Development and validation of decision rules to guide frequency of monitoring CD4 cell count in HIV-1 infection before starting antiretroviral therapy

Background Although CD4 cell count monitoring is used to decide when to start antiretroviral therapy in patients with HIV-1 infection, there are no evidence-based recommendations regarding its optimal frequency. It is common practice to monitor every 3 to 6 months, often coupled with viral load monitoring. We developed rules to guide frequency of CD4 cell count monitoring in HIV infection before starting antiretroviral therapy, which we validated retrospectively in patients from the Swiss HIV Cohort Study. Methodology/Principal Findings We built up two prediction rules (“Snap-shot rule” for a single sample and “Track-shot rule” for multiple determinations) based on a systematic review of published longitudinal analyses of CD4 cell count trajectories. We applied the rules in 2608 untreated patients to classify their 18 061 CD4 counts as either justifiable or superfluous, according to their prior ≥5% or <5% chance of meeting predetermined thresholds for starting treatment. The percentage of measurements that both rules falsely deemed superfluous never exceeded 5%. Superfluous CD4 determinations represented 4%, 11%, and 39% of all actual determinations for treatment thresholds of 500, 350, and 200×106/L, respectively. The Track-shot rule was only marginally superior to the Snap-shot rule. Both rules lose usefulness for CD4 counts coming near to treatment threshold. Conclusions/Significance Frequent CD4 count monitoring of patients with CD4 counts well above the threshold for initiating therapy is unlikely to identify patients who require therapy. It appears sufficient to measure CD4 cell count 1 year after a count >650 for a threshold of 200, >900 for 350, or >1150 for 500×106/L, respectively. When CD4 counts fall below these limits, increased monitoring frequency becomes advisable. These rules offer guidance for efficient CD4 monitoring, particularly in resource-limited settings.

Early antiretroviral therapy during primary HIV-1 infection results in a transient reduction of the viral setpoint upon treatment interruption

Long-term benefits of combination antiretroviral therapy (cART) initiation during primary HIV-1 infection are debated.

Improved virological outcome in White patients infected with HIV-1 non-B subtypes compared to subtype B

Antiretroviral compounds have been predominantly studied in human immunodeficiency virus type 1 (HIV-1) subtype B, but only ~10% of infections worldwide are caused by this subtype. The analysis of the impact of different HIV subtypes on treatment outcome is important.

Effect of early antiretroviral therapy during primary HIV-1 infection on cell-associated HIV-1 DNA and plasma HIV-1 RNA

Early initiation of combination antiretroviral therapy (ART) during primary HIV-1 infection may prevent the establishment of large viral reservoirs, possibly resulting in improved control of plasma viraemia rebound after ART cessation.

Comparative transcriptomics of extreme phenotypes of human HIV-1 infection and SIV infection in sooty mangabey and rhesus macaque

High levels of HIV-1 replication during the chronic phase of infection usually correlate with rapid progression to severe immunodeficiency. However, a minority of highly viremic individuals remains asymptomatic and maintains high CD4⁺ T cell counts. This tolerant profile is poorly understood and reminiscent of the widely studied nonprogressive disease model of SIV infection in natural hosts. Here, we identify transcriptome differences between rapid progressors (RPs) and viremic nonprogressors (VNPs) and highlight several genes relevant for the understanding of HIV-1-induced immunosuppression. RPs were characterized by a specific transcriptome profile of CD4⁺ and CD8⁺ T cells similar to that observed in pathogenic SIV-infected rhesus macaques. In contrast, VNPs exhibited lower expression of interferon-stimulated genes and shared a common gene regulation profile with nonpathogenic SIV-infected sooty mangabeys. A short list of genes associated with VNP, including CASP1, CD38, LAG3, TNFSF13B, SOCS1, and EEF1D, showed significant correlation with time to disease progression when evaluated in an independent set of CD4⁺ T cell expression data. This work characterizes 2 minimally studied clinical patterns of progression to AIDS, whose analysis may inform our understanding of HIV pathogenesis.

Ambiguous nucleotide calls from population-based sequencing of HIV-1 are a marker for viral diversity and the age of infection

The time passed since the infection of a human immunodeficiency virus (HIV)-infected individual (the age of infection) is an important but often only poorly known quantity. We assessed whether the fraction of ambiguous nucleotides obtained from bulk sequencing as done for genotypic resistance testing can serve as a proxy of this parameter.

HIV-1 Nef interferes with T-lymphocyte circulation through confined environments in vivo

HIV-1 negative factor (Nef) elevates virus replication and contributes to immune evasion in vivo. As one of its established in vitro activities, Nef interferes with T-lymphocyte chemotaxis by reducing host cell actin dynamics. To explore Nef's influence on in vivo recirculation of T lymphocytes, we assessed lymph-node homing of Nef-expressing primary murine lymphocytes and found a drastic impairment in homing to peripheral lymph nodes. Intravital imaging and 3D immunofluorescence reconstruction of lymph nodes revealed that Nef potently impaired T-lymphocyte extravasation through high endothelial venules and reduced subsequent parenchymal motility. Ex vivo analyses of transendothelial migration revealed that Nef disrupted T-lymphocyte polarization and interfered with diapedesis and migration in the narrow subendothelial space. Consistently, Nef specifically affected T-lymphocyte motility modes used in dense environments that pose high physical barriers to migration. Mechanistically, inhibition of lymph node homing, subendothelial migration and cell polarization, but not diapedesis, depended on Nef's ability to inhibit host cell actin remodeling. Nef-mediated interference with in vivo recirculation of T lymphocytes may compromise T-cell help and thus represents an important mechanism for its function as a HIV pathogenicity factor.

Role of retroviral restriction factors in the interferon-α-mediated suppression of HIV-1 in vivo

The antiviral potency of the cytokine IFN-α has been long appreciated but remains poorly understood. A number of studies have suggested that induction of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 (APOBEC3) and bone marrow stromal cell antigen 2 (BST-2/tetherin/CD317) retroviral restriction factors underlies the IFN-α-mediated suppression of HIV-1 replication in vitro. We sought to characterize the as-yet-undefined relationship between IFN-α treatment, retroviral restriction factors, and HIV-1 in vivo. APOBEC3G, APOBEC3F, and BST-2 expression levels were measured in HIV/hepatitis C virus (HCV)-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated IFN-α/ribavirin (IFN-α/riba) combination therapy. IFN-α/riba therapy decreased HIV-1 viral load by -0.921 (±0.858) log(10) copies/mL in HIV/HCV-coinfected patients. APOBEC3G/3F and BST-2 mRNA expression was significantly elevated during IFN-α/riba treatment in patient-derived CD4+ T cells (P < 0.04 and P < 0.008, paired Wilcoxon), and extent of BST-2 induction was correlated with reduction in HIV-1 viral load during treatment (P < 0.05, Pearson's r). APOBEC3 induction during treatment was correlated with degree of viral hypermutation (P < 0.03, Spearman's ρ), and evolution of the HIV-1 accessory protein viral protein U (Vpu) during IFN-α/riba treatment was suggestive of increased BST-2-mediated selection pressure. These data suggest that host restriction factors play a critical role in the antiretroviral capacity of IFN-α in vivo, and warrant investigation into therapeutic strategies that specifically enhance the expression of these intrinsic immune factors in HIV-1-infected individuals.

Minor protease inhibitor mutations at baseline do not increase the risk for a virological failure in HIV-1 subtype B infected patients

Background Minor protease inhibitor (PI) mutations often exist as polymorphisms in HIV-1 sequences from treatment-naïve patients. Previous studies showed that their presence impairs the antiretroviral treatment (ART) response. Evaluating these findings in a larger cohort is essential. Methods To study the impact of minor PI mutations on time to viral suppression and time to virological failure, we included patients from the Swiss HIV Cohort Study infected with HIV-1 subtype B who started first-line ART with a PI and two nucleoside reverse transcriptase inhibitors. Cox regression models were performed to compare the outcomes among patients with 0 and ≥1 minor PI mutation. Models were adjusted for baseline HIV-1 RNA, CD4 cell count, sex, transmission category, age, ethnicity, year of ART start, the presence of nucleoside reverse transcriptase inhibitor mutations, and stratified for the administered PIs. Results We included 1199 patients of whom 944 (78.7%) received a boosted PI. Minor PI mutations associated with the administered PI were common: 41.7%, 16.1%, 4.7% and 1.9% had 1, 2, 3 or ≥4 mutations, respectively. The time to viral suppression was similar between patients with 0 (reference) and ≥1 minor PI mutation (multivariable hazard ratio (HR): 1.1 [95% confidence interval (CI): 1.0–1.3], P = .196). The time to virological failure was also similar (multivariable HR:.9 [95% CI:.5–1.6], P = .765). In addition, the impact of each single minor PI mutation was analyzed separately: none was significantly associated with the treatment outcome. Conclusions The presence of minor PI mutations at baseline has no effect on the therapy outcome in HIV infected individuals.

Diagnostic performance of line-immunoassay based algorithms for incident HIV-1 infection

Background Serologic testing algorithms for recent HIV seroconversion (STARHS) provide important information for HIV surveillance. We have previously demonstrated that a patient's antibody reaction pattern in a confirmatory line immunoassay (INNO-LIA™ HIV I/II Score) provides information on the duration of infection, which is unaffected by clinical, immunological and viral variables. In this report we have set out to determine the diagnostic performance of Inno-Lia algorithms for identifying incident infections in patients with known duration of infection and evaluated the algorithms in annual cohorts of HIV notifications. Methods Diagnostic sensitivity was determined in 527 treatment-naive patients infected for up to 12 months. Specificity was determined in 740 patients infected for longer than 12 months. Plasma was tested by Inno-Lia and classified as either incident (< = 12 m) or older infection by 26 different algorithms. Incident infection rates (IIR) were calculated based on diagnostic sensitivity and specificity of each algorithm and the rule that the total of incident results is the sum of true-incident and false-incident results, which can be calculated by means of the pre-determined sensitivity and specificity. Results The 10 best algorithms had a mean raw sensitivity of 59.4% and a mean specificity of 95.1%. Adjustment for overrepresentation of patients in the first quarter year of infection further reduced the sensitivity. In the preferred model, the mean adjusted sensitivity was 37.4%. Application of the 10 best algorithms to four annual cohorts of HIV-1 notifications totalling 2'595 patients yielded a mean IIR of 0.35 in 2005/6 (baseline) and of 0.45, 0.42 and 0.35 in 2008, 2009 and 2010, respectively. The increase between baseline and 2008 and the ensuing decreases were highly significant. Other adjustment models yielded different absolute IIR, although the relative changes between the cohorts were identical for all models. Conclusions The method can be used for comparing IIR in annual cohorts of HIV notifications. The use of several different algorithms in combination, each with its own sensitivity and specificity to detect incident infection, is advisable as this reduces the impact of individual imperfections stemming primarily from relatively low sensitivities and sampling bias.

Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function

Background The Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF) region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region. Results The dysfunctional HIV group O8 nef allele was restored to the consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2) and non-canonical (B2 and C1422) HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF→AAA) and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef’s association with the cellular p21-activated kinase 2 (PAK2), the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN) were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck. Conclusion Based on these findings, we propose that this highly conserved three amino acid VGF motif together with the acidic cluster and the proline-rich motif form a previously unrecognized amphipathic surface on Nef. This surface appears to be essential for the majority of Nef functions and thus represents a prime target for the pharmacological inhibition of Nef.

CD4-guided scheduled treatment interruptions compared with continuous therapy for patients infected with HIV-1: results of the Staccato randomised trial

BACKGROUND: Stopping antiretroviral therapy in patients with HIV-1 infection can reduce costs and side-effects, but carries the risk of increased immune suppression and emergence of resistance. METHODS: 430 patients with CD4-positive T-lymphocyte (CD4) counts greater than 350 cells per muL, and viral load less than 50 copies per mL were randomised to continued therapy (n=146) or scheduled treatment interruptions (n=284). Median time on randomised treatment was 21.9 months (range 16.4-25.3). Primary endpoints were proportion of patients with viral load less than 50 copies per mL at the end of the trial, and amount of drugs used. Analysis was intention-to-treat. This study is registered at ClinicalTrials.gov with the identifier NCT00113126. FINDINGS: Drug savings in the scheduled treatment interruption group, compared with continuous treatment, amounted to 61.5%. 257 of 284 (90.5%) patients in the scheduled treatment interruption group reached a viral load less than 50 copies per mL, compared with 134 of 146 (91.8%) in the continued treatment group (difference 1.3%, 95% CI-4.3 to 6.9, p=0.90). No AIDS-defining events occurred. Diarrhoea and neuropathy were more frequent with continuous treatment; candidiasis was more frequent with scheduled treatment interruption. Ten patients (2.3%) had resistance mutations, with no significant differences between groups. INTERPRETATION: Drug savings with scheduled treatment interruption were substantial, and no evidence of increased treatment resistance emerged. Treatment-related adverse events were more frequent with continuous treatment, but low CD4 counts and minor manifestations of HIV infection were more frequent with scheduled treatment interruption.

Mortality of HIV-1-infected patients in the first year of antiretroviral therapy: comparison between low-income and high-income countries

BACKGROUND: Highly active antiretroviral therapy (HAART) is being scaled up in developing countries. We compared baseline characteristics and outcomes during the first year of HAART between HIV-1-infected patients in low-income and high-income settings. METHODS: 18 HAART programmes in Africa, Asia, and South America (low-income settings) and 12 HIV cohort studies from Europe and North America (high-income settings) provided data for 4810 and 22,217, respectively, treatment-naive adult patients starting HAART. All patients from high-income settings and 2725 (57%) patients from low-income settings were actively followed-up and included in survival analyses. FINDINGS: Compared with high-income countries, patients starting HAART in low-income settings had lower CD4 cell counts (median 108 cells per muL vs 234 cells per muL), were more likely to be female (51%vs 25%), and more likely to start treatment with a non-nucleoside reverse transcriptase inhibitor (NNRTI) (70%vs 23%). At 6 months, the median number of CD4 cells gained (106 cells per muL vs 103 cells per muL) and the percentage of patients reaching HIV-1 RNA levels lower than 500 copies/mL (76%vs 77%) were similar. Mortality was higher in low-income settings (124 deaths during 2236 person-years of follow-up) than in high-income settings (414 deaths during 20,532 person-years). The adjusted hazard ratio (HR) of mortality comparing low-income with high-income settings fell from 4.3 (95% CI 1.6-11.8) during the first month to 1.5 (0.7-3.0) during months 7-12. The provision of treatment free of charge in low-income settings was associated with lower mortality (adjusted HR 0.23; 95% CI 0.08-0.61). INTERPRETATION: Patients starting HAART in resource-poor settings have increased mortality rates in the first months on therapy, compared with those in developed countries. Timely diagnosis and assessment of treatment eligibility, coupled with free provision of HAART, might reduce this excess mortality.

Evaluation of p24-based antiretroviral treatment monitoring in pediatric HIV-1 infection: prediction of the CD4+ T-cell changes between consecutive visits

Worldwide, 700,000 infants are infected annually by HIV-1, most of them in resource-limited settings. Care for these children requires simple, inexpensive tests. We have evaluated HIV-1 p24 antigen for antiretroviral treatment (ART) monitoring in children. p24 by boosted enzyme-linked immunosorbent assay of heated plasma and HIV-1 RNA were measured prospectively in 24 HIV-1-infected children receiving ART. p24 and HIV-1 RNA concentrations and their changes between consecutive visits were related to the respective CD4+ changes. Age at study entry was 7.6 years; follow-up was 47.2 months, yielding 18 visits at an interval of 2.8 months (medians). There were 399 complete visit data sets and 375 interval data sets. Controlling for variation between individuals, there was a positive relationship between concentrations of HIV-1 RNA and p24 (P < 0.0001). While controlling for initial CD4+ count, age, sex, days since start of ART, and days between visits, the relative change in CD4+ count between 2 successive visits was negatively related to the corresponding relative change in HIV-1 RNA (P = 0.009), but not to the initial HIV-1 RNA concentration (P = 0.94). Similarly, we found a negative relationship with the relative change in p24 over the interval (P < 0.0001), whereas the initial p24 concentration showed a trend (P = 0.08). Statistical support for the p24 model and the HIV-1 RNA model was similar. p24 may be an accurate low-cost alternative to monitor ART in pediatric HIV-1 infection.

Modeling Breastmilk Infectivity in HIV-1 Infected Mothers

Estimation of breastmilk infectivity in HIV-1 infected mothers is difficult because transmission can occur while the fetus is in-utero, during delivery, or through breastfeeding. Since transmission can only be detected through periodic testing, however, it may be impossible to determine the actual mode of transmission in any individual child. In this paper we develop a model to estimate breastmilk infectivity as well as the probabilities of in-utero and intrapartum transmission. In addition, the model allows separate estimation of early and late breastmilk infectivity and individual variation in maternal infectivity. Methods for hypothesis testing of binary risk factors and a method for assessing goodness of fit are also described. Data from a randomized trial of breastfeeding versus formula feeding among HIV-1 infected mothers in Nairobi, Kenya are used to illustrate the methods.