IL-21 is associated with natural resistance to HIV-1 infection in a Colombian HIV exposed seronegative cohort

Higher IL-21 levels were associated with natural resistance to HIV infection in an Italian cohort. Thus we wanted to confirm such association in HIV exposed seronegative individuals (HESN) from Colombia. Cells from HESN were less susceptible to infection and expressed higher IL-21 mRNA levels than healthy controls at both baseline and 7-days post-infection; similar results were observed for IL-6, perforin, and granzyme. These results suggest that IL-21/IL-6 increase may be a distinctive quality in the profile of HIV-1 resistance, at least during sexual exposure. However, further studies are necessary to confirm the specific protective mechanisms of these cytokines.     

A Regulatory Polymorphism in HAVCR2 Modulates Susceptibility to HIV-1 Infection

The HAVCR2 gene encodes TIM-3, an immunoglobulin superfamily member expressed by exhausted CD8+ T cells during chronic viral infection. We investigated whether genetic variation at HAVCR2 modulates the susceptibility to HIV-1 acquisition; specifically we focused on a 3′ UTR variant (rs4704846, A/G) that represents a natural selection target. We genotyped rs4704846 in three independent cohorts of HIV-1 exposed seronegative (HESN) individuals with different geographic origin (Italy and Spain) and distinct route of exposure to HIV-1 (sexual and injection drug use). Matched HIV-1 positive subjects and healthy controls were also analyzed. In all case-control cohorts the minor G allele at rs4704846 was more common in HIV-1 infected individuals than in HESN, with healthy controls showing intermediate frequency. Results from the three association analyses were combined through a random effect meta-analysis, which revealed no heterogeneity among samples (Cochrane''s Q, p value =  0.89, I² =  0) and yielded a p value of 6.8 ×10⁻⁴. The minor G allele at rs4704846 was found to increase HAVCR2 expression after in vitro HIV-1 infection. Thus, a positively selected polymorphism in the 3′ UTR, which modulates HAVCR2 expression, is associated with the susceptibility to HIV-1 infection. These data warrant further investigation into the role of TIM-3 in the prevention and treatment of HIV-1/AIDS.     

Disparate associations of HLA class I markers with HIV-1 acquisition and control of viremia in an African population

Background

Acquisition of human immunodeficiency virus type 1 (HIV-1) infection is mediated by a combination of characteristics of the infectious and the susceptible member of a transmission pair, including human behavioral and genetic factors, as well as viral fitness and tropism. Here we report on the impact of established and potential new HLA class I determinants of heterosexual HIV-1 acquisition in the HIV-1-exposed seronegative (HESN) partners of serodiscordant Zambian couples.

Methodology/Principal Findings

We assessed the relationships of behavioral and clinically documented risk factors, index partner viral load, and host genetic markers to HIV-1 transmission among 568 cohabiting couples followed for at least nine months. We genotyped subjects for three classical HLA class I genes known to influence immune control of HIV-1 infection. From 1995 to December 2006, 240 HESNs seroconverted and 328 remained seronegative. In Cox proportional hazards models, HLA-A*68:02 and the B*42-C*17 haplotype in HESN partners were significantly and independently associated with faster HIV-1 acquisition (relative hazards = 1.57 and 1.55; p = 0.007 and 0.013, respectively) after controlling for other previously established contributing factors in the index partner (viral load and specific class I alleles), in the HESN partner (age, gender), or in the couple (behavioral and clinical risk score). Few if any previously implicated class I markers were associated here with the rate of acquiring infection.

Conclusions/Significance

A few HLA class I markers showed modest effects on acquisition of HIV-1 subtype C infection in HESN partners of discordant Zambian couples. However, the striking disparity between those few markers and the more numerous, different markers found to determine HIV-1 disease course makes it highly unlikely that, whatever the influence of class I variation on the rate of infection, the mechanism mediating that phenomenon is identical to that involved in disease control.     

A common polymorphism in TLR3 confers natural resistance to HIV-1 infection

TLR3 recognizes dsRNA and activates antiviral immune responses through the production of inflammatory cytokines and type I IFNs. Genetic association studies have provided evidence concerning the role of a polymorphism in TLR3 (rs3775291, Leu412Phe) in viral infection susceptibility. We genotyped rs3775291 in a population of Spanish HIV-1-exposed seronegative (HESN) individuals who remain HIV seronegative despite repeated exposure through i.v. injection drug use (IDU-HESN individuals) as witnessed by their hepatitis C virus seropositivity. The frequency of individuals carrying at least one 412Phe allele was significantly higher in IDU-HESN individuals compared with that of a matched control sample (odds ratio for a dominant model = 1.87; 95% confidence interval, 1.06-3.34; p = 0.023). To replicate this finding, we analyzed a cohort of Italian, sexually HESN individuals. Similar results were obtained: the frequency of individuals carrying at least one 412Phe allele was significantly higher compared with that of a matched control sample (odds ratio, 1.79; 95% confidence interval, 1.05-3.08; p = 0.029). In vitro infection assays showed that in PBMCs carrying the 412Phe allele, HIV-1(Ba-L) replication was significantly reduced (p = 0.025) compared with that of Leu/Leu homozygous samples and was associated with a higher expression of factors suggestive of a state of immune activation (IL-6, CCL3, CD69). Similarly, stimulation of PBMCs with a TLR3 agonist indicated that the presence of the 412Phe allele results in a significantly increased expression of CD69 and higher production of proinflammatory cytokines including IL-6 and CCL3. The data of this study indicate that a common TLR3 allele confers immunologically mediated protection from HIV-1 and suggest the potential use of TLR3 triggering in HIV-1 immunotherapy.     

Involvement of Neutrophil Hyporesponse and the Role of Toll-Like Receptors in Human Immunodeficiency Virus 1 Protection

Objectives

Neutrophils contribute to pathogen clearance through pattern recognition receptors (PRRs) activation. However, the role of PRRs in neutrophils in both HIV-1-infected [HIV-1(+)] and HIV-1-exposed seronegative individuals (HESN) is unknown. Here, a study was carried out to evaluate the level of PRR mRNAs and cytokines produced after activation of neutrophils from HIV-1(+), HESN and healthy donors.

Methods

The neutrophils were stimulated with specific agonists for TLR2, TLR4 and TLR9 in the presence of HIV-1 particles. Pro-inflammatory cytokine production, expression of neutrophil activation markers and reactive oxygen species (ROS) production were analyzed in neutrophils from HESN, HIV-1(+) and healthy donors (controls).

Results

We found that neutrophils from HESN presented reduced expression of PRR mRNAs (TLR4, TLR9, NOD1, NOD2, NLRC4 and RIG-I) and reduced expression of cytokine mRNAs (IL-1β, IL-6, IL-18, TNF-α and TGF-β). Moreover, neutrophils from HESN were less sensitive to stimulation through TLR4. Furthermore, neutrophils from HESN challenged with HIV-1 and stimulated with TLR2 and TLR4 agonists, produced significantly lower levels of reactive oxygen species, versus HIV-1(+).

Conclusions

A differential pattern of PRR expression and release of innate immune factors in neutrophils from HESN is evident. Our results suggest that lower neutrophil activation can be involved in protection against HIV-1 infection.     

Characteristics of HIV-1 serodiscordant couples enrolled in a clinical trial of antiretroviral pre-exposure prophylaxis for HIV-1 prevention

Introduction

Stable heterosexual HIV-1 serodiscordant couples in Africa have high HIV-1 transmission rates and are a critical population for evaluation of new HIV-1 prevention strategies. The Partners PrEP Study is a randomized, double-blind, placebo-controlled trial of tenofovir and emtricitabine-tenofovir pre-exposure prophylaxis to decrease HIV-1 acquisition within heterosexual HIV-1 serodiscordant couples. We describe the trial design and characteristics of the study cohort.

Methods

HIV-1 serodiscordant couples, in which the HIV-1 infected partner did not meet national guidelines for initiation of antiretroviral therapy, were enrolled at 9 research sites in Kenya and Uganda. The HIV-1 susceptible partner was randomized to daily oral tenofovir, emtricitabine-tenofovir, or matching placebo with monthly follow-up for 24–36 months.

Results

From July 2008 to November 2010, 7920 HIV-1 serodiscordant couples were screened and 4758 enrolled. For 62% (2966/4758) of enrolled couples, the HIV-1 susceptible partner was male. Median age was 33 years for HIV-1 susceptible and HIV-1 infected partners [IQR (28–40) and (26–39) respectively]. Most couples (98%) were married, with a median duration of partnership of 7.0 years (IQR 3.0–14.0) and recent knowledge of their serodiscordant status [median 0.4 years (IQR 0.1–2.0)]. During the month prior to enrollment, couples reported a median of 4 sex acts (IQR 2–8); 27% reported unprotected sex and 14% of male and 1% of female HIV-1 susceptible partners reported sex with outside partners. Among HIV-1 infected partners, the median plasma HIV-1 level was 3.94 log₁₀ copies/mL (IQR 3.31–4.53) and median CD4 count was 496 cells/µL (IQR 375–662); the majority (64%) had WHO stage 1 HIV-1 disease.

Conclusions

Couples at high risk of HIV-1 transmission were rapidly recruited into the Partners PrEP Study, the largest efficacy trial of oral PrEP. (ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00557245","term_id":"NCT00557245"}}NCT00557245)     

Microarray analysis of HIV resistant female sex workers reveal a gene expression signature pattern reminiscent of a lowered immune activation state

To identify novel biomarkers for HIV-1 resistance, including pathways that may be critical in anti-HIV-1 vaccine design, we carried out a gene expression analysis on blood samples obtained from HIV-1 highly exposed seronegatives (HESN) from a commercial sex worker cohort in Nairobi and compared their profiles to HIV-1 negative controls. Whole blood samples were collected from 43 HIV-1 resistant sex workers and a similar number of controls. Total RNA was extracted and hybridized to the Affymetrix HUG 133 Plus 2.0 micro arrays (Affymetrix, Santa Clara CA). Output data was analysed through ArrayAssist software (Agilent, San Jose CA). More than 2,274 probe sets were differentially expressed in the HESN as compared to the control group (fold change ≥1.3; p value ≤0.0001, FDR <0.05). Unsupervised hierarchical clustering of the differentially expressed genes readily distinguished HESNs from controls. Pathway analysis through the KEGG signaling database revealed a majority of the impacted pathways (13 of 15, 87%) had genes that were significantly down regulated. The most down expressed pathways were glycolysis/gluconeogenesis, pentose phosphate, phosphatidyl inositol, natural killer cell cytotoxicity and T-cell receptor signaling. Ribosomal protein synthesis and tight junction genes were up regulated. We infer that the hallmark of HIV-1 resistance is down regulation of genes in key signaling pathways that HIV-1 depends on for infection.     

The VNTR Polymorphism of the DC-SIGNR Gene and Susceptibility to HIV-1 Infection: A Meta-Analysis

Background

Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin related (DC-SIGNR) can bind to the human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein and is thus important for the host-pathogen interaction in HIV-1 infection. Studies of the association between the variable number tandem repeat (VNTR) polymorphism of the DC-SIGNR gene and HIV-1 susceptibility have produced controversial results.

Methods and Findings

We conducted a meta-analysis of the data contained in the literature to clarify these findings. In total, 10 studies consisting of 2683 HIV-1 patients and 3263 controls (2130 healthy controls and 1133 HIV-1 exposed but seronegative (HESN) controls) were included. Odds ratios (ORs) with 95% confidence intervals (95% CIs) were assessed in the main analyses. Further stratified analyses by ethnicity and sample size were performed. By dividing the controls into two groups, healthy controls and HIV-1 exposed but seronegative (HESN) controls, we explored different genetic models to detect any association between the VNTR polymorphism and predisposition to HIV-1 infection. The results showed that the 5-repeat allele carriers (OR = 0.84, 95% CI = 0.73–0.96) and the 5/5 homozygous (OR = 0.68, 95% CI = 0.50–0.93) had significantly reduced risk when using the HIV-1 exposed but seronegative (HESN) as controls. The stratified analyses by ethnicity and sample size confirmed these findings. However, a low to moderate degree of heterogeneity was also found across studies.

Conclusions

Our findings demonstrate that the VNTR polymorphism of the DC-SIGNR gene is associated with a moderate effect on host susceptibility to HIV-1 infection. Similar to the 32-bp deletion in the chemokine receptor-5 gene (CCR5Δ32), the DC-SIGNR VNTR 5-repeat allele might have a role in resistance to HIV infection, particularly in Asian populations.     

Human immunodeficiency virus type 1 superinfection was not detected following 215 years of injection drug user exposure

Evidence for human immunodeficiency virus type 1 (HIV-1) superinfection was sought among 37 HIV-1-positive street-recruited active injection drug users (IDUs) from the San Francisco Bay area. HIV-1 sequences from pairs of samples collected 1 to 12 years apart, spanning a total of 215 years of exposure, were generated at p17 gag, the V3-V5 region of env, and/or the first exon of tat and phylogenetically analyzed. No evidence of HIV-1 superinfection was detected in which a highly divergent HIV-1 variant emerged at a frequency >20% of the serum viral quasispecies. Based on the reported risk behavior of the IDUs and the HIV-1 incidence in uninfected subjects in the same cohort, a total of 3.4 new infections would have been expected if existing infection conferred no protection from superinfection. Adjusted for risk behaviors, the estimated relative risk of superinfection compared with initial infection was therefore 0.0 (95% confidence interval, 0.00, 0.79; P = 0.02), indicating that existing infection conferred a statistically significant level of protection against superinfection with an HIV-1 strain of the same subtype, which was between 21 and 100%.     

Haptoglobin phenotypes in HIV-1-seropositive patients in Ghana: decreased risk for Hp0 individuals

Plasma haptoglobin phenotypes were determined by polyacrylamide gel electrophoresis, followed by benzidine staining for 58 HIV-1 seropositive Ghanaians and 79 randomly selected age-matched controls. Hp0 was present in only 14% of HIV-1 seropositive individuals compared with more than 40% of the controls. The Hp0 individuals showed a highly significant reduced risk for HIV-1 infection (OR = 0. 21, 95% CI = 0.09-0.51, p = 0.0002). Hp0 may have a protective effect in HIV-1 infection.     

Altered Monocyte Phenotype in HIV-1 Infection Tends to Normalize with Integrase-Inhibitor-Based Antiretroviral Therapy

Background

Monocytes are increasingly implicated in the inflammatory consequences of HIV-1 disease, yet their phenotype following antiretroviral therapy (ART) initiation is incompletely defined. Here, we define more completely monocyte phenotype both prior to ART initiation and during 48 weeks of ART.

Methods

Cryopreserved peripheral blood mononuclear cells (PBMCs) were obtained at baseline (prior to ART initiation) and at weeks 12, 24, and 48 of treatment from 29 patients participating in ACTG clinical trial A5248, an open label study of raltegravir/emtricitibine/tenofovir administration. For comparison, cryopreserved PBMCs were obtained from 15 HIV-1 uninfected donors, each of whom had at least two cardiovascular risk factors. Thawed samples were stained for monocyte subset markers (CD14 and CD16), HLA-DR, CCR2, CX3CR1, CD86, CD83, CD40, CD38, CD36, CD13, and CD163 and examined using flow cytometry.

Results

In untreated HIV-1 infection there were perturbations in monocyte subset phenotypes, chiefly a higher frequency and density (mean fluorescence intensity–MFI) of HLA-DR (%-p = 0.004, MFI-p = .0005) and CD86 (%-p = 0.012, MFI-p = 0.005) expression and lower frequency of CCR2 (p = 0.0002) expression on all monocytes, lower CCR2 density on inflammatory monocytes (p = 0.045) when compared to the expression and density of these markers in controls’ monocytes. We also report lower expression of CX3CR1 (p = 0.014) on patrolling monocytes at baseline, compared to levels seen in controls. After ART, these perturbations tended to improve, with decreasing expression and density of HLA-DR and CD86, increasing CCR2 density on inflammatory monocytes, and increasing expression and density of CX3CR1 on patrolling monocytes.

Conclusions

In HIV-1 infected patients, ART appears to attenuate the high levels of activation (HLA-DR, CD86) and to increase expression of the chemokine receptors CCR2 and CX3CR1 on monocyte populations. Circulating monocyte phenotypes are altered in untreated infection and tend to normalize with ART; the role of these cells in the inflammatory environment of HIV-1 infection warrants further study.     

High Transcript Levels of Vitamin D Receptor Are Correlated with Higher mRNA Expression of Human Beta Defensins and IL-10 in Mucosa of HIV-1-Exposed Seronegative Individuals

Vitamin D (VitD) is an endogenous immunomodulator that could protect from HIV-1 infection reducing immune activation and inducing the expression of anti-HIV-1 peptides. To establish a correlation between VitD and natural resistance to HIV-1 infection, a case-control study using blood and mucosa samples of 58 HIV-1-exposed but seronegative (HESN) individuals, 43 HIV-1 seropositives (SPs) and 59 non-exposed healthy controls (HCs) was carried out. The VitD concentration in plasma was determined by ELISA, and mRNA relative units (RU) of VDR, IL-10, TGF-β, TNF-α and IL-1β in peripheral blood mononuclear cells (PBMCs), oral and genital mucosa was quantified by qRT-PCR. mRNA levels of human beta-defensin (HBD) -2 and -3 were previously reported and used for correlations. Significantly higher levels of VitD were found in plasma as well as higher mRNA RU of VDR in PBMCs, and in genital mucosa from HESN compared to HCs. In addition, higher mRNA RU of TNF-α, IL-1β and IL-10, and lower mRNA RU of TGF-β were found in PBMC from HESNs compared to HCs. We also observed higher IL-10 mRNA RU in genital mucosa of HESNs compared to HCs, and the mRNA levels of TNF-α in oral and genital mucosa of SPs were higher compared to HESNs. Furthermore, positive correlations between VDR and IL-10 mRNA RU in PBMCs and genital mucosa of HESNs were found. Finally, HBD-2 and HBD-3 mRNA RU were positively correlated with VDR mRNA expression in oral mucosa from HESNs. These results suggest that high levels of VitD and its receptor are associated with natural resistance to HIV-1 infection. Up-regulation of the anti-inflammatory IL-10, and the induction of anti-HIV-1 defensins in mucosa might be part of the mechanisms involved in this association. However, further studies are required to define causal associations.     

Infectious Cellular Load in Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals and Susceptibility of Peripheral Blood Mononuclear Cells from Their Exposed Partners to Non-Syncytium-Inducing HIV-1 as Major Determinants for HIV-1 Transmission in Homosexual Couples

To study risk factors for homosexual transmission of human immunodeficiency virus type 1 (HIV-1), we compared 10 monogamous homosexual couples between whom transmission of HIV-1 had occurred with 10 monogamous homosexual couples between whom HIV-1 transmission had not occurred despite high-risk sexual behavior. In the group of individuals who did not transmit virus, peripheral cellular infectious load was lower and the CD4⁺ T-cell counts were higher than in the group of transmitters. HIV-1 RNA levels in serum did not differ between transmitters and nontransmitters. Compared with peripheral blood mononuclear cells (PBMC) from normal healthy blood donors, 8 of 10 nonrecipients and only 3 of 8 recipients had PBMC with reduced susceptibility to in vitro infection with non-syncytium-inducing (NSI) HIV-1 variants isolated from either their respective partners or an unrelated individual. No difference in susceptibility was observed for infection with a syncytium-inducing variant. Among the individuals who had PBMC with reduced susceptibility, five nonrecipients and one recipient had PBMC that were equally or even less susceptible to NSI variants than PBMC that had low susceptibility and that were derived from healthy blood donors that were heterozygous for a 32-bp deletion in the CCR5 gene (CCR5 Δ32). Three of these individuals (all nonrecipients) had a CCR5 Δ32 heterozygous genotype themselves, confirming an association between low susceptibility to NSI variants and CCR5 Δ32 heterozygosity. All three recipients with less susceptible PBMC had partners with a high infectious cellular load; inversely, both nonrecipients with normally susceptible PBMC had partners with a very low infectious cellular load. These results suggest that a combination of susceptibility of target cells and inoculum size upon homosexual exposure largely determines whether HIV-1 infection is established.     

CC chemokine receptor 5 cell-surface expression in relation to CC chemokine receptor 5 genotype and the clinical course of HIV-1 infection.

CCR5 cell-surface expression was studied in relation to CCR5 genotype and clinical course of HIV-1 infection. HIV-1 infected CCR5+/+ individuals had higher percentages of CCR5-expressing CD4+ T cells as compared with HIV-1-infected CCR532/+ individuals. For both genotypic groups, the percentages of CCR5-expressing cells were higher than for the uninfected counterparts (CCR5+/+, HIV+ 28% and HIV- 15% (p < 0.0001); CCR532/+, HIV+ 21% and HIV- 10% (p = 0.001), respectively). In HIV-1-infected individuals, high percentages of CCR5-expressing cells were associated with low CD4+ T cell numbers (p = 0.001), high viral RNA load in serum (p = 0.046), and low T cell function (p = 0.054). As compared with nonprogressors with similar CD4+ T cell numbers, individuals who did progress to AIDS had a higher percentage of CCR5-expressing CD4+ T cells (32% vs 21% (p = 0.002). Longitudinal analysis of CCR5+/+ individuals revealed slight, although not statistically significant, increases in CCR5-expressing CD4+ T cells and CD4+ T cell subsets characterized by the expression of CD45 isoforms, during the course of HIV-1 infection. Preseroconversion, the percentage of CCR5-expressing CD4+ T cells was higher in individuals who subsequently developed AIDS (28%) than in those who did not show disease progression within a similar time frame (20%; p = 0.059). Our data indicate that CCR5 expression increases with progression of disease, possibly as a consequence of continuous immune activation associated with HIV-1 infection. In turn, CCR5 expression may influence the clinical course of infection.     

For protection from HIV-1 infection, more might not be better: a systematic analysis of HIV Gag epitopes of two alleles associated with different outcomes of HIV-1 infection

A subset of women in the Pumwani Sex Worker Cohort, established in 1985 in Nairobi, Kenya, remains uninfected despite repeated high-risk exposure (HIV-exposed, seronegative [HESN]) through active sex work. This HESN phenotype is associated with several alleles of human leukocyte antigens (HLAs) and specific CD8(+) and CD4(+) T cell responses to HIV-1. The associations of HLA alleles with differential HIV-1 infection are most likely due to their different abilities to present antigen and the different immune responses they induce. The characteristics of epitopes of HLA alleles associated with different outcomes of HIV-1 infection might therefore point to a vital clue for developing an effective vaccine. In this study, we systematically analyzed HIV-1 clade A and D Gag CD8(+) T cell epitopes of two HLA class I alleles associated with different outcomes of HIV-1 infection. Binding affinity and off-rates of the identified epitopes were determined. Gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assays with patient peripheral blood mononuclear cells (PBMCs) validated the epitopes. Epitope-specific CD8(+) T cells were further phenotyped for memory markers with tetramer staining. Our study showed that the protective allele A*01:01 recognizes only three Gag epitopes. By contrast, B*07:02, the allele associated with susceptibility, binds 30 epitope variants. These two alleles differ most importantly in the spectrum of Gag epitopes they can present and not in affinity, off-rates, the location of the epitopes, or epitope-specific Tem/Tcm frequencies. The binding of more epitopes and strong IFN-gamma ELISpot responses are associated with susceptibility to HIV-1 infection, while more focused antigen recognition of multiple subtypes is protective. Rational vaccine design should take these observations into account.     

A 6-amino acid insertion/deletion polymorphism in the mucin domain of TIM-1 confers protections against HIV-1 infection

We investigated whether a 6-amino acid insertion/deletion polymorphism in the mucin domain of TIM-1 (T-cell immunoglobulin and mucin domain 1), modulates susceptibility to HIV-1 infection. The polymorphism was genotyped in three case/control cohorts of HIV-1 exposed seronegative individuals (HESN) and HIV-1 infected subjects from Italy, Peru, and Colombia; data from a Thai population were retrieved from the literature. Across all cohorts, homozygosity for the short TIM-1 allele was more common in HESNs than in HIV-1 infected subjects. A meta-analysis of the four association analyses yielded a p value of 0.005. In vitro infection assays of CD4+ T lymphocytes indicated that homozygosity for the short allele is associated with lower rate of HIV-1 replication. These results suggest that the deletion allele protects from HIV-1 infection with a recessive effect.     

Functional correlation between a novel amino acid insertion at codon 19 in the protease of human immunodeficiency virus type 1 and polymorphism in the p1/p6 Gag cleavage site in drug resistance and replication fitness

Population-based sequence analysis revealed the presence of a variant of human immunodeficiency virus type 1 (HIV-1) containing an insertion of amino acid Ile in the protease gene at codon 19 (19I) and amino acid substitutions in the protease at codons 21 (E21D) and 22 (A22V) along with multiple mutations associated with drug resistance, M46I/P63L/A71V/I84V/I93L, in a patient who had failed protease inhibitor (PI) therapy. Longitudinal analysis revealed that the P63L/A71V/I93L changes were present prior to PI therapy. Polymorphisms in the Gag sequence were only seen in the p1/p6 cleavage site at the P1' position (Leu to Pro) and the P5' position (Pro to Leu). To characterize the role of these mutations in drug susceptibility and replication capacity, a chimeric HIV-1 strain containing the 19I/E21D/A22V mutations with the M46I/P63L/A71V/I84V/I93L and p1/p6 mutations was constructed. The chimera displayed high-level resistance to multiple PIs, but not to lopinavir, and grew to 30% of that of the wild type. To determine the relative contribution of each mutation to the phenotypic characteristic of the virus, a series of mutants was constructed using site-directed mutagenesis. A high level of resistance was only seen in mutants containing the 19I/A22V and p1/p6 mutations. The E21D mutation enhanced viral replication. These results suggest that the combination of the 19I/E21D/A22V mutations may emerge and lead to high-level resistance to multiple PIs. The combination of the 19I/A22V mutations may be associated with PI resistance; however, the drug resistance may be caused by the presence of a unique set of mutations in the p1/p6 mutations. The E21D mutation contributes to replication fitness rather than drug resistance.     

Early Combination Antiretroviral Therapy Limits Exposure to HIV-1 Replication and Cell-Associated HIV-1 DNA Levels in Infants

The primary aim of this study was to measure HIV-1 persistence following combination antiretroviral therapy (cART) in infants and children. Peripheral blood mononuclear cell (PBMC) HIV-1 DNA was quantified prior to and after 1 year of cART in 30 children, stratified by time of initiation (early, age <3 months, ET; late, age >3 months-2 years, LT). Pre-therapy PBMC HIV-1 DNA levels correlated with pre-therapy plasma HIV-1 levels (r = 0.59, p<0.001), remaining statistically significant (p = 0.002) after adjustment for prior perinatal antiretroviral exposure and age at cART initiation. PBMC HIV-1 DNA declined significantly after 1 year of cART (Overall: -0.91±0.08 log₁₀ copies per million PBMC, p<0.001; ET: -1.04±0.11 log₁₀ DNA copies per million PBMC, p<0.001; LT: -0.74 ±0.13 log₁₀ DNA copies per million PBMC, p<0.001) but rates of decline did not differ significantly between ET and LT. HIV-1 replication exposure over the first 12 months of cART, estimated as area-under-the-curve (AUC) of circulating plasma HIV-1 RNA levels, was significantly associated with PBMC HIV-1 DNA at one year (r = 0.51, p = 0.004). In 21 children with sustained virologic suppression after 1 year of cART, PBMC HIV-1 DNA levels continued to decline between years 1 and 4 (slope -0.21 log₁₀ DNA copies per million PBMC per year); decline slopes did not differ significantly between ET and LT. PBMC HIV-1 DNA levels at 1 year and 4 years of cART correlated with age at cART initiation (1 year: p = 0.04; 4 years: p = 0.03) and age at virologic control (1 and 4 years, p = 0.02). Altogether, these data indicate that reducing exposure to HIV-1 replication and younger age at cART initiation are associated with lower HIV-1 DNA levels at and after one year of age, supporting the concept that HIV-1 diagnosis and cART initiation in infants should occur as early as possible.     

Seroprevalence of and risk factors for HIV-1 infection in injection drug users in Montreal and Toronto: a collaborative study.

OBJECTIVE: To determine the prevalence of antibodies to HIV-1 and risk factors for HIV-1 infection among injection drug users. DESIGN: Questionnaire survey. A venous blood sample was taken for HIV-1 antibody testing. SETTING: Montreal and Toronto. PARTICIPANTS: A total of 810 subjects who had used injection drugs in the previous 6 months recruited mainly from treatment centres and from the street in Montreal (425 subjects) and from treatment centres in Toronto (385 subjects) between September 1988 and September 1990. The overall participation rate was 82%. OUTCOME MEASURES: HIV-1 seropositivity, sociodemographic and behavioural risk factors for HIV-1 infection. RESULTS: The overall seroprevalence rate of HIV-1 infection was 4.8% (95% confidence limits [CL] 3.5 and 6.5). In Montreal the rate was 8.2% (95% CL 6.0 and 11.2), and in Toronto 1.0% (95% CL 0.4 and 2.6) (p < 0.001). Seropositive subjects were significantly older (p = 0.041) and were more likely to have a history of imprisonment (p = 0.006) than seronegative subjects. In univariate analysis seropositivity was associated with the following behaviours: more frequent cocaine use (p < 0.001), injecting drugs in "shooting galleries" (p = 0.002), sharing equipment with a person known to be HIV-1 seropositive (p = 0.006), "booting" fresh blood (p = 0.004), homosexual or bisexual orientation (p = 0.006), engaging in prostitution (p < 0.001) and, for men, number of male sexual partners in the previous 6 months (p = 0.007). In multivariate analysis the determinants of HIV-1 seropositivity were Montreal as the city of recruitment (odds ratio [OR] 6.7, 95% CL 2.32 and 19.42), engaging in prostitution (OR 2.13, 95% CL 1.01 and 4.75), a history of imprisonment (OR 3.51, 95% CL 1.33 and 9.29) and sharing equipment with a person known to be HIV-1 seropositive (OR 4.43, 95% CL 1.43 and 13.74). CONCLUSIONS: Our findings show that HIV-1 is circulating among injection drug users in Montreal and Toronto and that both drug use and sexual behaviours are implicated in the transmission of infection in the populations studied. Adapted preventive programs should be developed to prevent further spread of HIV-1 infection in this population.     

1 alpha,25-dihydroxyvitamin D3 inhibits productive infection of human monocytes by HIV-1.

Pretreatment of freshly isolated human peripheral blood monocytes with the steroid hormone, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)D), markedly reduced (by 95%) productive infection of human monocytes by HIV-1. Equivalent concentrations (10nM) of 25-hydroxyvitamin D3 (25(OH)D), the biologic precursor of 1,25(OH)D, were ineffective at reducing either CD4 expression or HIV-1 production. Pretreatment was required for modulation of HIV-1 infection by 1,25(OH)D. Interestingly, 1,25(OH)D-mediated decreases in p24 antigen production were observed prior to any observed reduction in CD4 expression, suggesting that 1,25(OH)D treatment may modulate HIV-1 infection of monocytes through additional factors besides decreased HIV-1 binding. These data raise the possibility that 1,25(OH)D compounds may be important in host resistance to HIV-1.