Chemokine receptor CCR5 genotype influences the kinetics of human immunodeficiency virus type 1 infection in human PBL-SCID mice.

Individuals homozygous for a 32-bp deletion (delta 32) in the CCR5 gene encoding the coreceptor for macrophage-tropic human immunodeficiency virus type 1 (HIV-1) are resistant to virus infection, and heterozygous individuals show some slowing of disease progression. The impact of the CCR5 genotype on HIV-1 infection was assessed in vitro and in the human PBL-SCID (hu-PBL-SCID) model. Cells and hu-PBL-SCID mice from CCR5 delta 32/delta 32 donors were resistant to infection with macrophage-tropic HIV-1 and showed slower replication of dual-tropic HIV-1. hu-PBL-SCID mice derived from CCR5 delta 32/+ heterozygotes showed delayed replication of macrophage-tropic HIV-1 despite a small and variable effect of heterozygosity on viral replication in vitro. The level of CCR5 expression appears to limit replication of macrophage-tropic and dual-tropic HIV-1 strains in vivo.     

R5 human immunodeficiency virus type 1 infection of fetal thymic organ culture induces cytokine and CCR5 expression

Late-stage CCR5 tropic human immunodeficiency virus type 1 (HIV-1) isolates (R5 HIV-1) can deplete nearly all CD4+ thymocytes from human thymus/liver grafts, despite the fact that fewer than 5% of these cells express CCR5. To resolve this paradox, we studied the replication and cytopathic effects (CPE) of late-stage R5 HIV-1 biological clones from two progressors and two long-term nonprogressors (LTNP) in fetal thymic organ culture (FTOC) with and without added cytokines. We found that R5 HIV-1 clones from progressors but not LTNP were cytopathic in untreated FTOC. Moreover, R5 HIV-1 clones from progressors replicated to higher levels than LTNP-derived R5 HIV-1 clones in this system. In contrast, when FTOC was maintained in the presence of interleukin 2 (IL-2), IL-4, and IL-7, both progressor and LTNP clones exhibited similar replication and CPE, which were equal to or greater than the levels achieved by progressor-derived R5 HIV-1 clones in untreated FTOC. This finding was likely due to IL-2-induced CCR5 expression on CD4+ thymocytes in FTOC. R5 HIV-1 clones showed greater pathogenesis for CCR5+ cells but also showed evidence of CPE on CCR5- cells. Furthermore, infection of FTOC by R5 HIV-1 induced IL-10 and transforming growth factor beta (TGF-beta) expression. Both IL-10 and TGF-beta in turn induced CCR5 expression in FTOC. Induction of CCR5 expression via cytokine induction by R5 HIV-1 infection of CCR5+ thymocytes likely permitted further viral replication in newly CCR5+ thymocytes. CCR5 expression, therefore, is a key determinant of pathogenesis of R5 HIV-1 in FTOC.     

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.     

Role of chemokine and cytokine polymorphisms in the progression of HIV-1 disease

Allelic variants of the genes for chemokine receptors and their natural ligands, the chemokines, and cytokines can affect HIV-1 disease progression. This study investigates the level of expression of the CCR5-Δ32, CCR2b-641, RANTES In1.1C, SDF-1 3′A, IL-10-5′-592A and IL-4-589T alleles in two unique HIV-1 infected patient cohorts that represent the two distinct stages of disease progression, namely rapid progressors (RPs) and long term non-progressors (LTNPs) (n = 12/group) were recruited. Quantitation of the gene expression of CCR5-Δ32, CCR2b-641, RANTES In1.1C, SDF-1 3′A, IL-10-5′−592A and IL-4-589T in peripheral blood mononuclear leukocytes (PBML) isolated from patients was performed by real time, quantitative (Q)-PCR using DNA was isolated from PBML. We observed that expression of these HIV-protective alleles was generally greater in the LTNP cohort than the RP cohort. LTNPs expressed more of the protective chemokine, SDF-1α than RPs, and no statistically significant difference was observed in RANTES production between the LTNPs and RPs. The LTNPs expressed significantly less amounts of cytokines IL-10 and IL-4 as compared to the RPs. Our results demonstrate that gene polymorphisms for CCR5-Δ32, CCR2b-641, RANTES In1.1C, SDF-1 3′A, IL-10-5′−592A and IL-4-589T may be used as clinical markers to predict progression of HIV-1 infections.     

Distribution of the CCR5 gene 32-basepair deletion in 11 Chinese populations

A mutant allele of the chemokine receptor gene CCR5 bearing a 32-basepair deletion (delta 32CCR5) could increase the resistance to HIV-1 infection or delayed progression to AIDS. The frequency of this mutation is higher in Europeans than in Asians. To investigate the distribution of this polymorphism in China, 715 individuals from 11 Chinese populations were screened by PCR, including the Han and 10 other ethnic groups. The delta 32CCR5 gene was found in 16 individuals from 5 ethnic groups. All of them were heterozygous. The frequency of the mutant alleles of delta 32CCR5 is low in China and reflects (or might reflect) ancestral gene flow from Europe to Chinese ethnic groups and recent intermarriage within the ethnic groups.     

Nef alleles from human immunodeficiency virus type 1-infected long-term-nonprogressor hemophiliacs with or without late disease progression are defective in enhancing virus replication and CD4 down-regulation

Infection with human immunodeficiency virus (HIV)-encoding defective nef variants may contribute to a relatively benign course of disease in a minority of long-term nonprogressors (LTNP). We have examined the functions of nef alleles from six individuals belonging to the same cohort of hemophiliacs infected with HIV-1 prior to 1985 and classified as LTNP in 1995. Three out of six individuals have progressed to HIV disease (late progressors [LP]), whereas the three remainders have maintained their LTNP status at least up to 2003. The nef alleles were obtained from both plasma virus and peripheral blood mononuclear cells of all six individuals in 1995 and 1998. The proportion of sequences containing mutations not yielding Nef expression significantly diminished in 1998 versus that in 1995. Several previously defined functional regions of intact nef alleles were highly conserved. However, the major variant obtained in 1998 from plasma RNA of five out of six individuals significantly reduced HIV infectivity/replication and impaired Nef-mediated CD4 but not major histocompatibility complex class I antigen down-modulation from the cell surface. Thus, functional alterations of the nef gene are present in both LP and LTNP, suggesting that Nef defectiveness in vitro is not necessarily associated with the long-term maintenance of LTNP status. Of interest is the fact that isolates from three out of three LP showed a dual CCR5/CXCR4 coreceptor use (R5X4), in contrast to those from LTNP, which were exclusively R5. Thus, in vivo evolution of gp120 Env to CXCR4 use appears to be associated with HIV disease progression in individuals infected with nef-defective viruses.     

Comparison of HIV-1 nef and gag Variations and Host HLA Characteristics as Determinants of Disease Progression among HIV-1 Vertically Infected Kenyan Children

Objectives

Disease progression varies among HIV-1-infected individuals. The present study aimed to explore possible viral and host factors affecting disease progression in HIV-1-infected children.

Methods

Since 2000, 102 HIV-1 vertically-infected children have been followed-up in Kenya. Here we studied 29 children (15 male/14 female) who started antiretroviral treatment at <5 years of age (rapid progressors; RP), and 32 (17 male/15 female) who started at >10 years of age (slow progressors; SP). Sequence variations in the HIV-1 gag and nef genes and the HLA class I-related epitopes were compared between the two groups.

Results

Based on nef sequences, HIV-1 subtypes A1/D were detected in 62.5%/12.5% of RP and 66.7%/20% of SP, with no significant difference in subtype distribution between groups (p = 0.8). In the ten Nef functional domains, only the PxxP₃ region showed significantly greater variation in RP (33.3%) than SP (7.7%, p = 0.048). Gag sequences did not significantly differ between groups. The reportedly protective HLA-A alleles, A*74:01, A*32:01 and A*26, were more commonly observed in SP (50.0%) than RP (11.1%, p = 0.010), whereas the reportedly disease-susceptible HLA-B*45:01 was more common in RP (33.3%) than SP (7.4%, p = 0.045). Compared to RP, SP showed a significantly higher median number of predicted HLA-B-related 12-mer epitopes in Nef (3 vs. 2, p = 0.037), HLA-B-related 11-mer epitopes in Gag (2 vs. 1, p = 0.029), and HLA-A-related 9-mer epitopes in Gag (4 vs. 1, p = 0.051). SP also had fewer HLA-C-related epitopes in Nef (median 4 vs. 5, p = 0.046) and HLA-C-related 11-mer epitopes in Gag (median 1 vs. 1.5, p = 0.044) than RP.

Conclusions

Compared to rapid progressors, slow progressors had more protective HLA-A alleles and more HLA-B-related epitopes in both the Nef and Gag proteins. These results suggest that the host factor HLA plays a stronger role in disease progression than the Nef and Gag sequence variations in HIV-1-infected Kenyan children.     

Analysis of the 32 bp deletion in the CCR5 chemokine receptor gene in people from Moscow, infected with HIV-1

Chemokine receptors have recently been shown to mediate HIV-1 entry into cells. The chemokine receptor CCR5 plays a key role in this process. A 32-bp deletion within the coding region of the CCR5 gene generates a truncated nonfunctional receptor. In HIV-1-infected individuals homozygous for this mutation, disease progression is inhibited. We analyzed the frequencies of the deletion in HIV-1-infected seropositive individuals. No significant differences in allelic frequencies of the CCR5 gene between the control and general HIV-1-infected cohorts and within the latter group between the infected individuals and patients with AIDS symptoms were revealed.     

Autophagy plays an important role in the containment of HIV-1 in nonprogressor-infected patients

Recent in vitro studies have suggested that autophagy may play a role in both HIV-1 replication and disease progression. In this study we investigated whether autophagy protects the small proportion of HIV-1 infected individuals who remain clinically stable for years in the absence of antiretroviral therapy, these named long-term nonprogressors (LTNP) and elite controllers (EC). We found that peripheral blood mononuclear cells (PBMC) of the HIV-1 controllers present a significantly higher amount of autophagic vesicles associated with an increased expression of autophagic markers with respect to normal progressors. Of note, ex vivo treatment of PBMC from the HIV-1 controllers with the MTOR inhibitor rapamycin results in a more efficient autophagic response, leading to a reduced viral production. These data lead us to propose that autophagy contributes to limiting viral pathogenesis in HIV-1 controllers by targeting viral components for degradation.     

Combined effect of CCR5-Delta32 heterozygosity and the CCR5 promoter polymorphism -2459 A/G on CCR5 expression and resistance to human immunodeficiency virus type 1 transmission

Exposed seronegative individuals (ES) with persistent high-risk sexual behavior may be less susceptible to human immunodeficiency virus type 1 (HIV-1) infection because they carry the chemokine receptor (CR) gene alleles CCR5 open reading frame (ORF) Delta32, CCR5 promoter -2459G, or CCR2 ORF 64I (CCR2-64I), all of which have been found to diminish HIV-1 infectivity and/or disease progression. To investigate this, we determined the haplotypes for these three genetic loci in 93 ES and 247 low-risk control individuals. To test if protective haplotypes exert their effect by modulating CR expression, we measured the protein expression of CCR5 and CXCR4 on circulating CD4+ T cells and CD14+ monocytes in 71 ES and 92 controls. To avoid investigator bias, the analysis was performed without knowledge of each subject's risk and genotype. The CCR5 -2459G allele was significantly enriched in ES Caucasian men, who constituted the majority (84%) of the ES cohort, compared to the control Caucasian men (P = 0.02). This increase was mostly attributable to a higher frequency of the -2459 A/G versus the -2459 A/A genotype in individuals heterozygous for the delta32 allele (P = 0.012). No protective influence of the CCR2-64I allele was observed. The haplotypes CCR5 ORF delta32/CCR5 -2459A (in complete linkage disequilibrium) and CCR5 ORF wt/CCR5 -2459G had a cumulative negative effect on the expression of CCR5, since we measured significantly reduced CCR5 densities on both T-helper cells and monocytes only when both haplotypes were present. Densities of CCR5 on lymphocytes and monocytes were correlated (r = 0.59; P < 0.0001), indicating concordance of CCR5 expression patterns across different cell types. We conclude that the CCR5 ORF delta32/wt-CCR5 -2459 A/G genotype combination offers an advantage in resisting sexual HIV-1 transmission and that this effect is mediated by a relative paucity of CCR5 on potential target cells of HIV-1.     

Comparison CCR5de132 mutation in the CCR5 gene frequencies in Russians, Tuvinians, and in different groups of HIV-infected individuals

The 32-bp deletion (CCR5del32 mutation) in the CCR5 (chemokine (C-C motif) receptor 5) gene, encoding CCR5 chemokine receptor, is one of the factors determining natural resistance to human immunodeficiency virus (HIV-1) infection. In the present study, the samples of Russians (n = 107), Tuvinians (n = 50), and HIV-infected individuals were examined for the presence of CCR5del32 mutation in the CCR5 gene. The CCR5del32 allele frequency in Russians and Tuvinians constituted 7.84 and 2%, respectively. Among HIV-1 infected individuals, two groups, of macrophage-tropic HIV-1 strain- and T-cell-tropic HIV-1 strain-infected were distinguished. The CCR5del32 allele frequency in the first group (6.45%) was lower than in the second one (8.73%). Statistical treatment of the HIV-1 infected individuals typing data showed that the difference in the CCR5del32 allele frequencies between the groups of sexually (macrophage-tropic) and parenterally (T-cell-tropic) infected individuals observed was within the limit of random deviation.     

Primary Human Immunodeficiency Virus Type 2 (HIV-2) Isolates, Like HIV-1 Isolates, Frequently Use CCR5 but Show Promiscuity in Coreceptor Usage

Coreceptor usage of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to biological phenotype. The chemokine receptors CCR5 and CXCR4 are the major coreceptors that, together with CD4, govern HIV-1 entry into cells. Since CXCR4 usage determines the biological phenotype for HIV-1 isolates and is more frequent in patients with immunodeficiency, it may serve as a marker for viral virulence. This possibility prompted us to study coreceptor usage by HIV-2, known to be less pathogenic than HIV-1. We tested 11 primary HIV-2 isolates for coreceptor usage in human cell lines: U87 glioma cells, stably expressing CD4 and the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, and GHOST(3) osteosarcoma cells, coexpressing CD4 and CCR5, CXCR4, or the orphan receptor Bonzo or BOB. The indicator cells were infected by cocultivation with virus-producing peripheral blood mononuclear cells and by cell-free virus. Our results show that 10 of 11 HIV-2 isolates were able to efficiently use CCR5. In contrast, only two isolates, both from patients with advanced disease, used CXCR4 efficiently. These two isolates also promptly induced syncytia in MT-2 cells, a pattern described for HIV-1 isolates that use CXCR4. Unlike HIV-1, many of the HIV-2 isolates were promiscuous in their coreceptor usage in that they were able to use, apart from CCR5, one or more of the CCR1, CCR2b, CCR3, and BOB coreceptors. Another difference between HIV-1 and HIV-2 was that the ability to replicate in MT-2 cells appeared to be a general property of HIV-2 isolates. Based on BOB mRNA expression in MT-2 cells and the ability of our panel of HIV-2 isolates to use BOB, we suggest that HIV-2 can use BOB when entering MT-2 cells. The results indicate no obvious link between viral virulence and the ability to use a multitude of coreceptors.     

Effects of progesterone on the content of CCR5 and CXCR4 coreceptors in PBMCs of seropositive and exposed but uninfected Mexican women to HIV-1

CCR5 and CXCR4 play an important role in the establishment of HIV infection and disease progression. Caucasian people exposed to HIV but uninfected (EU) present a deletion of 32bp in CCR5 that has not been reported in EU Hispanics from Latin America. Therefore, other factors besides mutations should be involved in this phenomenon. Studies in healthy women have shown that sex hormones such as progesterone (P) can modulate CCR5/CXCR4 expression through an unknown mechanism. The aim of this paper was to determine the role of P in the regulation of CCR5 and CXCR4 in peripheral blood mononuclear cells (PBMCs) of HIV-1 infected and EU women. We analyzed HIV-1-infected women with stable highly active antiretroviral therapy (HAART) with CD4+ cell counts <400/mm(3) or diminution of 20%, EU and HIV-1 seronegative healthy controls. 5×10(6) PBMCs, from HIV-1 infected women, EU women and HIV-1 seronegative healthy controls were cultured and incubated with P (10 or 100 nM), RU486 (P antagonist, 1 μM) or P (100 nM)+RU486 (1 μM). CCR5/CXCR4 content was determined by Western blot. Densitometry data were analyzed using Mann-Whitney test. We found that CCR5 content was reduced by P in all groups. In contrast, CXCR4 content was increased by P in healthy controls and in HIV-1 infected women. Interestingly, CXCR4 content was reduced by P in EU. RU486 did not block P effects in any group. These findings suggest that P should participate in the acquisition and progression of HIV-1 infection by modulating CCR5 and CXCR4 expression. P could contribute to the resistance acquisition of HIV by EU through the down-regulation of both coreceptors.     

Prevalence of CCR5 and CCR2 HIV-coreceptor gene polymorphisms in Belgium

Polymorphisms of the chemokine receptor genes CCR5 and CCR2 are associated with resistance to HIV-1 infection or delayed progression to AIDS. Few data are available on their combined prevalence in healthy subjects; we therefore examined the occurrence of CCR5-Delta32 and CCR2-64I polymorphisms in a sample of 310 healthy Belgians. Allele frequencies were 0.119 and 0.074 for CCR5-Delta32 and CCR2-64I, respectively. Genotype distributions for both polymorphisms were found to be in accordance with Hardy-Weinberg equilibrium, but a significant (p = 0.002) linkage disequilibrium between CCR5-Delta32 and CCR2-64I was observed. The high prevalence of CCR5-Delta32 and CCR2-64I in Belgians may need to be taken into account in the design of studies of antiretroviral treatments.     

Comparison <Emphasis Type="Italic">CCR5del32</Emphasis> Mutation in the <Emphasis Type="Italic">CCR5</Emphasis> Gene Frequencies in Russians,Tuvinians, and in Groups of HIV-Infected Individuals

The 32-bp deletion (CCR5del32 mutation) in the CCR5 (chemokine (C-C motif) receptor 5) gene, encoding CCR5 chemokine receptor, is one of the factors determining natural resistance to human immunodeficiency virus (HIV-1) infection. In the present study, the samples of Russians (n = 102), Tuvinians (n = 50), and HIV-infected individuals (n = 107) were examined for the presence of CCR5del32 mutation in the CCR5 gene. The CCR5del32 allele frequency in Russians and Tuvinians constituted 7.84 and 2%, respectively. Among HIV-1 infected individuals, two groups, of macrophage-tropic HIV-1 strain- and T-cell-tropic HIV-1 strain-infected were distinguished. The CCR5del32 allele frequency in the first group (6.45%) was lower than in the second one (8.73%). Statistical treatment of the HIV-1 infected individuals typing data showed that the difference in the CCR5del32 allele frequencies between the groups of sexually (macrophage-tropic) and parenterally (T-cell-tropic) infected individuals observed was within the limit of random deviation.     

Heterozygous defect in HIV-1 coreceptor CCR5 and chemokine production

CC chemokine receptor 5 (CCR5) is a cell entry cofactor for macrophage-tropic isolates of human immunodeficiency virus 1 (HIV-1). An inactive CCR5 allele with a 32-nucleotide deletion (CCR5Delta32) has been described that confers resistance to HIV-1 infection in homozygotes and slows the rate of progression to AIDS in heterozygotes. We found the allele CCR5Delta32 to be not rare in 399 Swiss blood donors with a frequency of 0.080. To assess the influence of defective CCR5 on production of its ligands we determined the capacity to produce the chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and RANTES in comparison with the production of the CXC chemokine IL-8 which does not bind to CCR5. Production of chemokines was determined during endotoxin stimulation of whole-blood samples ex vivo. Both, basal and LPS-induced chemokine production in 32 blood donors heterozygous for CCR5Delta32 were not significantly different when compared with 55 blood donors who were homozygous for the wild type CCR5 allele.     

New class I and II HLA alleles strongly associated with opposite patterns of progression to AIDS.

The genetics of resistance to infection by HIV-1 cohort consists of 200 slow and 75 rapid progressors to AIDS corresponding to the extremes of HIV disease outcome of 20,000 Caucasians of European descent. A comprehensive analysis of HLA class I and class II genes in this highly informative cohort has identified HLA alleles associated with fast or slow progression, including several not described previously. A quantitative analysis shows an overall HLA influence independent of and equal in magnitude (for the protective effect) to the effect of the CCR5-Delta32 mutation. Among HLA class I genes, A29 (p = 0.001) and B22 (p < 0.0001) are significantly associated with rapid progression, whereas B14 (p = 0.001) and C8 (p = 0.004) are significantly associated with nonprogression. The class I alleles B27, B57, C14 (protective), and C16, as well as B35 (susceptible), are also influential, but their effects are less robust. Influence of class II alleles was only observed for DR11. These results confirm the influence of the immune system on disease progression and may have implications on peptide-based vaccine development.     

CCR5Δ32 Genotypes in a German HIV-1 Seroconverter Cohort and Report of HIV-1 Infection in a CCR5Δ32 Homozygous Individual

Background

Homozygosity (Δ32/Δ32) for the 32 bp deletion in the chemokine receptor 5 (CCR5) gene is associated with strong resistance against HIV infection. Heterozygosity is associated with protection of HIV-1 disease progression.

Methodology/Principal Findings

We genotyped a population of 737 HIV-positive adults and 463 healthy controls for the CCR5Δ32 deletion and found heterozygous frequencies of 16.2% (HIV-negative) and 17.5% (HIV-positive) among Caucasian individuals. Analysis of CCR5Δ32 influence on disease progression showed notably lower viral setpoints and a longer time to a CD4 count of <200 µl⁻¹ in seroconverters heterozygous for the deletion. Furthermore, we identified one HIV-positive man homozygous for the Δ32 deletion.

Conclusions/Significance

The protective effect of CCR5 Δ32 heterozygosity is confimed in a large cohort of German seroconverters. The HIV-infected CCR5 Δ32 homozygous individual, however, displays extremely rapid disease progression. This is the 12th case of HIV-infection in this genotype described worldwide.