Reconciling Longitudinal Naive T-Cell and TREC Dynamics during HIV-1 Infection

Naive T cells in untreated HIV-1 infected individuals have a reduced T-cell receptor excision circle (TREC) content. Previous mathematical models have suggested that this is due to increased naive T-cell division. It remains unclear, however, how reduced naive TREC contents can be reconciled with a gradual loss of naive T cells in HIV-1 infection. We performed longitudinal analyses in humans before and after HIV-1 seroconversion, and used a mathematical model to investigate which processes could explain the observed changes in naive T-cell numbers and TRECs during untreated HIV-1 disease progression. Both CD4⁺ and CD8⁺ naive T-cell TREC contents declined biphasically, with a rapid loss during the first year and a much slower loss during the chronic phase of infection. While naive CD8⁺ T-cell numbers hardly changed during follow-up, naive CD4⁺ T-cell counts continually declined. We show that a fine balance between increased T-cell division and loss in the peripheral naive T-cell pool can explain the observed short- and long-term changes in TRECs and naive T-cell numbers, especially if T-cell turnover during the acute phase is more increased than during the chronic phase of infection. Loss of thymic output, on the other hand, does not help to explain the biphasic loss of TRECs in HIV infection. The observed longitudinal changes in TRECs and naive T-cell numbers in HIV-infected individuals are most likely explained by a tight balance between increased T-cell division and death, suggesting that these changes are intrinsically linked in HIV infection.     

Naturally occurring CCR5 extracellular and transmembrane domain variants affect HIV-1 Co-receptor and ligand binding function.

Analysis of CCR5 variants in human immunodeficiency virus, type 1 (HIV-1), high risk cohorts led to the identification of multiple single amino acid substitutions in the amino-terminal third of the HIV-1 co-receptor CCR5 suggesting the possibility of protective and permissive genotypes; unfortunately, the low frequency of these mutations did not led to correlation with function. Therefore, we used analytical methods to assess the functional and structural significance of six of these variant receptors in vitro. These studies showed three categories of effects on CCR5 function. 1) Mutations in the first extracellular domain of CCR5 severely reduce specific ligand binding and chemokine-induced chemotaxis. 2) An extracellular domain variant, A29S, when co-expressed with CD4, supported HIV-1 infection whereas the others do not. 3) The transmembrane region variants of CCR5 support monotropic HIV-1 infection that is blocked by addition of some receptor agonists. Mutations in the first and second transmembrane domains increase RANTES (regulated on activation normal T-cell expressed) binding affinity but did not affect MIP1beta binding affinity presumably based on differences in ligand-receptor interaction sites. Furthermore, the CCR5 transmembrane mutants do not respond to RANTES with the classical bell-shaped chemotactic response curve, suggesting that they are resistant to RANTES-induced desensitization. These data demonstrate that single amino acid changes in the extracellular domains of CCR5 can have profound effects on both HIV-1 co-receptor and specific ligand-induced functions, whereas mutations in the transmembrane domain only affect the response to chemokine ligands.     

Predictors of Attrition and Immunological Failure in HIV-1 Patients on Highly Active Antiretroviral Therapy from Different Healthcare Settings in Mozambique

In Mozambique, the evaluation of retention in HIV care and ART programmes is limited. To assess rate and predictors of attrition (no retention in care) and HAART effectiveness in HIV-1 infected patients who pay for medication and laboratory testing in Mozambique, we conducted a multicenter survey of HIV-1-infected patients who started HAART during 2002–2006. Cox proportional hazard models were used to assess risk of attrition and of therapy failure. Overall, 142 patients from 16 healthcare centers located in the capital city Maputo were followed-up for 22.2 months (12.1–46.7). The retention rate was 75%, 48% and 37% after one, two and three years, respectively. Risk of attrition was lower in patients with higher baseline CD4 count (P = 0.022) and attending healthcare center 1 (HCC1) (P = 0.013). The proportion of individuals with CD4 count ≤200 cells/µL was 55% (78/142) at baseline and decreased to 6% (3/52) at 36 months. Among the patients with available VL, 86% (64/74) achieved undetectable VL levels. The rate of immunologic failure was 17.2% (95% CI: 12.6–22.9) per 100 person-years. Risk of failure was associated to higher baseline CD4 count (P = 0.002), likely reflecting low adherence levels, and decreased with baseline VL ≥10,000 copies/mL (P = 0.033). These results suggest that HAART can be effective in HIV-1 infected patients from Mozambique that pay for their medication and laboratory testing. Further studies are required to identify the causes for low retention rates in patients with low CD4 counts and to better understand the association between healthcare setting and attrition rate.     

Effectiveness of a Treatment Switch to Nevirapine plus Tenofovir and Emtricitabine (or Lamivudine) in Adults with HIV-1 Suppressed Viremia

Background

Switching subjects with persistently undetectable HIV-1 viremia under antiretroviral treatment (ART) to once-daily tenofovir/emtricitabine (or lamivudine) + nevirapine is a cost-effective and well-tolerated strategy. However, the effectiveness of this approach has not been established.

Methods

We performed a retrospective study evaluating the rates of treatment failure, virological failure (VF), and variables associated, in all subjects initiating this switch combination in our clinic since 2001. Analyses were performed by a modified intention to treat, where switch due to toxicity equalled failure. The main endpoint was plasma HIV-RNA < 50 copies/mL.

Results

341 patients were treated for a median of 176 (57; 308) weeks. At week 48, 306 (89.7%) subjects had HIV-1 RNA <50 copies/mL, 10 (2.9%) experienced VF, and 25 (7.4%) discontinued the treatment due to toxicity. During the whole follow-up 23 (6.7%) individuals (17 on lamivudine, 6 on emtricitabine; p = 0.034) developed VF and treatment modification due to toxicity occurred in 36 (10.7%). Factors independently associated with VF in a multivariate analysis were: intravenous drug use (HR 1.51; 95%CI 1.12, 2.04), time with undetectable viral load before the switch (HR 0.98; 0.97, 0.99), number of prior NRTIs (HR 1.49; 1.15, 1.93) or NNRTIs (HR 3.22; 1.64, 6.25), and previous NVP (HR 1.54; 1.10, 2.17) or efavirenz (HR 5.76; 1.11, 29.87) unscheduled interruptions. VF was associated with emergence of usual nevirapine mutations (Y181C/I/D, K103N and V106A/I), M184V (n = 16; 12 with lamivudine vs. 4 with emtricitabine, p = 0.04), and K65R (n = 7).

Conclusions

The rates of treatment failure at 48 weeks, or long-term toxicity or VF with this switch regimen are low and no unexpected mutations or patterns of mutations were selected in subjects with treatment failure.     

Accelerated In Vivo Proliferation of Memory Phenotype CD4+ T-cells in Human HIV-1 Infection Irrespective of Viral Chemokine Co-receptor Tropism

CD4⁺ T-cell loss is the hallmark of HIV-1 infection. CD4 counts fall more rapidly in advanced disease when CCR5-tropic viral strains tend to be replaced by X4-tropic viruses. We hypothesized: (i) that the early dominance of CCR5-tropic viruses results from faster turnover rates of CCR5⁺ cells, and (ii) that X4-tropic strains exert greater pathogenicity by preferentially increasing turnover rates within the CXCR4⁺ compartment. To test these hypotheses we measured in vivo turnover rates of CD4⁺ T-cell subpopulations sorted by chemokine receptor expression, using in vivo deuterium-glucose labeling. Deuterium enrichment was modeled to derive in vivo proliferation (p) and disappearance (d*) rates which were related to viral tropism data. 13 healthy controls and 13 treatment-naive HIV-1-infected subjects (CD4 143–569 cells/ul) participated. CCR5-expression defined a CD4⁺ subpopulation of predominantly CD45R0⁺ memory cells with accelerated in vivo proliferation (p = 2.50 vs 1.60%/d, CCR5⁺ vs CCR5⁻; healthy controls; P<0.01). Conversely, CXCR4 expression defined CD4⁺ T-cells (predominantly CD45RA⁺ naive cells) with low turnover rates. The dominant effect of HIV infection was accelerated turnover of CCR5⁺CD45R0⁺CD4⁺ memory T-cells (p = 5.16 vs 2.50%/d, HIV vs controls; P<0.05), naïve cells being relatively unaffected. Similar patterns were observed whether the dominant circulating HIV-1 strain was R5-tropic (n = 9) or X4-tropic (n = 4). Although numbers were small, X4-tropic viruses did not appear to specifically drive turnover of CXCR4-expressing cells (p = 0.54 vs 0.72 vs 0.44%/d in control, R5-tropic, and X4-tropic groups respectively). Our data are most consistent with models in which CD4⁺ T-cell loss is primarily driven by non-specific immune activation.     

The Individualized Genetic Barrier Predicts Treatment Response in a Large Cohort of HIV-1 Infected Patients

The success of combination antiretroviral therapy is limited by the evolutionary escape dynamics of HIV-1. We used Isotonic Conjunctive Bayesian Networks (I-CBNs), a class of probabilistic graphical models, to describe this process. We employed partial order constraints among viral resistance mutations, which give rise to a limited set of mutational pathways, and we modeled phenotypic drug resistance as monotonically increasing along any escape pathway. Using this model, the individualized genetic barrier (IGB) to each drug is derived as the probability of the virus not acquiring additional mutations that confer resistance. Drug-specific IGBs were combined to obtain the IGB to an entire regimen, which quantifies the virus'' genetic potential for developing drug resistance under combination therapy. The IGB was tested as a predictor of therapeutic outcome using between 2,185 and 2,631 treatment change episodes of subtype B infected patients from the Swiss HIV Cohort Study Database, a large observational cohort. Using logistic regression, significant univariate predictors included most of the 18 drugs and single-drug IGBs, the IGB to the entire regimen, the expert rules-based genotypic susceptibility score (GSS), several individual mutations, and the peak viral load before treatment change. In the multivariate analysis, the only genotype-derived variables that remained significantly associated with virological success were GSS and, with 10-fold stronger association, IGB to regimen. When predicting suppression of viral load below 400 cps/ml, IGB outperformed GSS and also improved GSS-containing predictors significantly, but the difference was not significant for suppression below 50 cps/ml. Thus, the IGB to regimen is a novel data-derived predictor of treatment outcome that has potential to improve the interpretation of genotypic drug resistance tests.     

Low Resistance to First and Second Line Anti-Tuberculosis Drugs among Treatment Naive Pulmonary Tuberculosis Patients in Southwestern Uganda

BackgroundThere are limited data on region-specific drug susceptibility of tuberculosis (TB) in Uganda. We performed resistance testing on specimens collected from treatment-naive patients with pulmonary TB in Southwestern Uganda for first and second line anti-TB drugs. We sought to provide data to guide regional recommendations for empiric TB therapy.MethodsArchived isolates, obtained from patients at Mbarara Regional Referral Hospital from February 2009 to February 2013, were tested for resistance to isoniazid and rifampicin using the MTBDRplus and Xpert MTB/RIF assays. A subset of randomly selected isolates was tested for second line agents, including fluoroquinolones (FQs), aminoglycosides, cyclic peptides, and ethambutol using the MTBDRsl assay. We performed confirmatory testing for FQ resistance using repeated MTBDRsl, the Mycobacteria growth indicator tube (MGIT) assay, and sequencing of the gyrA and gyrB genes.ResultsWe tested isolates from 190 patients. The cohort had a median age of 33 years (IQR 26-43), 69% (131/190) were male, and the HIV prevalence was 42% (80/190). No isolates (0/190) were rifampicin-resistant and only 1/190 (0.5%) was isoniazid-resistant. Among 92 isolates tested for second-line drug resistance, 71 (77%) had interpretable results, of which none were resistant to aminoglycosides, cyclic peptides or ethambutol. Although 7 (10%) initially tested as resistant to FQs by the MTBDRsl assay, they were confirmed as susceptible by repeat MTBDRsl testing as well as by MGIT and gyrase gene sequencingConclusionWe found no MDR-TB and no resistance to ethambutol, FQs, or injectable anti-TB drugs in treatment naïve patients with pulmonary TB in Southwestern Uganda. Standard treatment guidelines for susceptible TB should be adequate for most patients with TB in this population. Where possible, molecular susceptibility testing methods should be routinely validated by culture methods.     

南宁市HIV-1/AIDS人群治疗前pol区遗传特性及蛋白结构分析

为探讨南宁市某县艾滋病病毒1型(HIV-1)感染人群中治疗前pol区遗传特性及蛋白结构变化情况,本研究通过RT-PCR扩增pol区部分序列并进行测序,将序列同源比对构建系统进化树;分型确定毒株亚型和斯坦福大学HIV耐药性数据库比对,分析耐药相关位点;SWISS-MODEL蛋白质同源数据库进行建模分析氨基酸的突变对蛋白质结构和功能的影响。本研究在90份HIV-1标本中获得46个pol区有效序列,共发现4种亚型,其中CRF01_AE占76.08%(35/46)、CRF08_BC占15.22%(7/46)、CRF07_BC占(3/46)6.52%、CRF59_01B1占2.17%(1/46);46个序列中有4例(8.69%)出现耐药突变位点,没有针对核苷酸反转录酶抑制剂(NRTI)的耐药突变;针对蛋白酶类抑制剂(PIs)1例,PR蛋白酶的柔性部位I47V位点发生突变,β折叠结构的I84V位点发生突变,都是异亮氨酸突变为缬氨酸;针对非核苷酸反转录酶抑制剂(NNRTI)有3例,2例位于活性中心的Y181C位点由酪氨酸突变为半胱氨酸,1例位于转角处的E138G位点由谷氨酸突变为甘氨酸。研究表明,南宁市某县HIV-1病毒CRF01_AE重组亚型比例最大,未经抗病毒治疗HIV1感染者中已经出现pol区耐药突变株,突变位点主要位于活性中心及柔性部位,传播水平已经处于中等流行状态。深入分析蛋白质与抑制剂相互作用机制,有助于为艾滋病抗病毒及耐药性监测方案提供科学依据。     

Predictors of Death during Tuberculosis Treatment in TB/HIV Co-Infected Patients in Malaysia

Background

Mortality among TB/HIV co-infected patients is still high particularly in developing countries. This study aimed to determine the predictors of death in TB/HIV co-infected patients during TB treatment.

Methods

We reviewed medical records at the time of TB diagnosis and subsequent follow-up of all newly registered TB patients with HIV co-infection at TB clinics in the Institute of Respiratory Medicine and three public hospitals in the Klang Valley between January 2010 and September 2010. We reviewed these medical records again twelve months after their initial diagnosis to determine treatment outcomes and survival. We analysed using Kaplan-Meier and conducted multivariate Cox proportional hazards analysis to identify predictors of death during TB treatment in TB/HIV co-infected patients.

Results

Of the 227 patients studied, 53 (23.3%) had died at the end of the study with 40% of deaths within two months of TB diagnosis. Survival at 2, 6 and 12 months after initiating TB treatment were 90.7%, 82.8% and 78.8% respectively. After adjusting for other factors, death in TB/HIV co-infected patients was associated with being Malay (aHR 4.48; 95%CI 1.73-11.64), CD4 T-lymphocytes count < 200 cells/µl (aHR 3.89; 95% CI 1.20-12.63), three or more opportunistic infections (aHR 3.61; 95% CI 1.04-12.55), not receiving antiretroviral therapy (aHR 3.21; 95% CI 1.76-5.85) and increase per 10³ total white blood cell count per microliter (aHR 1.12; 95% CI 1.05-1.20)

Conclusion

TB/HIV co-infected patients had a high case fatality rate during TB treatment. Initiation of antiretroviral therapy in these patients can improve survival by restoring immune function and preventing opportunistic infections.     

Frequency of HLA-A*03 associates with HIV-1 infection in a Chinese cohort

During the early mid-1990s, a number of rural farmers across central China were employed to the unregulated plasmaselling-activity and many of them were infected by HIV-1. However, AIDS progression in the former blood donors (FBDs) is various. The aim of this study is to assess human leukocyte antigen (HLA) class I allele distribution in FBDs and evaluate its association with HIV-1 infection and disease progression. A total of 353 FBDs were enrolled in the cohort including 294 ART naïve HIV-1 seropositive and 59 HIV-1 seronegative age-matched subjects. The viral load and CD4/CD8 T cell counts were assessed in all subjects. Compared with HIV-seropositive group, the frequency of HLA-A*03 in control was significantly higher. After classifying the HLA-B alleles of the subjects according to the presence of Bw4/Bw6 serological epitopes, detrimental effect of HLA Bw6/ Bw6 homozygosity was also confirmed in the HIV-seropositive subjects. This study provides novel evidence on HLA class I allele distribution and association of HLA-A*03 frequency with HIV-1 infection and viremia in the HIV-1 infected FBDs, which may throw light on intervention strategy for the HIV-1 infection and our understanding how host immunity and genetic background affect HIV infection and AIDS progression.     

Resistance Mutations and CTL Epitopes in Archived HIV-1 DNA of Patients on Antiviral Treatment: Toward a New Concept of Vaccine

Eleven patients responding successfully to first-line antiretroviral therapy (ART) were investigated for proviral drug resistance mutations (DRMs) in RT by ultra-deep pyrosequencing (UDPS). After molecular typing of the class I alleles A and B, the CTL epitopes in the Gag, Nef and Pol regions of the provirus were sequenced and compared to the reference HXB2 HIV-1 epitopes. They were then matched with the HLA alleles with determination of theoretical affinity (TA). For 3 patients, the results could be compared with an RNA sample of the circulating virus at initiation of therapy. Five out of 11 patients exhibited DRMs by UDPS. The issue is whether a therapeutic switch is relevant in these patients by taking into account the identity of the archived resistance mutations. When the archived CTL epitopes were determined on the basis of the HLA alleles, different patterns were observed. Some epitopes were identical to those reported for the reference with the same TA, while others were mutated with a decrease in TA. In 2 cases, an epitope was observed as a combination of subpopulations at entry and was retrieved as a single population with lower TA at success. With regard to immunological stimulation and given the variability of the archived CTL epitopes, we propose a new concept of curative vaccine based on identification of HIV-1 CTL epitopes after prior sequencing of proviral DNA and matching with HLA class I alleles.     

Helminth-Associated Systemic Immune Activation and HIV Co-receptor Expression: Response to Albendazole/Praziquantel Treatment

Background

It has been hypothesized that helminth infections increase HIV susceptibility by enhancing systemic immune activation and hence contribute to elevated HIV-1 transmission in sub-Saharan Africa.

Objective

To study systemic immune activation and HIV-1 co-receptor expression in relation to different helminth infections and in response to helminth treatment.

Methods

HIV-negative adults with (n = 189) or without (n = 57) different helminth infections, as diagnosed by Kato-Katz, were enrolled in Mbeya, Tanzania. Blinded to helminth infection status, T cell differentiation (CD45RO, CD27), activation (HLA-DR, CD38) and CCR5 expression was determined at baseline and 3 months after Albendazole/Praziquantel treatment. Plasma cytokine levels were compared using a cytometric bead array.

Results

Trichuris and Ascaris infections were linked to increased frequencies of “activated” CD4 and/or CD8 T cells (p<0.05), whereas Hookworm infection was associated with a trend towards decreased HLA-DR⁺ CD8 T cell frequencies (p = 0.222). In Trichuris infected subjects, there was a linear correlation between HLA-DR⁺ CD4 T cell frequencies and the cytokines IL-1β and IL-10 (p<0.05). Helminth treatment with Albendazole and Praziquantel significantly decreased eosinophilia for S. mansoni and Hookworm infections (p<0.005) but not for Trichuris infection and only moderately modulated T cell activation. CCR5 surface density on memory CD4 T cells was increased by 1.2-fold during Trichuris infection (p-value: 0.053) and reduced after treatment (p = 0.003).

Conclusions

Increased expression of T cell activation markers was associated with Trichuris and Ascaris infections with relatively little effect of helminth treatment.