shown to be an antiviral cytokine inhibiting infection of several viruses, among which HIV-1, HIV-2, HSV, HCV, HBV and Influenza virus. We have already reported that IL-27 induces HIV-1 inhibition in macrophages and dendritic cells in an IFN-independent manner, and that the cytokine enhances potential reactive oxygen species induction from cells. In this study, we demonstrated for the first time that IL-27 triggers autophagy induction in human AB serum-induced macrophages, while other IL-12 cytokine family members did not have such effect on these cells; IL-27 was able to induce autophagy both in terminally differentiated macrophages, causing a 2 to 5-fold increase in autophagosome accumulation, as well as during monocyte-to-macrophage differentiation, where IL-27 effect was even stronger (generating a 20-fold increase in autophagosome formation). Furthermore, we also determined that two key autophagy proteins, mTOR and LC3, were not involved in this phenomenon: while we were able to assess autophagosome accumulation in IL-27-treated cells via electron microscopy and immunofluorescence, this was not linked to either mTOR dephosphorylation nor LC3 lipidation, implying that IL-27 induces autophagy through a novel non-canonical pathway. Finally, we established that blocking IL-27 signaling during macrophage differentiation using both Ruxoliotinib or Tofacitinib, respectively JAK1/2 and JAK3 inhibitors, successfully inhibited autophagy triggering together with STAT3 phosphorylation, which suggests that the JAK-STAT pathway is responsible for regulating IL-27-induced autophagy.

H5N1 avian influenza represents an episodic zoonotic disease with the potential to cause a pandemic, and antiviral resistance is of considerable concern. We sought to generate high-titer H5N1 antibodies in healthy volunteers for the purpose of developing hyperimmune intravenous immunoglobulin.We conducted a dose-escalating, unblinded clinical trial involving 75 subjects aged 18-59 years. Three cohorts of twenty-five subjects were enrolled sequentially and received 90, 120, or 180 microg of H5N1 A/Vietnam/1203/04 vaccine in 4 doses administered approximately 28 days apart.No statistically significant dose-related increases in the geometric mean titers (GMTs) of serum hemagglutination inhibition antibody were observed when the 90-microg, 120-microg, and 180-microg cohorts were compared. When the cohorts were analyzed together to determine the effect of additional vaccinations, the GMTs of hemagglutination inhibition antibody after the first, second, third, and fourth vaccinations were 1:15.7, 1:22.2, 1:36.0, and 1:32.0, respectively (first vaccination vs. baseline, P.001; second vs. first vaccination, P=.02; and third vs. second vaccination, P.001). The microneutralization GMTs after the first, second, third, and fourth vaccinations were 1:17.5, 1:33.1, 1:55.7, and 1:68.4, respectively (P.001 for all comparisons).The results of our study suggest that a third and fourth dose of the H5N1 A/Vietnam/1203/04 vaccine may result in higher hemagglutination inhibition and microneutralization GMTs, compared with the GMTs resulting from fewer doses. There was no benefit to increasing the dose of the vaccine.Clinical Trials.gov identifier: NCT00383071.

Multiple HIV-1 subtypes and circulating recombinant forms (CRFs) are known to cocirculate in Africa. In West Africa, the high prevalence of CRF02_AG, and cocirculation of subtype A, CRF01_AE, CRF06_cpx, and other complex intersubtype recombinants has been well documented. Mali, situated in the heart of West Africa, is likely to be affected by the spread of recombinant subtypes. However, the dynamics of the spread of HIV-1 recombinant subtypes as well as nonrecombinant HIV-1 group M subtypes in this area have not been systematically assessed. Herein, we undertook genetic analyses on full-length env sequences derived from HIV-1-infected individuals living in the capital city of Mali, Bamako. Of 23 samples we examined, 16 were classified as CRF02_AG and three had a subsubtype A3. Among the remaining HIV-1 strains, CRF06_cpx and CRF09_cpx were each found in two patients. Comparison of phylogenies for six matched pol and full-length env sequences revealed that two strains had discordant subtype/CRF designations between the pol and env regions: one had A3(pol)CRF02_AG(env) and the other had CRF02_AG(pol)A3(env). Taken together, our study demonstrated the high prevalence of CRF02_AG and complexity of circulating HIV-1 strains in Mali. It also provided evidence of ongoing virus evolution of CRF02_AG, as illustrated by the emergence of more complex CRF02_AG/A3 intersubtype recombinants in this area.

HIV infection is characterized by a brisk immune activation that plays an important role in the CD4 depletion and immune dysfunction of patients with AIDS. The mechanism underlying this activation is poorly understood. In the current study, we tested the hypothesis that this activation is the net product of two distinct pathways: the inflammatory response to HIV infection and the homeostatic response to CD4 T cell depletion. Usingex vivoBrdU incorporation of PBMCs from 284 patients with different stages of HIV infection, we found that CD4 proliferation was better predicted by the combination of CD4 depletion and HIV viral load (R2= 0.375,P< 0.001) than by either parameter alone (CD4 T cell counts,R2= 0.202,P< 0.001; HIV viremia,R2= 0.302,P< 0.001). Interestingly, CD8 T cell proliferation could be predicted by HIV RNA levels alone (R2= 0.334,P< 0.001) and this predictive value increased only slightly (R2= 0.346,P< 0.001) when CD4 T cell depletion was taken into account. Consistent with the hypothesis that CD4 T cell proliferation is driven by IL-7 as a homeostatic response to CD4 T cell depletion, levels of phosphorylated STAT-5 were found to be elevated in naive subsets of CD4 and CD8 T cells from patients with HIV infection and in the central memory subset of CD4 T cells. Taken together these data demonstrate that at least two different pathways lead to immune activation of T cells in patients with HIV infection and these pathways differentially influence CD4 and CD8 T cell subsets.

Administration of interleukin (IL)-2 to human immunodeficiency virus (HIV)-infected patients leads to significant increases in CD4 T cell counts. We previously have shown that IL-2 induces increased proliferation and survival of CD4 T cells. Deuterium labeling studies were undertaken to study the relationship between IL-2-induced increases in the CD4 T cell count and the effects of IL-2 on cell proliferation and survival. A strong inverse correlation was noted between the rate of decay of the label in CD4 cells and increases in CD4 cell counts (R =or- 0.67; P

Idiopathic CD4+ lymphocytopenia (ICL) is a rare non–HIV-related syndrome with unclear natural history and prognosis. This prospective natural history cohort study describes the clinical course, CD4 T lymphocyte kinetics, outcome, and prognostic factors of ICL. Thirty-nine patients (17 men, 22 women) 25 to 85 years old with ICL were evaluated between 1992 and 2006, and 36 were followed for a median of 49.5 months. Cryptococcal and nontuberculous mycobacterial infections were the major presenting opportunistic infections. Seven patients presented with no infection. In 32, CD4 T-cell counts remained less than 300/mm3 throughout the study period and in 7 normalized after an average of 31 months. Overall, 15 (41.6%) developed an opportunistic infection in follow-up, 5 (13.8%) of which were “AIDS-defining clinical conditions,” and 4 (11.1%) developed autoimmune diseases. Seven patients died, 4 from ICL-related opportunistic infections, within 42 months after diagnosis. Immunologic analyses revealed increased activation and turnover in CD4 but not CD8 T lymphocytes. CD8 T lymphocytopenia (< 180/mm3) and the degree of CD4 T cell activation (measured by HLA-DR expression) at presentation were associated with adverse outcome (opportunistic infection-related death; P = .003 and .02, respectively). This trial is registered at http://clinicaltrials.gov as #NCT00001319.

A human immunodeficiency virus type 1 (HIV-1) derivative (HIVNL-DT5R) containing sequences encoding a 7-amino-acid segment of CA and the entirevifgene from simian immunodeficiency virus (SIV) was previously shown to establish spreading infections in cultured macaque peripheral blood mononuclear cells. To assess its replicative and disease-inducing properties in vivo, HIVNL-DT5Rwas inoculated into pig-tailed macaques. HIVNL-DT5Rgenerated plasma viremia in all five of the monkeys and elicited humoral responses against all of the HIV-1 structural proteins but did not cause CD4+T-lymphocyte depletion or clinical disease. Additional adaptation will be required to optimize infectivity in vivo.

Recent studies have demonstrated that cell populations intended for therapeutic purposes that are cultured in heterologous animal products can acquire xenoantigens, potentially limiting their utility. In investigations of the immune response to murine embryonic stem cells, we found that a strong antibody response was generated after the second infusion. Both polyclonal and monoclonal antibody responses, derived from immunized mice, were found to be specific for bovine apolipoprotein B-100, which binds to abundant low-density lipoprotein receptors on the cell surface and is internalized. Here we show that in the majority of patients administered 3 different types of cell-based therapies using cells grown in fetal calf serum-containing media, an antibody response to bovine apolipoprotein B-100 develops after the second infusion and is the dominant specificity. The known and potential clinical effects of such antibodies are discussed.

Human immunodeficiency virus and simian immunodeficiency virus (SIV) induce a slow progressive disease, characterized by the massive loss of memory CD4 + T cells during the acute infection followed by a recovery phase in which virus replication is partially controlled. However, because the initial injury is so severe and virus production persists, the immune system eventually collapses and a symptomatic fatal disease invariably occurs. We have assessed CD4 + T-cell dynamics and disease progression in 12 SIV-infected rhesus monkeys for nearly 2 years. Three macaques exhibiting a rapid progressor phenotype experienced rapid and irreversible loss of memory, but not naïve, CD4 + T lymphocytes from peripheral blood and secondary lymphoid tissues and died within the first 6 months of virus inoculation. In contrast, SIV-infected conventional progressor animals sustained marked but incomplete depletions of memory CD4 + T cells and continuous activation/proliferation of this T-lymphocyte subset. This was associated with a profound loss of naïve CD4 + T cells from peripheral blood and secondary lymphoid tissues, which declined at rates that correlated with disease progression. These data suggest that the persistent loss of memory CD4 + T cells, which are being eliminated by direct virus killing and activation-induced cell death, requires the continuous differentiation of naïve into memory CD4 + T cells. This unrelenting replenishment process eventually leads to the exhaustion of the naïve CD4 + T-cell pool and the development of disease.

In December 2002, a decision was reached between the South African Military Health Service of the South African National Defence Force and the US Ambassador to South Africa that the USA and the Republic of South Africa would explore the possibility of initiating a collaborative effort to build the capacity to conduct clinical research within the South African military environment and that the area of initial focus would be HIV/AIDS. Three and a half years later, a clinical research program now known as Project Phidisa has enrolled over 4000 patients on Institutional Review Board-approved protocols, and provided antiretroviral therapy to approximately 1400 members of the South African National Defence Force and their dependents. The purpose of this article is to present some of the challenges faced in the development of this program and provide the lessons learned as these challenges were and continue to be addressed.

Objectives: After DNA or RNA virus infection, cytosolic foreign DNA or RNA derived from the infecting viruses is recognized by intracellular pathogen recognition receptors (PRRs) and induces activation of the innate immune system. Transfection of DNA has been used as an experimental model for DNA virus-mediated innate responses. We have previously reported that DNA transfection preferentially induces Type-III IFN (IFN-λ1) rather than Type-I IFN (IFN-β). In this study, we compared the DNA-mediated immune response between healthy controls and HIV-1 infected patients with undetectable viral loads and assessed potential innate immune responses in these patients. Methods: The study consisted of 50 HIV-1 negative healthy donors, 46 patients on combination antiretroviral therapy with HIV-1 viral loads

Hemagglutination inhibition (HAI) antibody responses to anti–influenza virus hyperimmune intravenous immunoglobulin (hIVIG) were characterized. Thirty-one patients with influenza during the 2013–2014 season were randomly assigned to receive 0.25 g/kg of hIVIG (n = 16) or placebo (n = 15). For hIVIG recipients, the ratio of geometric mean titers (1 hour after infusion/before infusion) was 4.00 (95% confidence interval [CI], 2.61–6.13) for 2009 pandemic influenza A(H1N1) and 1.76 (95% CI, 1.33–2.32) for influenza A(H3N2) and influenza B. Among patients with 2009 pandemic influenza A(H1N1), ratios for hIVIG (n = 9) versus placebo (n = 8) were higher 1 hour after infusion (3.9 [95% CI, 2.3–6.7]) and sustained through day 3 (2.0 [95% CI, 1.0–4.0]). hIVIG administration significantly increases HAI titer levels among patients with influenza, supporting the need to perform a clinical outcomes study. Clinical trials registration: NCT02008578.

Background: Intermittent administration of interleukin (IL)-2 to HIV infected patients leads to CD4 T-cell expansions that are associated with decreased CD4 T-cell turnover. IL-2 is under evaluation in antiretroviral therapy (ART) interruption studies, but it is unclear how the emergence of viremia may affect CD4 expansions. Methods: CD4 T-cell responses were evaluated in 27 HIV infected patients on long-term intermittent IL-2 therapy who underwent ART interruption immediately after an IL-2 cycle (‘IL-2/off’) and compared with responses from a previous IL-2 cycle while on continuous ART (‘IL-2/on’). Immunophenotypic analysis, including intracellular Ki67 staining, of cryopreserved peripheral blood mononuclear cells was performed. Results: CD4 T-cell increases, in naive and central memory CD4 T-cell subsets, were observed in the IL-2/on (106 and 327 cells/μl, respectively) and IL-2/off (84 and 184 cells/μl, respectively) cycles 1 month following IL-2 administration. These increases were greater during the IL-2/on cycle (P = 0.05, P = 0.01, respectively). In both cycles, the change in CD4 T-cell count correlated with the change in CD4/CD25 T cells. In the IL-2/off cycle, the change in the proportion of CD4 T cells expressing Ki67 was associated with both the changes in viral load (r = 0.64, P = 0.001) and the changes in CD4 T cells (r = −0.56, P = 0.01). Conclusions: IL-2 administration followed by ART interruption led to significant, although blunted, CD4 T-cell increases. IL-2 induced CD4 T-cell increases in the setting of emergent viremia were associated with decreased CD4 T-cell activation that counteracted the viremia-induced increases in CD4 T-cell activation.

HIV infection leads to decreases in the number of CD4+ T lymphocytes and an increased risk for opportunistic infections and neoplasms. The administration of intermittent cycles of IL-2 to HIV-infected patients can lead to profound increases (often greater than 100%) in CD4 cell number and percentage. Using in vivo labeling with 2H-glucose and BrdU, we have been able to demonstrate that, although therapy with IL-2 leads to high levels of proliferation of CD4 as well as CD8 lymphocytes, it is a remarkable preferential increase in survival of CD4 cells (with half-lives that can exceed 3 years) that is critical to the sustained expansion of these cells. This increased survival was time-dependent: the median half-life, as determined by semiempirical modeling, of labeled CD4 cells in 6 patients increased from 1.7 weeks following an early IL-2 cycle to 28.7 weeks following a later cycle, while CD8 cells showed no change in the median half-life. Examination of lymphocyte subsets demonstrated that phenotypically naive (CD27+CD45RO–) as well as central memory (CD27+CD45RO+) CD4 cells were preferentially expanded, suggesting that IL-2 can help maintain cells important for host defense against new antigens as well as for long-term memory to opportunistic pathogens.

Administration of IL-2 to HIV-infected patients leads to expansion of a unique subset of CD4+CD45RO–CD25+ cells. In this study, the origin, clonality, and function of these cells were investigated. Analysis of TCR excision circles revealed that the CD4+CD45RO–CD25+ cells were the product of peripheral expansion but remained polyclonal as determined by TCR repertoire analysis. Phenotypically, these cells were distinct from naturally occurring Tregs; they exhibited intermediate features, between those of memory and naive cells, and had lower susceptibility to apoptosis than CD45RO–CD25– or memory T cells. Studies of intracellular cytokine production and proliferation revealed that cytokine-expanded naive CD25+ cells had low IL-2 production and required costimulation for proliferation. Despite elevated expression of forkhead transcription factor P3 (foxP3), they exerted only weak suppression compared with CD45RO+CD25+high cells (Tregs). In summary, in vivo IL-2 administration to HIV-infected patients leads to peripheral expansion of a population of long-lived CD4+CD45RO–CD25+ cells that express high levels of foxP3 but exert weak suppressive function. These CD4+CD25+ cytokine-expanded naive cells, distinct from antigen-triggered cells and Tregs, play a role in the maintenance of a state of low turnover and sustained expansion of the CD4+ T cell pool.

Interleukin-2 administration induces CD4 T cell expansion in HIV-infected patients, however, toxicity can limit dosing. BAY 50-4798 is a recombinant IL-2 analog with >1000-fold specificity for the high-affinity IL-2 receptor. The effects of this compound on unstimulated human PBMC were evaluated. PBMC from HIV(-) and HIV(+) donors were cultured in vitro with incremental doses of BAY 50-4798 or aldesleukin. CD25 expression and proliferation were evaluated with flow cytometry. Cytokine levels were measured by ELISA in culture supernatants. BAY 50-4798 induced dose-dependent increases in CD25 expression and proliferation of T cells, NK, and B cells and showed selectivity for CD4 T cells expressing CD25. Induction of pro-inflammatory cytokines was also dose-dependent and was observed at the concentrations of BAY 50-4798 with the highest biologic activity. These data suggest that BAY 50-4798 can induce proliferation of unstimulated T cells but loss of T cell selectivity and induction of pro-inflammatory cytokines occur at concentrations exerting the highest biologic activity.

Administration of interleukin 2 (IL-2) leads to selective and sustained CD4+ T-cell expansions in patients infected with HIV. It has been hypothesized that persistent CD4+ T-cell proliferation is the primary mechanism maintaining these expansions. T-cell proliferation was studied by ex vivo bromodeoxyuridine (BrdU) incorporation and intracellular Ki67 staining in HIV-infected patients treated with antiretroviral therapy (ART) with or without IL-2. In contrast to the tested hypothesis, HIV-infected patients treated with IL-2 had lower CD4+ T-cell proliferation compared to patients treated with ART alone. Independently of viral load changes, administration of IL-2 led to a decrease in basal CD4+ T-cell proliferation. Total numbers of CD4+ T cells with naive and recall, but not effector, memory phenotype were increased. The degree of CD4+ T-cell expansion correlated with the decreases in proliferation and a strong association was seen between these decreases and the expansion of the CD4+/CD25+ subset. Intermittent IL-2 in HIV-infected patients leads to expansions of CD4+/CD25+ T cells with naive and recall memory phenotypes that strongly correlate with decreases in proliferation. These data suggest that decreased T-cell proliferation is central in the CD4+ T-cell expansions induced by IL-2.

In vivo, the principal target of human immunodeficiency virus type 1 (HIV-1) is the CD4+ T cell. Over time, virus-induced elimination and/or dysfunction of this T-lymphocyte subset, whether caused directly or indirectly, leads to clinical disease in infected individuals (10). Like all other lentiviruses, HIV-1 is also able to infect macrophage, a property recognized since the beginning of the AIDS epidemic (21, 50). The pathogenic consequences of HIV-1-infected macrophages is best exemplified by the AIDS dementia complex in which viral RNA expressed in microglia within the central nervous system may cause severe neuronal injury leading to encephalopathy (24). In addition, because of their reported resistance to the cytopathic effects elicited by HIV-1 and long life span, tissue macrophages have also been considered to be an important reservoir of virus (36). In this regard, macrophages have been reported to be a prominent source of virus during the late stages of disease, especially in conjunction with opportunistic infections (35). HIV-1 infection of macrophages in vivo has been logistically difficult to study. For example, it is not currently known whether monocyte precursors are initially infected in the bone marrow and only begin to produce virus after their migration to and differentiation in specific tissues or whether they become infected after they reach their final tissue of residence. Current understanding about the dynamics of virus production by tissue macrophage is similarly limited: are progeny virions generated as a result of de novo infections or by the continuous release of particles by long-lived cells? Consequently, most present knowledge about HIV-1 infection of macrophage accrues from an in vitro surrogate: monocyte-derived macrophage (MDM). MDM have been useful for identifying so-called macrophage-tropic (M-tropic) HIV-1 strains. M-tropic strains infect MDM and primary CD4+ T lymphocytes but not most human T-cell lines in vitro, fail to induce syncytium formation, and can be recovered from infected persons during all phases of their disease (7, 13, 42). Prototypic M-tropic strains of HIV-1 use the CCR5 chemokine receptor for cell entry (1, 11). From studies of highly pathogenic SHIVs that irreversibly and systemically deplete CD4+ T lymphocytes in rhesus monkeys within weeks of virus inoculation, we previously reported that tissue macrophages are able to sustain high levels of plasma viremia (>106 RNA copies/ml) in the virtual absence of CD4+ T cells (16). It was subsequently found that viral variants, circulating in the plasma during the macrophage phase of SHIV infections, carried gp120 V2 changes (specific double amino acid deletions and the loss of a conserved glycosylation site) (19). Some of these SHIV variants had also acquired the capacity to infect alveolar macrophages (AM). In the present study, the gp120 structure, cell tropism, and coreceptor utilization properties of macrophage-phase simian immunodeficiency virus/HIV chimeras (SHIVs), isolated directly from lymphoid tissues, were examined. In contrast to plasma virus, the tissue-associated SHIVs bore envelope glycoproteins that were genetically more heterogeneous and contained specific changes primarily within V1 and V2. Small molecule coreceptor-targeted inhibitors, specific for CCR5 or CXCR4, were used to assess the chemokine receptor usage by (i) the starting highly pathogenic, T-cell-deleting SHIVs and the (ii) late-phase M-tropic SHIV variants. Blockade of CXCR4 potently suppressed infection of rhesus monkey PBMC by both viruses, whereas CCR5 targeted inhibitors had little if any effect. Infection of macaque AM by M-tropic SHIVs was also suppressed by CXCR4 not by CCR5 specific inhibitors. Thus, the acquisition of macrophage tropism by SHIVs, present in late-phase monkeys, is not accompanied by a switch in chemokine coreceptor usage.

The effect of length of therapy on the safety and efficacy profile of continuous intravenous (CIV) interleukin-2 (IL-2) in combination with antiretroviral therapy (ART) was evaluated in 81 HIV-seropositive patients with CD4(+) T-cell counts of 100-300/mm(3). Patients were randomized to CIV IL-2 (12 mIU/day) for 3, 4, or 5 days plus ART every 8 weeks for six cycles, or to ART alone. The mean percent increase in CD4(+) T-cell counts was 24.5% for IL-2 recipients compared with a mean percent decrease of 30.5% for control patients (P = 0.005). Increasing duration of CIV IL-2 therapy resulted in improved CD4(+) T-cell response. The most frequent clinical adverse events and laboratory abnormalities were predominantly of grade 1 or 2 severity. However, grade 3 or 4 events were reported in 57%, 60%, and 84% of the 3-, 4-, and 5-day CIV IL-2 patients, respectively. Serious adverse events, mainly due to the requirement of hospitalization, occurred in 20% of IL-2 recipients, compared with 10% of control patients. Viral load during the course of the study was not different among the treatment groups. IL-2 therapy in cycles of 5 days resulted in an optimal increase in CD4(+) T-cell counts and is the preferred cycle length for IL-2 therapy geared toward increasing CD4(+) T-cell numbers.

Naive CD4+ T cells use L-selectin (CD62L) expression to facilitate immune surveillance. However, the reasons for its expression on a subset of memory CD4+ T cells are unknown. We show that memory CD4+ T cells expressing CD62L were smaller, proliferated well in response to tetanus toxoid, had longer telomeres, and expressed genes and proteins consistent with immune surveillance function. Conversely, memory CD4+ T cells lacking CD62L expression were larger, proliferated poorly in response to tetanus toxoid, had shorter telomeres, and expressed genes and proteins consistent with effector function. These findings suggest that CD62L expression facilitates immune surveillance by programming CD4+ T cell blood and lymph node recirculation, irrespective of naive or memory CD4+ T cell phenotype.

The long-term immunologic effects of intermittent interleukin 2 (IL-2) therapy were evaluated in a cross-sectional study by comparing 3 groups: HIV-seronegative volunteers, HIV-infected (HIV+) patients receiving highly active antiretroviral therapy (HAART), and HIV+ patients receiving HAART and intermittent IL-2. Whole-blood immunophenotyping was performed to study expression of the IL-2 receptor chains on T lymphocytes and natural killer cells and to further characterize CD4+/CD25+ T cells. Increased CD25 expression, especially in CD4+ T cells but also in CD8+ T cells, without increases in expression of the β and γ chains of the IL-2 receptor was detected in the IL-2 group. Up to 79% of naive CD4+ T cells (median, 61%) from patients in the IL-2 group expressed CD25, and the number of naive CD4+/CD25+ T cells correlated positively with both the total and naive CD4+ T-cell counts. A discrete population of CD45 double intermediate RA+/RO+CD4+ cells was also preferentially expanded in the IL-2 group, and the number of these cells strongly correlated with the total CD4+ count. Despite increases in CD25 expression, T lymphocytes from patients treated with IL-2 did not have increased expression of early (CD69) or late (CD95) activation markers or evidence of recent proliferation (Ki67). Both CD4+/CD25+ and CD4+/CD25− cells from IL-2–treated HIV+ patients proliferated in response to mitogens, specific antigens, and T-cell-receptor–mediated stimuli. Thus, intermittent administration of IL-2 in HIV+ patients leads to preferential expansion of a unique subset of CD4+ T cells that may represent a critical population in T-cell homeostasis.

Treatment advances have led to dramatic clinical improvements for patients with HIV-1 infection. These clinical improvements reflect treatment-related improvements in immune function, which are most striking in individuals who develop exaggerated immune inflammatory responses to occult opportunistic infections. The mechanisms accounting for these exaggerated immune responses are unknown. To gain insight into these mechanisms, we intensively studied a subject untreated for disseminated tuberculosis and HIV-1 coinfection who then began treatment for both diseases. We examined the changing frequencies of Mycobacterium tuberculosis (MTB)-specific CD4(+) T cells that produced interferon gamma (IFN-gamma) after short-term stimulation with MTB antigen, and we compared these frequencies with those in HIV-1-seronegative subjects with and without prior exposure to MTB antigens. For the HIV-1/MTB-coinfected subject, the proportion of peripheral blood CD4(+) T cells expressing MTB-specific IFN-gamma was 8.6% at 11 days, 11% at 33 days, and 33% at 95 days after starting treatment for HIV-1. CD4(+) IFN-gamma(+) T cells had a CD45RA(-)CD62L(-) (effector memory) phenotype and most coexpressed interleukin 2. Median frequencies of CD4(+) IFN-gamma(+) T cells from six subjects without and nine subjects with prior exposure to MTB antigens were 0.06 and 0.46%, respectively. We conclude that individuals starting treatment for disseminated tuberculosis and HIV-1 coinfection can accumulate remarkably large numbers of MTB-specific CD4(+) T cells in the peripheral blood. The rapid expansion of antigen-specific effector CD4(+) T cells is one mechanism to explain immediate improvements in clinical immunity after HIV-1 treatment. This mechanism provides a theoretical framework to understand the unusual inflammatory responses recently reported to occur after starting HIV-1 treatment.

The effect of intermittent courses of recombinant interleukin-2 (rIL-2) on HIV-1 load in patients receiving combination antiretroviral therapy remains uncertain. CPCRA 059 was an open-label, randomized, multicenter trial in which 511 patients with HIV-1 infection and CD4 + cell counts of ≥300/mm 3 who were receiving antiretroviral therapy were assigned to receive no rIL-2 (255 patients [controls]) or subcutaneous rIL-2 in dosages of 4.5 MIU (130) or 7.5 MIU (126) twice daily for 5-day courses every 8 weeks to maintain CD4 + cell counts that were twice the baseline value or ≥1,000/mm 3 . The primary objective of this study was to compare the effects of the two doses of rIL-2 and no rIL-2 on viral load and CD4 + cell counts over 12 months. There was no difference in the following viral load measurements between the rIL-2 treatment groups and the control treatment group: percentage of patients with viral loads of

IL-7 regulates homeostatic mechanisms that maintain the overall size of the T cell pool throughout life. We show that under steady state conditions, IL-7 signaling is principally mediated by activation of Signal Transducers and Activators of Transcription 5 (STAT5). In contrast, under lymphopenic conditions there is a modulation of STAT1 expression resulting in an IL-7-dependent STAT1 and STAT5 activation. Consequently, the IL-7-induced transcriptome is altered with enrichment of interferon (IFN) stimulated genes (ISGs). Moreover, STAT1 overexpression was associated with reduced survival in CD4 T cells undergoing Lymphopenia-Induced Proliferation (LIP). We propose a model in which T cells undergoing LIP upregulate STAT1 protein, “switching on” an alternate IL-7-dependent program. This mechanism could be a physiological process to “sense space” regulating the size of the CD4 T cell pool. During HIV infection the virus could exploit this pathway leading to the homeostatic dysregulation of the T cell pools observed in these patients.

Combination antiretroviral therapy can suppress human immunodeficiency virus (HIV) infection but cannot completely eradicate the virus. A major obstacle in the quest for a cure is the difficulty in targeting and measuring latently infected cells. To date, a single person seems to have been cured of HIV. Hematopoietic stem cell transplantation (HSCT) preceded this cancer patient's long-term sustained HIV remission, but researchers have been unable to replicate this cure, and the mechanisms that led to HIV remission remain to be established. In February 2014, the National Institute of Allergy and Infectious Diseases sponsored a workshop that provided a venue for in-depth discussion of whether HSCT could be exploited to cure HIV in cancer patients requiring such procedures. Participants also discussed how HSCT might be applied to a broader community of HIV-infected persons in whom the risks of HSCT currently outweigh the likelihood and benefits of HIV cure.

Objective IL-27 is an immunomodulatory cytokine with potent anti-HIV properties in PBMCs, CD4+ T cells, macrophages and immature dendritic cells. Previous smaller studies have suggested that HIV-1 infection may alter IL-27 and influence HIV-1 pathogenesis. The aim of this study was to examine the relationship between plasma IL-27 levels in a well-characterised cohort of HIV-1 infected patients. Methods Patients were stratified into four groups based on HIV-1 viral load and matched according to age, gender and those receiving antiretroviral treatment. IL-27 levels and C-reactive protein (CRP) were measured using electrochemiluminescence assays. D-dimer and CD4+ T cell counts were measured using an Enzyme Linked Fluorescence Assay and FACS, respectively. sCD14 and sCD163 were measured using ELISA. HIV-1 viral load was measured by bDNA or qRT-PCR assays. Results Plasma IL-27 levels were measured in 505 patients (462 HIV+, 43 controls). The mean level (±SEM) of IL-27 in controls was 2990.7±682.1 pg/ml, in the 100,000 copies/ml group it was 1590.1±223.7 pg/ml. No statistically significant difference in IL-27 levels between groups were seen. There were no correlations noted between IL-27 and HIV-1 viral load or CD4+ T cell counts. There was a small correlation noted between D-dimer and IL-27 (Spearman r = 0.09, p = 0.03) and sCD163 and IL-27 (Spearman r = 0.12, p = 0.005). No correlation was observed between IL-27 and CRP or sCD14 levels. Conclusions This is the largest study examining the levels of plasma IL-27 in HIV-1 infection. While IL-27 levels are not significantly altered in HIV-1 infection compared to uninfected controls there may be a small association between IL-27 and D-dimer levels and IL-27 and sCD163 levels.

Objectives: The optimal individualized selection of antiretroviral drugs in resource-limited settings is challenging because of the limited availability of drugs and genotyping. Here we describe the development of the latest computational models to predict the response to combination antiretroviral therapy without a genotype, for potential use in such settings. Methods: Random forest models were trained to predict the probability of a virological response to therapy (

We examined the effects of human immunodeficiency virus infection on the turnover of CD4 and CD8 T lymphocytes in 17 HIV-infected patients by 30 min in vivo pulse labeling with bromodeoxyuridine (BrdU). The percentage of labeled CD4 and CD8 T lymphocytes was initially higher in lymph nodes than in blood. Labeled cells equilibrated between the two compartments within 24 h. Based on mathematical modeling of the dynamics of BrdU-labeled cells in the blood, we identified rapidly and slowly proliferating subpopulations of CD4 and CD8 T lymphocytes. The percentage, but not the decay rate, of labeled CD4 or CD8 cells in the rapidly proliferating pool correlated significantly with plasma HIV RNA levels for both CD4 (r = 0.77, P < 0.001) and CD8 (r = 0.81, P < 0.001) T cells. In six patients there was a geometric mean decrease of greater than 2 logs in HIV levels within 2 to 6 mo after the initiation of highly active antiretroviral therapy; this was associated with a significant decrease in the percentage (but not the decay rate) of labeled cells in the rapidly proliferating pool for both CD4 (P = 0.03) and CD8 (P < 0.001) T lymphocytes. Neither plasma viral levels nor therapy had an effect on the decay rate constants or the percentage of labeled cells in the slowly proliferating pool. Monocyte production was inversely related to viral load (r = −0.56, P = 0.003) and increased with therapy (P = 0.01). These findings demonstrate that HIV does not impair CD4 T cell production but does increase CD4 and CD8 lymphocyte proliferation and death by inducing entry into a rapidly proliferating subpopulation of cells.