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

Abstract

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.

FIG. 1.

Association between CCR5 ORF plus −2459 genotype combinations and CCR5 expression. Stored peripheral blood CD4⁺ T cells and CD14⁺ monocytes from 163 individuals were blinded and analyzed by FACS for the mean number of CCR5 surface receptors per CCR5⁺ cell and the percentage of CCR5-expressing cells. CCR5 genotypes were determined by restriction fragment length polymorphism analysis. Stratification of CCR5 densities and percentages by CCR5 ORF plus −2459 genotype combinations was performed after completion of all FACS analyses. Horizontal bars represent population medians. P values were determined by Mann-Whitney test. wt, wild type.

FIG. 2.

Correlation between T-helper cell and monocyte surface CCR5 densities. Peripheral blood CD4⁺ T cells and CD14⁺ monocytes from 163 individuals were analyzed by FACS for the mean number of CCR5 surface receptors per CCR5⁺ cell. Line fit was done by linear regression. r, Spearman correlation coefficient.

FIG. 3.

HIV-1 infectivity of CD4⁺ lymphoblasts. CD4-selected, PHA-stimulated lymphoblasts from 102 individuals were infected with HIV-1_JR-CSF at MOI of 0.006 (not shown) and 0.003 (shown). p24 concentrations in culture supernatants were determined 7 days after infection. Stratification of p24 values by CCR5 genotype combinations was performed after completion of all infection assays. Numbers next to symbols are the mean number of CCR5 surface receptors per cell measured on peripheral blood CCR5⁺ CD4⁺ T cells from the same individuals but obtained at different clinic visits from those used to obtain the cells used for the infectivity assays. nd, not done.