An intensive, prospective, open-label pharmacokinetic (PK) study in a subset of HIV-infected mothers and their uninfected infants enrolled in the Breastfeeding, Antiretroviral, and Nutrition study was performed to describe drug exposure and antiviral response.
Women using Combivir®[zidovudine (ZDV)+ lamivudine (3TC)]+Aluvia®[lopinavir/ritonavir(LPV/RTV)] were enrolled. Breast milk (BM) and mother and infant plasma (MP, IP) samples were obtained over 6hrs after observed dosing at 6, 12, or 24wks post-partum for drug concentrations and HIV RNA.
30 mother/infant pairs (10 each at 6, 12,and 24wks post-partum) were enrolled. Relative to MP, BM concentrations of ZDV and 3TC were 35% and 21% higher, while LPV and RTV were 80% lower. Only 3TC was detected in IP with concentrations 96% and 98% lower than MP and BM, respectively. Concentrations in all matrices were similar at 6-24wks. The majority (98.3%) of BM concentrations were >HIVwt IC50, with one having detectable virus. There was no association between PK parameters and MP or BM HIV RNA.
ZDV and 3TC concentrated in BM while LPV and RTV did not, possibly due to protein binding and drug transporter affinity. Undetectable to low ARV concentrations in IP suggests prevention of transmission while breast feeding may be due to ARV effects on systemic or BM HIV RNA in the mother. Low IP 3TC exposure may predispose an infected infant to HIV resistance, necessitating testing and treating infants early.
Weight losses in lifestyle interventions are variable, yet prediction of long-term success is difficult.
We examined the utility of using various weight loss thresholds in the first 2 months of treatment for predicting 1-year outcomes.
Design and Methods
Participants included 2327 adults with type 2 diabetes (BMI:35.8±6.0) randomized to the intensive lifestyle intervention (ILI) of the Look AHEAD trial. ILI included weekly behavioral sessions designed to increase physical activity and reduce caloric intake. 1-month, 2-month, and 1-year weight changes were calculated.
Participants failing to achieve a ≥2% weight loss at Month 1 were 5.6 (95% CI:4.5,7.0) times more likely to also not achieve a ≥10% weight loss at Year 1, compared to those losing ≥2% initially. These odds were increased to 11.6 (95% CI:8.6,15.6) when using a 3% weight loss threshold at Month 2. Only 15.2% and 8.2% of individuals failing to achieve the ≥2% and ≥3% thresholds at Months 1 and 2 respectively, go on to achieve a ≥10% weight loss at Year 1.
Given the association between initial and 1-year weight loss, the first few months of treatment may be an opportune time to identify those who are unsuccessful and utilize rescue efforts.
weight loss; behavioral treatment; lifestyle intervention
Recommendations for diabetes prevention in patients with prediabetes include lifestyle modification and metformin. However, the significance of early weight loss and glucose measurements when monitoring response to these proven interventions is unknown.
To quantify the relationship between early measures of weight and glucose and subsequent diabetes in patients undergoing diabetes prevention interventions.
Analysis of results from a randomized controlled trial in 27 academic medical centers in the United States.
3,041 adults with hyperglycemia randomized to lifestyle (n = 1,018), metformin (n = 1,036), or placebo (n = 987) with complete follow-up in The Diabetes Prevention Program.
Independent variables were weight loss at 6 and 12 months; fasting glucose (FG) at 6 months; hemoglobin A1c (HbA1c) at 6 months; and post-load glucose at 12 months. The main outcome was time to diabetes diagnosis.
After 6 months, 604 participants developed diabetes in the lifestyle (n = 140), metformin (n = 206), and placebo (n = 258) arms over 2.7 years. In the lifestyle arm, 6-month weight loss predicted decreased diabetes risk in a graded fashion: adjusted HR (95 % CI) 0.65 (0.35–1.22), 0.62 (0.33–1.18), 0.46 (0.24–0.87), 0.34 (0.18–0.64), and 0.15 (0.07–0.30) for 0–<3 %, 3–<5 %, 5–<7 %, 7–<10 %, and ≥10 % weight loss, respectively (reference: weight gain). Attainment of optimal 6-month FG and HbA1c and 12-month post-load glucose predicted >60 % lower diabetes risk across arms. We found a significant interaction between 6-month weight loss and FG in the lifestyle arm (P = 0.038).
Weight and glucose at 6 and 12 months strongly predict lower subsequent diabetes risk with a lifestyle intervention; lower FG predicts lower risk even with substantial weight loss. Early reduction in glycemia is a stronger predictor of future diabetes risk than weight loss for metformin. We offer the first evidence to guide clinicians in making interval management decisions for high-risk patients undertaking measures to prevent diabetes.
Electronic supplementary material
The online version of this article (doi:10.1007/s11606-013-2548-4) contains supplementary material, which is available to authorized users.
diabetes prevention; diabetes risk; type 2 diabetes
The qualitative Roche HIV-1 DNA Amplicor assay has been used for the past 20 years to diagnose HIV infection in infants and young children but is being phased out; hence, alternative assays must be found. The Gen-Probe Aptima qualitative HIV-1 RNA assay is currently the only FDA-cleared HIV-1 nucleic acid assay approved for diagnosis, but data on the use of this assay with infant plasma are limited. We assessed Aptima's performance using control material for reproducibility and limit of detection and 394 plasma samples (0.2 to 0.5 ml) from HIV-exposed infected and uninfected infants and children for analytical sensitivity and specificity. Assays to assess within-run repeatability and between-run reproducibility indicated that the controls with 10,000 (5 of 5), 200 (5 of 5), 100 (16 of 16), 50 (12 of 12), and 25 (20 of 20) HIV-1 RNA copies/ml (cp/ml) were always positive, and negatives were always negative (20 of 20). The limit of detection was 14 cp/ml, as determined by probit analysis. The analytic sensitivity of the assay was 99.5% (189/190 samples; 95% confidence interval [CI], 97.1 to 99.9%) and specificity was 99.5% (199/200 samples; 95% CI, 97.2 to 99.9%). These results suggest that the assay is suitable for early infant diagnosis of HIV-1.
Sickle cell disease is recognized as a hypercoagulable state; however
the frequency and characteristics of venous thromboembolism in sickle cell
patients have not been well-defined.
To establish the prevalence and risk factors for venous
thromboembolism in a large cohort of patients with sickle cell disease and
determine the relationship between venous thromboembolism and mortality.
We performed a cross-sectional study of 404 sickle cell disease
patients cared for at the Sickle Cell Center for Adults at Johns Hopkins.
Demographic, sickle cell disease-specific comorbidity, and venous
thromboembolism data were collected on all patients.
101 patients (25%) had a history of venous thromboembolism
with a median age at diagnosis of 29.9 years. A history of
non-catheter-related venous thromboembolism was found in 18.8% of
patients. Sickle variant genotypes conferred a higher risk of
non-catheter-related venous thromboembolism compared to sickle cell anemia
genotypes (SS/Sβ0) (relative risk (RR) 1.77, 95%
confidence interval (CI) 1.18–2.66). Tricuspid regurgitant jet
velocity ≥ 2.5 m/s was also associated with non-catheter-related
venous thromboembolism (RR 1.65, CI 1.12–2.45). Thirty patients
(7.4%) died during the study period. Adjusting for all variables,
non-catheter-related venous thromboembolism was independently correlated
with death (RR 3.63, CI 1.66–7.92).
Venous thromboembolism is common in adults with sickle cell disease.
Sickle variant genotypes and tricuspid regurgitant jet velocity ≥
2.5m/s are associated with non-catheter-related venous thromboembolism. In
addition, non-catheter-related venous thromboembolism appears to be an
independent risk factor for death in our cohort. These results suggest that
disease-specific prophylaxis and treatment strategies for venous
thromboembolism should be investigated in sickle cell disease patients.
The p16INK4a tumor suppressor gene is a mediator of cellular senescence and has been suggested to be a biomarker of ‘molecular’ age in several tissues including T-cells. To determine the association of both active and suppressed HIV infection with T-cell aging, T-cell p16INK4a expression was compared between 60 HIV+ suppressed subjects, 23 HIV+ untreated subjects, and 18 contemporaneously collected HIV-negative controls, as well as 148 HIV-negative historical samples. Expression did not correlate with chronologic age in untreated HIV+ patients, consistent with an effect of active HIV replication on p16INK4a expression. In patients on cART with suppressed viral loads, however, p16INK4a levels were similar to uninfected controls and correlated with chronologic age, with a trend toward an inverse correlation with CD4 count. These data show that p16INK4a is a reliable biomarker of T cell aging in HIV+ patients with suppressed viral loads and suggest that poor CD4 cell recovery on cART may be associated with increased T-cell expression of p16INK4a, a marker of cellular senescence.
Determine the reliability of two different modified (MOD1 and MOD2) testing methods compared to a standard method (ST) for testing trunk flexion and extension endurance.
Twenty‐eight healthy individuals (age 26.4 ± 3.2 years, height 1.75 ± m, weight 71.8 ± 10.3 kg, body mass index 23.6 ± 3.4 m/kg2).
Trunk endurance time was measured in seconds for flexion and extension under the three different stabilization conditions. The MOD1 testing procedure utilized a female clinician (70.3 kg) and MOD2 utilized a male clinician (90.7 kg) to provide stabilization as opposed to the ST method of belt stabilization.
No significant differences occurred between flexion and extension times. Intraclass correlations (ICCs3,1) for the different testing conditions ranged from .79 to .95 (p <.000) and are found in Table 3. Concurrent validity using the ST flexion times as the gold standard coefficients were .95 for MOD1 and .90 for MOD2. For ST extension, coefficients were .91 and .80, for MOD1 and MOD2 respectively (p <.01).
These methods proved to be a reliable substitute for previously accepted ST testing methods in normal college‐aged individuals. These modified testing procedures can be implemented in athletic training rooms and weight rooms lacking appropriate tables for the ST testing.
Level of Evidence:
Core; stabilization; trunk endurance
It is well known that antioxidants have protective effects against oxidative stress. Unfortunately, in the presence of transition metals, antioxidants including polyphenols with potent antioxidant activities may also exhibit pro-oxidant effects, which may irreversibly damage DNA. Therefore, antioxidants with strong free radical scavenging abilities and devoid of pro-oxidant effects would be of immense biological importance. We report two antioxidant dendrimers with a surface rich in multiple phenolic hydroxyl groups, benzylic hydrogens and electron donating ring substituents that contribute to their potent free radical quenching property. In order to minimize their pro-oxidant effects, the dendrimers were designed with a metal chelating tris(2-aminoethyl)amine (TREN) core. The dendritic antioxidants were prepared by attachment of six syringaldehyde or vanillin molecules to TREN by reductive amination. They exhibited potent radical scavenging properties: 5 times stronger than quercetin and 15 times more potent than Trolox according to the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The antioxidant dendrimers also protected low-density lipoprotein, lysozyme and DNA against 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced free radical damage. More importantly, unlike quercetin and Trolox, the two TREN antioxidant dendrimers did not damage DNA via their pro-oxidant effects when incubated with physiological amounts of copper ions. The dendrimers also showed no cytotoxicity towards Chinese hamster ovary cells.
Polyphenolic dendrimer; antioxidant; pro-oxidant; lipoprotein; lysozyme; DNA
We assessed whether 7 days of zidovudine+lamivudine postpartum with single-dose nevirapine at labor decreases nevirapine resistance in HIV-infected women in Malawi.
HIV-infected pregnant women receiving intrapartum single-dose nevirapine and 7 days of zidovudine+lamivudine (n=132), and women receiving intrapartum single-dose nevirapine alone (n=66) were followed from an antenatal visit through 6 weeks postpartum. Plasma specimens at 2 and 6 weeks postpartum were tested for genotypic resistance to nevirapine by population sequencing and sensitive real-time PCR. Poisson regression was used to determine predictors of postpartum nevirapine resistance.
Median HIV RNA was similar at entry (4.27 log vs. 4.35 log, p=0.87), differed at 2 weeks postpartum (2.67 log vs. 3.58 log, p<0.0001), but not at 6 weeks postpartum (4.49 log vs. 4.40 log, p=0.79), between single-dose nevirapine/zidovudine+lamivudine and single-dose nevirapine groups, respectively. Nevirapine resistance, measured by population sequencing and sensitive real-time PCR, was significantly less common in those receiving single-dose nevirapine/zidovudine+lamivudine compared to single-dose nevirapine, respectively, at 2 weeks (10% (4/40) vs. 74% (31/42), p<0.0001) and 6 weeks postpartum [10% (11/115) vs. 64% (41/64), p<0.0001; adjusted relative risk=0.18, 95% confidence interval (0.10–0.34)].
The significant decrease in nevirapine resistance conferred by one week of zidovudine+lamivudine should help policymakers optimize peripartum HIV prophylaxis recommendations.
The ability of an AEF (axolemma-enriched fraction) to influence the proliferation, survival and differentiation of OPC (oligodendrocyte progenitor cells) was evaluated. Following addition of AEF to cultured OPC, the AEF associated with the outer surface of OPC so that subsequent metabolic events were likely mediated by direct AEF-OPC contact. Addition of AEF to the cultured OPC resulted in a dose- and time-dependent increase in proliferation that was partially dependent on Akt (protein kinase B) and MAPK (mitogen-activated protein kinase) activation. The major mitogen in an AEF-SE (soluble 2.0 M NaCl extract of the AEF) was identified as aFGF (acidic fibroblast growth factor) and accounted for 50% of the mitogenicity. The remaining 50% of the mitogenicity had properties consistent with bFGF (basic fibroblast growth factor) but was not unequivocally identified. Under conditions that limit the survival of OPC in culture, AEF treatment prolonged the survival of the OPC. Antigenic and morphological examination of the AEF-treated OPC indicated that the AEF treatment helped the OPC survive in a more immature state. The potential downstream metabolic pathways potentially activated in OPC by AEF and the consequences of these activated pathways are discussed. The results of these studies are consistent with the view that direct contact of axons with OPC stimulates their proliferation and survival while preventing their differentiation.
acidic fibroblast growth factor (aFGF); axolemma-enriched fraction (AEF); axonal–oligodendrocyte signalling; CNS trophic factors; multiple sclerosis; oligodendrocyte differentiation; oligodendrocyte progenitor cell; AEF, axolemma-enriched fraction; AEF-SE, soluble 2.0 M NaCl extract of the AEF; aFGF, acidic fibroblast growth factor; Akt, protein kinase B; BCA, bicinchoninic acid; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; CNPase, 2′,3′-cyclic nucleotide 3′-phosphodiesterase; CNS, central nervous system; CREB, cAMP-response-element-binding protein; DAPI, 4′,6-diamidino-2-phenylindole; F-12, Ham's F12 nutrient medium; DMEM–F12, Dulbecco's modified Eagle medium nutrient mixture F-12; DSHB, Developmental Studies Hybridoma Bank; ERK, extracellular-signal-regulated kinase; FCS, fetal calf serum; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; GalC, galactosylcerebroside; GFAP, glial fibrillary acidic protein; LIF, leukaemia inhibitory factor; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NRG, neuregulin; OPC, oligodendrocyte progenitor cell; RIPA buffer, radio immunoprecipitation assay buffer
Autophagy is a homeostatic mechanism of lysosomal degradation. Defective autophagy has been linked to various disorders such as impaired control of pathogens and neurodegeneration. Autophagy is regulated by a complex array of signaling pathways that act upstream of autophagy proteins. Little is known about the role of altered regulatory signaling in disorders associated with defective autophagy. In particular, it is not known if pathogens inhibit autophagy by modulation of upstream regulatory pathways. Cells infected with HIV-1 blocked rapamycin-induced autophagy and CD40-induced autophagic killing of Toxoplasma gondii in bystander (non-HIV-1 infected) macrophage/monocytic cells. Blockade of autophagy was dependent on Src-Akt and STAT3 triggered by HIV-1 Tat and IL-10. Neutralization of the upstream receptors VEGFR, β-integrin or CXCR4, as well as of HIV-1 Tat or IL-10 restored autophagy in macrophage/monocytic cells exposed to HIV-1-infected cells. Defective autophagic killing of T. gondii was detected in monocyte-derived macrophages from a subset of HIV-1+ patients. This defect was also reverted by neutralization of Tat or IL-10. These studies revealed that a pathogen can impair autophagy in non-infected cells by activating counter-regulatory pathways. The fact that pharmacologic manipulation of cell signaling restored autophagy in cells exposed to HIV-1-infected cells raises the possibility of therapeutic manipulation of cell signaling to restore autophagy in HIV-1 infection.
Over 150 000 Malawians have started antiretroviral therapy (ART), in which first-line therapy is stavudine/lamivudine/nevirapine. We evaluated drug resistance patterns among patients failing first-line ART on the basis of clinical or immunological criteria in Lilongwe and Blantyre, Malawi.
Patients meeting the definition of ART failure (new or progressive stage 4 condition, CD4 cell count decline more than 30%, CD4 cell count less than that before treatment) from January 2006 to July 2007 were evaluated. Among those with HIV RNA of more than 1000 copies/ml, genotyping was performed. For complex genotype patterns, phenotyping was performed.
Ninety-six confirmed ART failure patients were identified. Median (interquartile range) CD4 cell count, log10 HIV-1 RNA, and duration on ART were 68 cells/μl (23–174), 4.72 copies/ml (4.26–5.16), and 36.5 months (26.6–49.8), respectively. Ninety-three percent of samples had nonnucleoside reverse transcriptase inhibitor mutations, and 81% had the M184V mutation. The most frequent pattern included M184V and nonnucleoside reverse transcriptase inhibitor mutations along with at least one thymidine analog mutation (56%). Twenty-three percent of patients acquired the K70E or K65R mutations associated with tenofovir resistance; 17% of the patients had pan-nucleoside resistance that corresponded to K65R or K70E and additional resistance mutations, most commonly the 151 complex. Emergence of the K65R and K70E mutations was associated with CD4 cell count of less than 100 cells/μl (odds ratio 6.1) and inversely with the use of zidovudine (odds ratio 0.18). Phenotypic susceptibility data indicated that the nucleoside reverse transcriptase inhibitor backbone with the highest activity for subsequent therapy was zidovudine/lamivudine/tenofovir, followed by lamivudine/tenofovir, and then abacavir/didanosine.
When clinical and CD4 cell count criteria are used to monitor first-line ART failure, extensive nucleoside reverse transcriptase inhibitor and nonnucleoside reverse transcriptase inhibitor resistance emerges, with most patients having resistance profiles that markedly compromise the activity of second-line ART.
Africa; antiretroviral failure; public health approach; resistance; resource-limited setting
Transmitted drug resistance (TDR) limits antiretroviral options, complicating management of HIV-positive patients. HIV disproportionately affects the Southern United States (US), but available national estimates of TDR prevalence principally reflect large metropolitan centers outside this region.
The Duke/UNC Acute HIV Program has collected data on acute or recent HIV infections (ARHI) in North Carolina (NC) since 1998. Acute infections represent antibody-negative, RNA-positive subjects; recent infection was determined by history of HIV testing, or concordance between detuned ELISA and antibody avidity assays. Genotypic sequence data from the earliest collected pre-treatment plasma sample were analyzed with the Stanford HIV Database and screened for Surveillance Drug Resistance Mutations (SDRMs).
253 individuals with ARHI between 1998 and May 2007 had complete genotypic sequence data for analysis; 39.5% were acute infections, 78.7% were male, 64.8% were non-white, and 53.8% were men who have sex with men. The overall prevalence of TDR was 17.8%, with SDRMs for non-nucleoside reverse transcriptase inhibitors (NNRTIs) in 9.5% of the cohort. Mutations for nucleos(t)ide RT inhibitors (NRTIs) were detected in 7.5%, and for protease inhibitors (PIs) in 3.2%. K103N was the most common mutation (7.5%). Thymidine analogue mutations were found in 4.7% of samples; the most common PI SDRM was L90M (2.4%). Dual-or triple-class antiretroviral resistance was rare, encountered in only six samples (2.4%).
The prevalence of TDR in NC is similar to estimates from US metropolitan areas. These findings have implications for initial regimen selection and secondary prevention efforts outside of large, metropolitan HIV epicenters.
HIV Infections/epidemiology; HIV Infections/transmission; North Carolina/epidemiology; Drug resistance, viral; Antiretroviral therapy, highly active
Dried blood spots that had been stored ambiently for 3 to 6 years lost approximately 1 log10 of human immunodeficiency virus type 1 (HIV-1) RNA, but the majority could still be genotyped for resistance. Nevirapine resistance was found in 7/16 (43.5%) HIV-1-positive HIVNET 024 infants at 4 to 6 weeks, but no resistance was found at other time points.
The Gen-Probe Aptima human immunodeficiency virus type 1 (HIV-1) RNA assay was adapted for the diagnosis of HIV infection in infants by using dried blood spots. The assay was 99% sensitive (128/129) and 100% specific (162/162). This may prove useful in resource-limited settings, since it precludes the need for a phlebotomist and maintenance of a cold chain.
Modified protocols of two rapid tests were compared with a less sensitive (LS) (detuned) enzyme immunoassay (EIA) for their abilities to distinguish recent human immunodeficiency virus (HIV) seroconversion from long-term infections. The results for samples from 100 HIV-positive patient that had previously been tested by the Vironostika LS EIA had a 97% concordance with the results of the Determine HIV 1/2 assay and 93% concordance with those of the OraQuick HIV 1/2 assay.
Using DNA heteroduplex tracking assays, we characterized human immunodeficiency virus type 1 env V4/V5 genetic populations in multiple blood plasma samples collected over an average of 7 months from 24 chronically infected human subjects. We observed complex and dynamic V4/V5 genetic populations in most subjects. Comparisons of V4/V5 and V1/V2 population changes over the course of the study showed that major shifts in genetic populations frequently occurred in one region but not the other, and these observations were independently confirmed in one subject by single-genome sequencing. These results suggest that the V1/V2 and V4/V5 regions of env often evolve independently during chronic infection.
We examined the rates of variant population turnover of the V1-V2 and V4-V5 hypervariable domains of the human immunodeficiency virus type 1 (HIV-1) gp120 molecule in longitudinal plasma samples from 14 men with chronic HIV-1 infection using heteroduplex tracking assays (HTA). Six men had high rates of CD4+ T-cell loss, and eight men had low rates of CD4+ T-cell loss over 2.5 to 8 years of infection. We found that V1-V2 and V4-V5 env populations changed dramatically over time in all 14 subjects; the changes in these regions were significantly correlated with each another over time. The subjects with rapid CD4 loss had significantly less change in their env populations than the subjects with slow CD4 loss. The two subjects with rapid CD4 loss and sustained low CD4 counts (<150/μl for at least 2 years) showed stabilization of their V1-V2 and V4-V5 populations as reflected by low levels of total change in HTA pattern and low HTA indices (a novel measure of the emergence of new bands and band distribution); this stabilization was not observed in other subjects. The stabilization of env variant populations at low CD4 counts following periods of rapid viral evolution suggests that selective pressure on env, likely from new immune responses, is minimal when CD4 counts drop dramatically and remain low for extended periods of time.
The initiation of drug therapy or the addition of a new drug to preexisting therapy can have a significant impact on human immunodeficiency virus type 1 (HIV-1) populations within a person. Drug therapy directed at reverse transcriptase and protease can result in dramatic decreases in virus load, causing a contraction in the virus population that represents a potential genetic bottleneck as a subset of virus with genomes carrying resistance mutations repopulate the host. While this bottleneck exerts an effect directly on the region that is being targeted by the drugs, it also affects other regions of the viral genome. We have applied heteroduplex tracking assays (HTA) specific to variable regions 1 and 2 (V1/V2) and variable region 3 (V3) of the HIV-1 env gene to analyze the effect of a genetic bottleneck created by the selection of resistance to ritonavir, a protease inhibitor. Subjects were classified into groups on the basis of the extent of the initial drop in virus load and the duration of virus load reduction. Subjects with a strong initial drop in virus load exhibited a loss of heterogeneity in the env region at virus load rebound; in contrast, subjects with a weak initial drop in virus load exhibited little to no loss of heterogeneity at virus load rebound in either region of env examined. The duration of virus load reduction also affected env populations. Subjects that had prolonged reductions exhibited slower population diversification and the appearance of new V1/V2 species after rebound. The longer reduction of virus load in these subjects may have allowed for improved immune system function, which in turn could have selected for new escape mutants. Subjects with rapid rebound quickly reequilibrated the entry env variants back into the resistant population. When the pro gene developed further resistance mutations subsequent to virus load rebound, no changes were observed in V1/V2 or V3 populations, suggesting that the high virus loads allowed the env populations to reequilibrate rapidly. The rapid equilibration of env variants during pro gene sequence transitions at high virus load suggests that recombination is active in defining the HIV-1 sequence population. Conversely, part of the success of suppressive antiviral therapy may be to limit the potential for evolution through recombination, which requires dually infected cells.
We recently isolated from an infant an X4-syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) variant (92US143-T8) that was able to infect CD8+ lymphocytes independently of CD4. Although it was CD4 independent, the 92US143-T8 isolate also maintained the ability to infect CD4+ cells. In the present study, we investigated the role of CXCR4 in the infection of CD4+ and CD8+ cells by this primary isolate. The expression of CXCR4 was down modulated in CD8+ lymphocytes after infection with the 93US143-T8 isolate. Infection of CD8+ lymphocytes by the 93US143-T8 isolate was prevented by treatment with AMD3100, a specific antagonist for CXCR4, indicating CXCR4-dependent infection. Interestingly, AMD3100 treatment had no inhibitory role in the infection of purified CD4+ lymphocytes by the same isolate. Furthermore, AMD3100 treatment failed to prevent infection of known CD4+ CXCR4+ T-cell lines (MT-2 and CEM) by the 93US143-T8 isolate. In fact, virus replication in the CD4+ cells was often enhanced in the presence of AMD3100. Viruses produced from the infected CD4+ cells in the presence of AMD3100 maintained an unchanged envelope genotype and an SI phenotype. For the first time, these results provide evidence of CXCR4-dependent infection of CD8+ lymphocytes by a primary HIV-1 isolate. This study also shows a different mode of infection for the CD4+ and CD8+ lymphocytes by the same HIV-1 variant. Finally, our findings suggest that a more careful evaluation is necessary before the random use of AMD3100 as a new entry inhibitor in patients harboring SI HIV-1 strains.
Human immunodeficiency virus type 1 (HIV-1) exists as a complex population of multiple genotypic variants in persons with chronic infection. However, acute HIV-1 infection via sexual transmission is a low-probability event in which there is thought to be low genetic complexity in the initial inoculum. In order to assess the viral complexity present during primary HIV-1 infection, the V1/V2 and V3 variable regions of the env gene were examined by using a heteroduplex tracking assay (HTA) capable of resolving these genotypic variants. Blood plasma samples from 26 primary HIV-1-infected subjects were analyzed for their level of diversity. Half of the subjects had more than one V1/V2 viral variant during primary infection, indicating the frequent transmission of multiple variants. This observation is inconsistent with the idea of infrequent transmission based on a small transmitting inoculum of cell-free virus. In chronically infected subjects, the complexity of the viral populations was even greater in both the V1/V2 and the V3 regions than in acutely infected subjects, indicating that in spite of the presence of multiple variants in acute infection, the virus does pass through a genetic bottleneck during transmission. We also examined how well the infecting virus penetrated different anatomical compartments by using the HTA. Viral variants detected in blood plasma were compared to those detected in seminal plasma and/or cerebral spinal fluid of six individuals. The virus in each of these compartments was to a large extent identical to virus in blood plasma, a finding consistent with rapid penetration of the infecting variant(s). The low-probability transmission of multiple variants could be the result of transient periods of hyperinfectiousness or hypersusceptibility. Alternatively, the inefficient transfer of a multiply infected cell could account for both the low probability of transmission and the transfer of multiple variants.
Previous studies have established the existence of CD4-independent simian immunodeficiency virus, human immunodeficiency virus type 2 (HIV-2), and laboratory strains of HIV-1. However, whether CD4-independent viruses may also exist in HIV-1-infected patients has remained unclear. We have recently reported the isolation of viruses from an AIDS patient that were able to infect CD8+ cells independent of CD4, using CD8 as a receptor. Using a similar approach, here we examined viruses from 12 randomly selected patients (obtained from the AIDS Research and Reference Program, National Institutes of Health) for the presence of CD4-independent HIV-1. CD4-independent variants were isolated from infected CD8+ cells from the viral quasispecies of 7 of 12 patients. The CD4-independent isolates were able to infect primary CD8+ cells as well as a CD4− CD8+ T-cell line. Soluble CD4 and blocking anti-CD4 or -CD8 antibody had no effect on infection of CD8+ cells. Remarkably, two of the seven CD4-independent isolates, but not their parental bulk viruses, induced syncytia and caused acute death of infected CD8+ cells. Some of the CD4-independent variants were also able to infect U87 cells that were negative for CD4, CD8, and common HIV coreceptors, suggesting a novel entry mechanism for these isolates. The CD4-independent isolates were derived from adults and children infected with subtypes A, B, and D. Although no common motif for CD4 independence was found, novel sequence changes were observed in critical areas of the envelopes of the CD4-independent viruses. These results demonstrate that HIV-1-infected patients can frequently harbor viruses that are able to mediate CD4-independent infection of CD8+ cells. In addition, this study also provides evidence of primary HIV-1 variants that are syncytium inducing and acutely cytopathic for CD8+ lymphocytes.
The env gene of human immunodeficiency virus type 1 (HIV-1) includes some of the most genetically diverse regions of the viral genome, which are called variable regions 1 through 5 (V1 through V5). We have developed a heteroduplex tracking assay to detect changes in variable regions 1 and 2 of env (V1/V2-HTA). Using sequences from two molecular clones as probes, we have studied the nature of longitudinal virus population changes in a cohort of HIV-1-infected subjects. Viral sequences present in 21 subjects with late-stage HIV-1 infection were initially screened for stability of the virus population by V1/V2-HTA. The virus populations at entry comprised an average of five coexisting V1/V2 genotypic variants (as identified by HTA). Eight of the 21 subjects were examined in detail because of the dynamic behavior of their env variants over an approximately 9-month period. In each of these cases we detected a single discrete transition of V1/V2 genotypes based on monthly sampling. The major V1/V2 genotypes (those present at >10% abundance) from the eight subjects were cloned and sequenced to define the nature of V1/V2 variability associated with a discrete transition. Based on a comparison of V1/V2 genotypic variants present at entry with the newly emerged variants we categorized the newly emerged variants into two groups: variants without length differences and variants with length differences. Variants without length differences had fewer nucleotide substitutions, with the changes biased to either V1 or V2, suggestive of recent evolutionary events. Variants with length differences included ones with larger numbers of changes that were distributed, suggestive of recall of older genotypes. Most length differences were located in domains where the codon motif AVT (V = A, G, C) had become enriched and fixed. Finally, recombination events were detected in two subjects, one of which resulted in the reassortment of V1 and V2 regions. We suggest that turnover in V1/V2 populations was largely driven by selection on either V1 or V2 and that escape was accomplished either through changes focused in the region under selection or by the appearance of a highly divergent variant.