Chemokines and their receptors play a critical role in HIV-1 binding and entry. Chemokine receptors function as viral coreceptors with CD4 to permit binding and entry of HIV-1 into macrophage and T cells. The principal chemokine receptors in HIV-1 transmission and progression are CCR5 for the R5 (macrophage tropic) and CXCR4 for the X4 (T-cell tropic) viruses. Other chemokine receptors, including CCR2, may act as secondary coreceptors.1
The chemokine RANTES is one of 3 natural ligands for CCR5. Chemokines interfere with the spread of HIV-1 by 2 mechanisms: by competitively binding to their respective receptors, they block binding of HIV envelope glycoprotein, gp120, and by inducing internalization of the bound receptor, thereby reducing coreceptor availability.2 Studies have shown a decline in RANTES levels with HIV disease progression3 and increased production of β-chemokines, including RANTES, among HIV-exposed uninfected individuals.4-7
Genetic epidemiologic cohort studies have shown that polymorphisms in the genes encoding these chemokines and chemokine receptors are associated with altered rates of disease progression after HIV-1 infection. Genetic variants of CCR2, CCR5, and its ligand, RANTES, have been associated with both delayed disease progression (CCR5-Δ32, CCR2-64I, and RANTES −403A) and accelerated disease progression (RANTES In1.C and CCR5-P1).8-16
Although this epidemiologic evidence has aided our understanding of the importance of chemokines and chemokine receptors in HIV-1 disease progression, the effect of RANTES variant alleles on HIV-1 RNA levels has not been demonstrated. Clinical studies of the CCR5 genotypes have shown decreased early HIV-1 plasma RNA for individuals heterozygous for CCR5-Δ32.17-19 The underlying mechanism of the 2- to 4-year delay in progression to AIDS-defining conditions afforded by CCR5-Δ32 in heterozygotes may result from a decrease in available CCR5.
The CCR5-P1 promoter haplotype has been associated with accelerated progression to AIDS in both European and African Americans.8,20 Unlike CCR5Δ32, which is rare or absent in non-Europeans, CCR5-P1 is quite frequent in all tested populations (frequency between 10% and 35%).20,21 In African Americans, the effect of CCR5-P1 is codominant, suggesting that the CCR5 promoter haplotypes differentially regulate transcription levels of CCR5. A single nucleotide polymorphism (SNP) within the CCR5-P1 haplotype has been associated with increased transcription of the CCR5 gene.22
SNPs in the RANTES regulatory regions have been shown to modify RANTES transcription in reporter assay systems.9,11 Two variant alleles, −28G and −403A, in the promoter have been shown to upregulate RANTES transcription in reporter assays and to modify rates of HIV-1 progression in some studies. A variant allele in the 3′-untranslated region 3′222C) has been suggested as an HIV/AIDS genetic modifier.9 Another variant allele, In1.1C, occurs in an enhancing regulatory element in the first intron of the RANTES gene and was found to downregulate transcription in reporter assays.9 Because −28G, −403A, In1.1C, and 3′222C are in strong positive disequilibrium and both In1.1C and −28G always occur on a −403A-bearing haplotype, it is difficult to separate the independent effects of the 4 variant alleles, although functional assays suggest that the In1.1T/C site is likely causal. All these alleles are in strong positive linkage disequilibrium and form 4 common haplotypes, with the most frequent being the R1 haplotype with the common allele at positions −403, −28, In1.1, and 3′222.
A key prognostic indicator of HIV-1 disease progression is the initial level of HIV-1 plasma RNA. Elevated levels of HIV-1 plasma RNA are associated with fast progression to AIDS in this cohort and others.23,24
In this study, we sought to determine whether genetic variants in the CCR5-promoter and in the RANTES gene encoding the CCR5 ligand altered the initial HIV-1 plasma RNA in a cohort of African American seroincident injection drug users (IDUs). Here, we test the hypothesis that the CCR5-promoter P1 haplotype and RANTES haplotypes previously shown to modify HIV-1 progression to AIDS are associated with HIV-1 plasma RNA levels in a group of seroincident IDUs.
As previously described, from February 1988 to March 1989, IDUs from the Baltimore community were recruited and screened for HIV antibodies and invited to join a prospective cohort study on the natural history of HIV, the AIDS Link to the Intravenous Experience (ALIVE).25 Individuals were considered eligible if they were at least 18 years old, had engaged in injection drug use within the previous 10 years, and had not experienced an AIDS-defining illness. The Institutional Review Board of the Johns Hopkins Bloomberg School of Public Health approved all study and consent procedures.
The early viral load was defined as the first viral load measurement within 2 years after the estimated date of seroconversion. There were 250 participants in the study who seroconverted during the study period and had initial plasma HIV-1 RNA. Of these, 11 were ineligible, because the initial viral load measurement was more than 2 years after seroconversion. Additionally, 15 individuals were not African American and were excluded because of the potential for confounding as a result of population heterogeneity. Of the 224 individuals with HIV-1 seroconversion who met the criteria for inclusion, 198 were genotyped at the Laboratory of Genomic Diversity, Frederick, Maryland, for each of the following genotypes: CCR5-Δ32; CCR2-64I; CCR5 P1; and RANTES −28G, −403A, In1.1C, and 3′222. Haplotypes were inferred using the maximum likelihood method. For comparability, we describe the CCR5 variants using both the Laboratory of Genomic Diversity and Tri-Service HIV-1 Natural History Study (TSS) nomenclature.20,26
Antibodies to HIV-1 were detected using an enzyme-linked immunosorbent assay (ELISA) (Genetic Systems, Seattle, WA) with confirmation of positive ELISA tests by Western blot analysis (DuPont, Wilmington, DE). HIV-1 viral load was assessed using the reverse transcriptase polymerase chain reaction (RT-PCR) assay (Roche Molecular Systems, Branch-burg, NJ). The minimum detectable level of HIV-1 RNA was 400 copies/mL; levels that were read as undetectable were coded for analysis as 200 copies/mL. PCR restriction fragment length polymorphism (RFLP) and 5′ nuclease PCR (TaqMan) assays were used for genotyping. Genotyping was performed as previously described.8,9,14,16,27
The seroconversion date was estimated as the midpoint between the last HIV-negative visit date and the first HIV-positive visit date. HIV-1 RNA measurements were transformed to log10, and early HIV-1 plasma RNA was used as the outcome. The RANTES haplotypes (Table 1) were determined using an expectation maximization (EM) algorithm that first recursively computes the expectation of the haplotype and then maximizes the expected likelihood until a convergence criterion is met to determine population-based haplotypes, as previously described.9 The CCR2-CCR5P1-CCR5 haplotype was determined by counting and resulted in 4 haplotypes (Table 2). Individuals homozygous for CCR2-64V, CCR5-P1, and CCR5+ are also referred to as E/E (TSS nomenclature). The Wilcoxon rank sum test was used for comparisons of continuous variables. The χ2 test was used for comparisons of categoric variables. General linear model regression statistics were used to assess the association of individual genotypes or haplotypes with plasma HIV-1 RNA levels. To assess confounding, gender, age at seroconversion, seroconversion date, time from seroconversion to early viral load measurement, and other genotypes (possible genotypes included CCR5Δ32, CCR2V64I, CCR5-P1, RANTES −403, RANTES −28, RANTES In1.1, and RANTES3′222) were included as covariates in a multivariate model. All statistical analyses were done using STATA, version 6.0 (College Station, TX).
Of the 198 individuals with both early plasma HIV-1 RNA and genotype information, 151 were men (76%) and 47 were women (24%), which is consistent with the gender distribution among all seroincident cases in this cohort. The median early HIV-1 RNA for men (4.62, interquartile range [IQR]: 4.04-5.0) and women (4.23, IQR: 3.86-4.64) differed by 0.40 log10 units, which is consistent with previous studies showing a lower initial viral load for women compared with men.28-30 Individuals with higher early HIV-1 plasma RNA levels progressed to AIDS faster than individuals with lower early HIV-1 plasma RNA measurements (data not shown), as seen in this and other cohorts previously.23,24
There were 4 haplotypes of CCR2-V64I: CCR5 P1/other: CCR5+/Δ32 alleles (see Table 2). Additionally, there were 4 previously established RANTES haplotypes from the alleles (−403, −28, In1.1, and 3′222) that were also included (see Table 1).9 Simple linear regression of initial HIV-1 plasma RNA on individual genotypes for each marker identified RANTES 403A and RANTES In1.1C as having an effect on HIV-1 plasma RNA (Table 3). Because these alleles are in strong or complete linkage disequilibrium with one another, a haplotype analysis was necessary to determine their combined effect. The −28 site, polymorphic in both Asians and Europeans, is not polymorphic in African Americans. Our cohort of African Americans did not exhibit any variance at the RANTES −28C/G promoter allele (100% were −28C/C), so this SNP was not included in the haplotype analysis.
Univariate linear regression of early HIV-1 plasma RNA and RANTES haplotypes identified the homozygous RANTES R1 haplotype (carrying the most common allele at the 4 sites) as having a lower viral load by almost one-half log10 unit compared with that with any other RANTES haplotype (−0.43, 95% confidence interval [CI]: −0.74, −0.12; Table 4). A separate analysis by gender showed similar trends in men (−0.48, 95% CI: −0.82, −0.13) and women (−0.21, 95% CI: −0.88, 0.46), although statistical power was considerably less among women because of the small number (n = 47). Additionally, after adjusting for potential confounders, including the CCR5Δ32, CCR2 V64I, and CCR5 P1 promoter haplotypes, gender, age, seroconversion date, and the time from seroconversion to viral load measurement, the strength of the association was even stronger for the RANTES R1 haplotype (−0.49, 95% CI: −0.80, −0.17; Table 5). The R1 homozygous individuals have lower HIV-1 plasma RNA levels than individuals carrying at least 1 R2, R3, or R4 haplotype.
The CCR5 promoter analysis did not identify any single haplotype/diplotype with significant influence on initial HIV-1 plasma RNA. Additionally, we analyzed the CCR2 64I allele, which has been associated with modest influences on HIV-1 disease progression and is located in close chromosomal proximity to CCR5. The CCR2 variant did not alter the initial HIV-1 plasma RNA in our cohort, which may reflect a diminished role of this genetic variant on HIV-1 plasma RNA.
In this cross-sectional study, we analyzed early HIV-1 plasma RNA according to known modifying haplotypes in the chemokine coreceptor CCR5 promoter, CCR2, and RANTES genes. We identified 0.49-log10 lower initial HIV-1 plasma RNA for those individuals homozygous for the RANTES R1 haplotype carrying the most frequent allele at the 4 polymorphic sites. Because RANTES is a critical ligand for CCR5, it was important to adjust for the effects of the CCR5-Δ32 mutation and its promoter haplotypes, because both the CCR5-Δ32 deletion and the CCR5 promoter haplotypes have been shown to modify CCR5 surface expression and thereby interfere with HIV-1 replication kinetics. In univariate and multivariate analysis, the R1 haplotype maintained a lower initial viral load. Because the homozygous haplotype frequencies of R2 to R4 were quite small, we could not independently determine which allele might be responsible for the increased initial HIV-1 plasma RNA.
Earlier studies have shown increased rates of RANTES transcription with the −28G allele in a Japanese population.11 In a study of at-risk HIV-seronegative individuals, those with the RANTES haplotype −403A, −28C had increased infection compared with those with the RANTES haplotype −403G, −28C. Additionally, seroconverters had a significantly slower progression to AIDS (under the 1993 criteria) for those carrying the RANTES −403A allele, suggesting that the RANTES −403A allele is a risk factor for acquiring HIV but may be protective in progression.12 A global survey of RANTES −28C and −403A identified both as risk factors for HIV infection and accelerated progression to AIDS among European Americans homozygous for the RANTES promoter alleles −403A, −28C, but this association was not found in African Americans.31 A recent study by An and colleagues9 extended the haplotype region to include the RANTES SNP In1.C, which always occurs with the RANTES SNP −403A. In a survival analysis of 962 individuals (including ALIVE participants), the effect of the In1.1C allele and the composite RANTES R3 haplotype was associated with an accelerated progression to AIDS (relative hazard [RH] = 1.7; P = 0.05) and AIDS-related death (RH = 2.3; P = 0.02) among African Americans.9 Further functional analysis determined that the In1.1C allele results in decreased rates of RANTES transcription and that relative to the In1.1T allele, the In1.1C allele downregulates RANTES transcription by approximately 4-fold.9 We have recently shown that the In1.1T/C site occurs within a regulatory element that enhances transcription of RANTES and that this enhancement is abrogated by the In1.1C allele.9 It is likely that the association of the RANTES R1 haplotype with lower HIV-1 RNA is a result of more abundant RANTES for CCR5 binding. Our finding validates the previously described association of accelerated disease progression to AIDS among those with genetic variants in RANTES by showing a correlation with increased initial HIV-1 plasma RNA.9,11,12 In addition, these findings suggest that a potential mechanism for increased or decreased HIV disease progression is the level of RANTES expression controlled by these RANTES haplotypes. We did not determine if primary cells from individuals with different RANTES haplotypes produced different amounts of RANTES with antigenic stimulation, however. We would expect that R1 RANTES homozygotes would secrete more RANTES than cells from non-R1 RANTES individuals. This warrants further research to determine if the genetic differences in RANTES haplotypes can translate into significant differences in protein secretion over time.
Our analysis of the CCR5 promoter P1 haplotype and its diplotypes (both haplotypes at a gene or locus) did not show a statistically significant difference on initial HIV-1 plasma RNA, unlike previous analyses. A viral load analysis of 341 white seroincident participants in the Multicenter AIDS cohort study (MACS) revealed a higher mean plasma HIV-1 RNA among E/E carriers (64V/P1/+) in the initial 42 months after seroconversion.32 Additionally, African-American serocon-verters with the E/E genotype had elevated HIV-1 RNA levels compared with other genotypes. In an additional study of predominantly African American women in the Reaching for Excellence in Adolescent Care and Health (REACH) study, 207 women tested seroprevalent and the E/E genotype was also identified as being associated with a higher plasma HIV-1 RNA level.33
We could not validate any influence of the E/E diplotype on initial HIV-1 plasma RNA or any effect of the CCR5 P1 promoter allele. The accelerated disease progression noted in numerous cohort studies may be influenced by another variant in linkage disequilibrium with the CCR5 P1 to P4 haplotypes. Although we detected an association with RANTES, we may have lacked adequate statistical power to identify a weaker association with CCR5 P1. It is also plausible that the increased rates of progression to HIV-1 infection may not be driven by an increase in HIV-1 plasma RNA but through another mechanism altogether.
One limitation of this study is an incomplete understanding of the effects of genetic variants when there are differences in viral tropism. Individuals may be infected with R5 or X4 tropic virus, which can alter the utilization and significance of the RANTES chemokine. Within the ALIVE cohort, 46 seroconverters have been sequenced and/or characterized for viral tropism. Only 1 CXCR4 virus (2%) and 3 dual-tropic (CCR5 and CXCR4) viruses were detected. The remaining 42 seroconverters (91%) only had CCR5 virus detected. Even though not all the seroconverters in the ALIVE cohort were sequenced, these results suggest that this cohort of IDUs does exhibit a bias toward R5 transmission and would most likely use the RANTES chemokine.
Both a strength and a limitation of our study is that it is composed 100% of African Americans; the frequency of alleles or patterns of linkage disequilibrium differ between races with different population histories.34,35 An and colleagues9 have shown that European Americans have a higher frequency of the RANTES R1 haplotype as compared with African Americans (frequency = 0.77 and 0.57, respectively), which may make the effects of the RANTES haplotypes on elevated HIV-1 plasma RNA more prominent among African Americans. The association of the RANTES R1 haplotype with HIV-1 plasma RNA may differ between populations because of the differences in allele frequencies and haplotype structure both for the RANTES gene and at other known and unknown AIDS-modifying genes, however. As an example, the protective factor CCR5 Δ32 is carried by approximately 20% of people of European origin and is virtually absent from all other geographic groups. These studies affirm the necessity of investigating the influence of genetic factors in human diseases in populations of different geographic origins.
Implications of measurable differences in HIV-1 viral load by genetic variants are important for a better understanding of HIV-1 pathogenesis and the design of therapeutics and a vaccine. Because RANTES can alter the level of HIV-1 plasma RNA in newly infected individuals, it should be considered as a potential treatment. Current studies into the use of RANTES derivatives as HIV suppressor agents should be encouraged by the apparent protective effect of RANTES R1 haplotypes on initial HIV-1 plasma RNA as seen here. Further cohort studies are necessary to identify the complete effects of the RANTES haplotypes so that targeted treatment strategies can be developed.
The authors thank the participants of the ALIVE cohort.
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Keywords:© 2005 Lippincott Williams & Wilkins, Inc.
chemokines; RANTES; HIV-1 plasma RNA; HIV