Kaposi sarcoma-associated herpes virus and response to antiretroviral therapy: a prospective study of HIV-infected adults

BACKGROUND The possible impact of coinfection with the Kaposi sarcoma-associated herpes virus (KSHV) on the response to antiretroviral therapy (ART) is unknown. Prospective studies are rare, particularly in Africa. METHODS We enrolled a prospective cohort of HIV-infected adults initiating ART in Johannesburg, South Africa. The subjects were defined as seropositive to KSHV if they were reactive to either KSHV lytic K8.1 or latent Orf73 antigen or to both. The subjects were followed from ART initiation until 18 months of treatment. HIV viral load and CD4 counts were tested 6 monthly. Linear generalized estimating and log-binomial regression models were used to estimate the effect of KSHV infection on immunologic recovery and response and HIV viral load suppression within 18 months after ART initiation. RESULTS Three hundred eighty-five subjects initiating ART from November 2008 to March 2009 were considered to be eligible including 184 (48%) KSHV+. The KSHV+ group was similar to the KSHV- in terms of age, gender, initiating CD4 count, body mass index, tuberculosis, and hemoglobin levels. The KSHV+ group gained a similar number of cells at 6 [difference of 10 cells per cubic millimeter, 95% confidence interval (CI): -11 to 31], 12 (3 cells per cubic millimeter, 95% CI: -19 to 25), and 18 months (24 cells per cubic millimeter, 95% CI: -13 to 61) compared with that gained by the KSHV- group. Adjusted relative risk of failure to suppress viral load to <400 copies per milliliter (1.03; 95% CI: 0.90 to 1.17) were similar for KSHV+ and KSHV- by 6 months on treatment. CONCLUSIONS In a population with a high KSHV prevalence, HIV-positive adults coinfected with KSHV achieved similar immunologic and virologic responses to ART early after treatment initiation compared with those with KSHV-.

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.

Marked increase of the astrocytic marker S100B in the cerebrospinal fluid of HIV-infected patients on LPV/r-monotherapy

Objective: To determine changes of cerebrospinal fluid (CSF) biomarkers of patients on monotherapy with lopinavir/ritonavir. Design: The Monotherapy Switzerland/Thailand study (MOST) trial compared monotherapy with ritonavir-boosted lopinavir with continued therapy. The trial was prematurely stopped due to virological failure in six patients on monotherapy. It, thus, offers a unique opportunity to assess brain markers in the early stage of HIV virological escape. Methods: Sixty-five CSF samples (34 on continued therapy and 31 on monotherapy) from 49 HIV-positive patients enrolled in MOST. Using enzyme-linked immunosorbent assay, we determined the CSF concentration of S100B (astrocytosis), neopterin (inflammation), total Tau (tTau), phosphorylated Tau (pTau), and amyloid-β 1–42 (Aβ), the latter three indicating neuronal damage. Controls were CSF samples of 29 HIV-negative patients with Alzheimer dementia. Results: In the CSF of monotherapy, concentrations of S100B and neopterin were significantly higher than in continued therapy (P = 0.006 and P = 0.013, respectively) and Alzheimer dementia patients (P < 0.0001 and P = 0.0005, respectively). In Alzheimer dementia, concentration of Aβ was lower than in monotherapy (P = 0.005) and continued therapy (P = 0.016) and concentrations of tTau were higher than in monotherapy (P = 0.019) and continued therapy (P = 0.001). There was no difference in pTau among the three groups. After removal of the 16 CSF with detectable viral load in the blood and/or CSF, only S100B remained significantly higher in monotherapy than in the two other groups. Conclusion: Despite full viral load-suppression in blood and CSF, antiretroviral monotherapy with lopinavir/ritonavir can raise CSF levels of S100B, suggesting astrocytic damage.

PCR identification of HIV-1 DNA sequences in brain tissue of patients with AIDS encephalopathy.

We analyzed brain tissue from 39 patients for the presence of proviral HIV-1 sequences, using the polymerase chain reaction (PCR) for the amplification of segments of the viral LTR and gag genes. A novel primer extension procedure allowed the detection of a single HIV-1 copy in 1 micrograms DNA. We detected proviral HIV-1 DNA in 16 of 25 brain samples from AIDS patients. Semiquantitative evaluation of the amplified DNAs indicated considerable variation in viral load. Highest levels of proviral DNA were present in brain samples from six patients with clinical evidence of HIV-associated cognitive/motor complex and the histopathologic correlate of HIV leukoencephalopathy or HIV encephalitis. An additional 11 brain samples contained smaller amounts of proviral DNA. In these patients, clinical data were inconclusive regarding the diagnosis of HIV-1 encephalopathy and histopathologically there was no evidence of HIV-1-induced tissue lesions. In nine of 25 seropositive patients with AIDS (36%), brain samples scored negative or did not contain an unequivocal signal indicating the presence of proviral DNA. HIV-1 sequences were not detected in any of 14 control brain samples from HIV-1 seronegative patients. Our data indicate that HIV-1 is present in the central nervous system of the majority (two thirds) of AIDS patients and that the highest levels of proviral DNA in brain tissue are associated with HIV encephalopathy.

Loss to follow-up of HIV-infected women after delivery: The Swiss HIV Cohort Study and the Swiss Mother and Child HIV Cohort Study.

INTRODUCTION HIV-infected pregnant women are very likely to engage in HIV medical care to prevent transmission of HIV to their newborn. After delivery, however, childcare and competing commitments might lead to disengagement from HIV care. The aim of this study was to quantify loss to follow-up (LTFU) from HIV care after delivery and to identify risk factors for LTFU. METHODS We used data on 719 pregnancies within the Swiss HIV Cohort Study from 1996 to 2012 and with information on follow-up visits available. Two LTFU events were defined: no clinical visit for >180 days and no visit for >360 days in the year after delivery. Logistic regression analysis was used to identify risk factors for a LTFU event after delivery. RESULTS Median maternal age at delivery was 32 years (IQR 28-36), 357 (49%) women were black, 280 (39%) white, 56 (8%) Asian and 4% other ethnicities. One hundred and seven (15%) women reported any history of IDU. The majority (524, 73%) of women received their HIV diagnosis before pregnancy, most of those (413, 79%) had lived with diagnosed HIV longer than three years and two-thirds (342, 65%) were already on antiretroviral therapy (ART) at time of conception. Of the 181 women diagnosed during pregnancy by a screening test, 80 (44%) were diagnosed in the first trimester, 67 (37%) in the second and 34 (19%) in the third trimester. Of 357 (69%) women who had been seen in HIV medical care during three months before conception, 93% achieved an undetectable HIV viral load (VL) at delivery. Of 62 (12%) women with the last medical visit more than six months before conception, only 72% achieved an undetectable VL (p=0.001). Overall, 247 (34%) women were LTFU over 180 days in the year after delivery and 86 (12%) women were LTFU over 360 days with 43 (50%) of those women returning. Being LTFU for 180 days was significantly associated with history of intravenous drug use (aOR 1.73, 95% CI 1.09-2.77, p=0.021) and not achieving an undetectable VL at delivery (aOR 1.79, 95% CI 1.03-3.11, p=0.040) after adjusting for maternal age, ethnicity, time of HIV diagnosis and being on ART at conception. CONCLUSIONS Women with a history of IDU and women with a detectable VL at delivery were more likely to be LTFU after delivery. This is of concern regarding their own health, as well as risk for sexual partners and subsequent pregnancies. Further strategies should be developed to enhance retention in medical care beyond pregnancy.

The Cost-Effectiveness of Monitoring Strategies for Antiretroviral Therapy of HIV Infected Patients in Resource-Limited Settings: Software Tool.

BACKGROUND The cost-effectiveness of routine viral load (VL) monitoring of HIV-infected patients on antiretroviral therapy (ART) depends on various factors that differ between settings and across time. Low-cost point-of-care (POC) tests for VL are in development and may make routine VL monitoring affordable in resource-limited settings. We developed a software tool to study the cost-effectiveness of switching to second-line ART with different monitoring strategies, and focused on POC-VL monitoring. METHODS We used a mathematical model to simulate cohorts of patients from start of ART until death. We modeled 13 strategies (no 2nd-line, clinical, CD4 (with or without targeted VL), POC-VL, and laboratory-based VL monitoring, with different frequencies). We included a scenario with identical failure rates across strategies, and one in which routine VL monitoring reduces the risk of failure. We compared lifetime costs and averted disability-adjusted life-years (DALYs). We calculated incremental cost-effectiveness ratios (ICER). We developed an Excel tool to update the results of the model for varying unit costs and cohort characteristics, and conducted several sensitivity analyses varying the input costs. RESULTS Introducing 2nd-line ART had an ICER of US$1651-1766/DALY averted. Compared with clinical monitoring, the ICER of CD4 monitoring was US$1896-US$5488/DALY averted and VL monitoring US$951-US$5813/DALY averted. We found no difference between POC- and laboratory-based VL monitoring, except for the highest measurement frequency (every 6 months), where laboratory-based testing was more effective. Targeted VL monitoring was on the cost-effectiveness frontier only if the difference between 1st- and 2nd-line costs remained large, and if we assumed that routine VL monitoring does not prevent failure. CONCLUSION Compared with the less expensive strategies, the cost-effectiveness of routine VL monitoring essentially depends on the cost of 2nd-line ART. Our Excel tool is useful for determining optimal monitoring strategies for specific settings, with specific sex-and age-distributions and unit costs.

Hepatitis B viral load in dried blood spots: A validation study in Zambia

Background: Access to hepatitis B viral load (VL) testing is poor in sub-Saharan Africa (SSA) due toeconomic and logistical reasons.Objectives: To demonstrate the feasibility of testing dried blood spots (DBS) for hepatitis B virus (HBV)VL in a laboratory in Lusaka, Zambia, and to compare HBV VLs between DBS and plasma samples.Study design: Paired plasma and DBS samples from HIV-HBV co-infected Zambian adults were analyzedfor HBV VL using the COBAS AmpliPrep/COBAS TaqMan HBV test (Version 2.0) and for HBV genotypeby direct sequencing. We used Bland-Altman analysis to compare VLs between sample types and bygenotype. Logistic regression analysis was conducted to assess the probability of an undetectable DBSresult by plasma VL.Results: Among 68 participants, median age was 34 years, 61.8% were men, and median plasma HBV VLwas 3.98 log IU/ml (interquartile range, 2.04–5.95). Among sequenced viruses, 28 were genotype A1 and27 were genotype E. Bland–Altman plots suggested strong agreement between DBS and plasma VLs. DBSVLs were on average 1.59 log IU/ml lower than plasma with 95% limits of agreement of −2.40 to −0.83 logIU/ml. At a plasma VL ≥2,000 IU/ml, the probability of an undetectable DBS result was 1.8% (95% CI:0.5–6.6). At plasma VL ≥20,000 IU/ml this probability reduced to 0.2% (95% CI: 0.03–1.7).

Predictors of HIV-1 slow progression among vertically infected children in Botswana

HIV-1 infected children display a highly variable rate of progression to AIDS. Data about reasons underlying the variable progression to AIDS among vertically-infected children is sparse, and the few studies that have examined this important question have almost exclusively been done in the developed world. This is despite the fact that Sub-Saharan Africa is home to over 90% of all HIV infected children around the world.^ The main objective of this study was to examine predictors of HIV-1 slow progression among vertically infected children in Botswana, using a case control design. Cases (slow progressors) and controls (rapid progressors) were drawn from medical records of HIV-1 infected children being followed up for routine care and treatment at the BBCCCOE between February 2003 and February 2011. Univariate and Multivariate Logistic Regression Analyses were performed to identify independent predictors of slow disease progression and control for confounding respectively. ^ The study population comprised of 152 cases and 201 controls with ages ranging from 6 months to 16 years at baseline. Low baseline HIV-1 RNA viral load was the strongest independent predictor of slow progression (adjusted OR = 5.52, 95% CI = 2.75-11.07; P <0.001). Other independent predictors of slow disease progression identified were: lack of history of PMTCT with single dose Nevirapine plus Zidovudine (adjusted OR = 4.45, 95% CI = 1.45-13.69; P = 0.009) and maternal vital status (alive) (adjusted OR = 2.46, 95% CI = 1.51-4.01; P < 0.00 ).^ The results of this study may help clinicians and policy-makers in resource-limited settings to identify, at baseline, which children are at highest risk of rapid progression to AIDS and thus prioritize them for immediate intervention with HAART and other measures that would mitigate disease progression. At the same time HAART may be delayed among children who are at lower risk of disease progression. This would enable the highly affected, yet impoverished, Sub-Saharan African countries to use their scarce resources more efficiently which may in turn ensure that their National Antiretroviral Therapy Programs become more sustainable. Delaying HAART among the low-risk children would also lower the occurrence of adverse drug reactions associated with antiretroviral drugs exposure.^ Keywords. Slow Progressors, Rapid Progressors, HIV-1, Predictors, Children, Vertical Transmission, Sub-Saharan Africa^

Lysis of HIV-1-infected cells and inhibition of viral replication by universal receptor T cells

Increasing evidence suggests that HIV-1-specific cytotoxic T lymphocytes (CTLs) are a key host immune response to HIV-1 infection. Generation of CTL responses for prevention or therapy of HIV-1 infection has several intrinsic technical barriers such as antigen expression and presentation, the varying HLA restrictions between different individuals, and the potential for viral escape by sequence variation or surface molecule alteration on infected cells. A strategy to circumvent these limitations is the construction of a chimeric T cell receptor containing human CD4 or HIV-1-specific Ig sequences linked to the signaling domain of the T cell receptor ζ chain (universal T cell receptor). CD8+ CTLs transduced with this universal receptor can then bind and lyse infected cells that express surface HIV-1 gp120. We evaluated the ability of universal-receptor-bearing CD8+ cells from a seronegative donor to lyse acutely infected cells and inhibit HIV-1 replication in vitro. The kinetics of lysis and efficiency of inhibition were comparable to that of naturally occurring HIV-1-specific CTL clones isolated from infected individuals. Further study will be required to determine the utility of these cells as a therapeutic strategy in vivo.

Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy

Although highly active antiretroviral therapy (HAART) in the form of triple combinations of drugs including protease inhibitors can reduce the plasma viral load of some HIV-1-infected individuals to undetectable levels, it is unclear what the effects of these regimens are on latently infected CD4+ T cells and what role these cells play in the persistence of HIV-1 infection in individuals receiving such treatment. The present study demonstrates that highly purified CD4+ T cells from 13 of 13 patients receiving HAART with an average treatment time of 10 months and with undetectable (<500 copies HIV RNA/ml) plasma viremia by a commonly used bDNA assay carried integrated proviral DNA and were capable of producing infectious virus upon cellular activation in vitro. Phenotypic analysis of HIV-1 produced by activation of latently infected CD4+ T cells revealed the presence in some patients of syncytium-inducing virus. In addition, the presence of unintegrated HIV-1 DNA in infected resting CD4+ T cells from patients receiving HAART, even those with undetectable plasma viremia, suggests persistent active virus replication in vivo.

Peptide inhibitors of HIV-1 protease and viral infection of peripheral blood lymphocytes based on HIV-1 Vif

We recently reported that HIV-1 Vif (virion infectivity factor) inhibits HIV-1 protease in vitro and in bacteria, suggesting that it may serve as the basis for the design of new protease inhibitors and treatment for HIV-1 infection. To evaluate this possibility, we synthesized peptide derivatives from the region of Vif, which inhibits protease, and tested their activity on protease. In an assay of cleavage of virion-like particles composed of HIV-1 Gag precursor polyprotein, full-length recombinant Vif, and a peptide consisting of residues 21–65 of Vif, but not a control peptide or BSA, inhibited protease activity. Vif21–65 blocked protease at a molar ratio of two to one. We then tested this peptide and a smaller peptide, Vif41–65, for their effects on HIV-1 infection of peripheral blood lymphocytes. Both Vif peptides inhibited virus expression below the limit of detection, but control peptides had no effect. To investigate its site of action, Vif21–65 was tested for its effect on Gag cleavage by protease during HIV-1 infection. We found that commensurate with its reduction of virus expression, Vif21–65 inhibited the cleavage of the polyprotein p55 to mature p24. These results are similar to those obtained by using Ro 31–8959, a protease inhibitor in clinical use. We conclude that Vif-derived peptides inhibit protease during HIV-1 infection and may be useful for the development of new protease inhibitors.

Cytotoxic T lymphocytes and viral turnover in HIV type 1 infection

To understand the role of the immune system in limiting HIV type 1 replication, it is critical to know to what extent the rapid turnover of productively infected cells is caused by viral cytopathicity or by immune-mediated lysis. We show that uncultured peripheral blood mononuclear cells of many patients contain cytotoxic T lymphocytes (CTL) that lyse target cells—at plausible peripheral blood mononuclear cell-to-target ratios—with half-lives of less than 1 day. In 23 patients with CD4 counts ranging from 10 to 900 per μl, the average rate of CTL-mediated lysis corresponds to a target cell half-life of 0.7 day. We develop mathematical models to calculate the turnover rate of infected cells subjected to immune-mediated lysis and viral cytopathicity and to estimate the fraction of cells that are killed by CTL as opposed to virus. The models provide new interpretations of drug treatment dynamics and explain why the observed rate of virus decline is roughly constant for different patients. We conclude that in HIV type 1 infection, CTL-mediated lysis can reduce virus load by limiting virus production, with small effects on the half-life of infected cells.

Inhibition of HIV type 1 infectivity by constrained α-helical peptides: Implications for the viral fusion mechanism

Linear peptides derived from the membrane proximal region of the gp41 ectodomain are effective inhibitors of HIV type 1 (HIV-1)-mediated fusion events. These inhibitory peptides lack structure in solution, rendering mechanistic interpretation of their activity difficult. Using structurally constrained analogs of these molecules, we demonstrate that the peptides inhibit infectivity by adopting a helical conformation. Moreover, we show that a specific face of the helix must be exposed to block viral infectivity. Recent crystal structures show that the region of gp41 corresponding to the inhibitory peptides is helical and uses the analogous face to pack against a groove formed by an N-terminal coiled-coil trimer. Our results provide a direct link between the inhibition of HIV-1 infectivity by these peptides and the x-ray structures, and suggest that the conformation of gp41 observed by crystallography represents the fusogenic state. Other agents that block HIV-1 infectivity by binding to this groove may hold promise for the treatment of AIDS.

Catalytic efficiency and vitality of HIV-1 proteases from African viral subtypes

The vast majority of HIV-1 infections in Africa are caused by the A and C viral subtypes rather than the B subtype prevalent in the United States and Western Europe. Genomic differences between subtypes give rise to sequence variations in the encoded proteins, including the HIV-1 protease. Because some amino acid polymorphisms occur at sites that have been associated with drug resistance in the B subtype, it is important to assess the effectiveness of protease inhibitors that have been developed against different subtypes. Here we report the enzymatic characterization of HIV-1 proteases with sequences found in drug-naïve Ugandan adults. The A protease used in these studies differs in seven positions (I13V/E35D/M36I/R41K/R57K/H69K/L89M) in relation to the consensus B subtype protease. Another protease containing a subset of these amino acid polymorphisms (M36I/R41K/H69K/L89M), which are found in subtype C and other HIV subtypes, also was studied. Both proteases were found to have similar catalytic constants, kcat, as the B subtype. The C subtype protease displayed lower Km values against two different substrates resulting in a higher (2.4-fold) catalytic efficiency than the B subtype protease. Indinavir, ritonavir, saquinavir, and nelfinavir inhibit the A and C subtype proteases with 2.5–7-fold and 2–4.5-fold weaker Kis than the B subtype. When all factors are taken into consideration it is found that the C subtype protease has the highest vitality (4–11 higher than the B subtype) whereas the A subtype protease exhibits values ranging between 1.5 and 5. These results point to a higher biochemical fitness of the A and C proteases in the presence of existing inhibitors.