ROLE OF CCR5 CHEMOKINE RECEPTOR GENE IN VERTICAL HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 TRANSMISSION AND DISEASE PROGRESSION (1998)

Several chemokine receptors serve as membrane coreceptors for primate immunodeficiency lentiretroviruses. The two most widely used coreceptors thus far recognized, CCR5 and CXCR4, are expressed by activated T lymphocytes and by mononuclear phagocytes. CCR5, a regulated on activation, normal T cell expressed and secreted, macrophage-inflammatory protein 1-alpha and macrophage-inflammatory protein 1-beta receptor, is used preferentially by macrophage-tropic HIV-1 and HIV-2 strains and by simian immunodeficiency virus, whereas CXCR4, a receptor for the C-X-C chemokine SDF-1, is used by T cell-tropic variants of HIV-1 and HIV-2, but not by simian immunodeficiency virus.1 A 32-nucleotide deletion (CCR5Δ32) may be present in one or both alleles of the CCR5 gene. There are discordant data about the role of CCR5Δ32 in HIV-1-infected patients. Some studies in adult patients have shown that the deletion on both alleles is associated with protection from HIV-1 infection,2, 3 whereas other reports have shown that HIV-1 infection can occur even in adults with a homozygous genotype.4, 5 In children the deleted allele in the homozygous state has not been associated with any protective effect against vertical HIV-1 transmission.6-10 Studies in adults and in children have suggested a possible protective effect of CCR5Δ32 heterozygosity on HIV-1 transmission and/or disease progression,2, 9-12 whereas other findings have suggested that the heterozygous condition is not associated with protection from HIV-1 infection and/or delayed progression.3-8 To clarify the impact of CCR5Δ32 on perinatal HIV-1 transmission and subsequent disease progression, we analyzed the polymorphism of CCR5 gene in a cohort of vertically HIV-1-infected and vertically HIV-1-exposed but uninfected children. HIV-1 infected and uninfected children. Blood samples were collected from 208 Caucasian children, who were born between 1983 and 1996. Eighty-three children were vertically HIV-1-infected, and 125 were perinatally HIV-1-exposed but uninfected. All children's parents were Caucasian. The CCR5 genotype was analyzed in all the patients; CD4+ cell counts and plasma viremia were also measured in HIV-1-infected children. Patients were classified according to the criteria of the 1994 CDC classification for children.13 On the basis of the previously published guidelines,14 according to the pattern of disease progression HIV-1-infected children were stratified as: (1) long term resistant host: children with absent disease progression or mild symptoms, ≥8 years, ≥500 CD4+/μl and ≥25% CD4+ in the 2 years preceding the study; (2) slow progressors: children ≥8 years with moderate symptomatology and/or moderate CD4+ depletion; and (3) progressors: children ≥8 years with severe clinical disease and/or severe CD4+ depletion. Informed consent was obtained from the parents or legal guardian of all the children. CCR-5gene amplification and sequencing. A portion of the CCR-5 gene was amplified by PCR from genomic DNA and analyzed using 10% polyacrylamide gel electrophoresis. Primers SP4.760 and PM6.942 flanking the 32-base pair deletion were used to obtain the wild type (183 base pairs) and deleted (151 base pairs) fragments. The PCR reaction mixture and the thermal profile have been described elsewhere.15 A control amplification containing no added DNA was included in each set of amplifications. Lymphocyte subsets and quantitation of HIV-1 RNA. Diagnostic monoclonal antibodies were used to evaluate lymphocyte subsets by fluorocytometry with Coulter (Coulter Electronics, Inc., Miami Lakes, FL) or Ortho (Ortho Diagnostic Systems) fluorocytometers. Plasma HIV-1 RNA of vertically HIV-1-infected children was measured in parallel samples using a branched chain signal amplification assay (Quantiplex® HIV-1 RNA assay kit Version 2.0; Chiron Diagnostic Laboratories, Emeryville, CA), according to the manufacturer's instructions; the test has a limit of sensitivity of 500 copies/ml. Statistical analysis. Proportions were compared by the chi square test or Fisher's exact test for expected values below 5. Values of CD4+ cell counts and HIV-1 RNA concentrations were compared by the Mann-Whitney test. Results.Table 1 shows the distribution of CCR5 gene polymorphism in all the patients and summarizes the clinical and laboratory characteristics of HIV-1-infected children in relation to their genotype. Allele frequencies of the entire study group were 89.9% for the wild type allele and 10.1% for the mutated allele. No child in either the HIV-1-infected group or in the exposed but uninfected group was homozygous for CCR5Δ32. The prevalence of the heterozygotes into the infected and uninfected groups was not significantly different (P = 0.95). Allele frequencies in children did not differ according to maternal route of infection (P = 0.95). Among the 83 HIV-1-infected children, no statistically significant differences were observed in the distribution of CCR5 gene polymorphism in the different clinical and immunologic CDC classes (CDC clinical classes N + A vs. B + C, P = 0.69; CDC immunologic classes 1 + 2 vs. 3, P = 0.71). Moreover, values of CD4+ absolute and percentage cell counts and viral load were not significantly different in wild type and heterozygous conditions (P = 0.78 and 0.55 for CD4+ absolute and percentage cell counts, respectively; P = 0.69 for HIV-1 RNA).TABLE 1: Distribution of CCR5 gene polymorphism in all the patients and clinical and laboratory characteristics of HIV-1-infected children As regards pattern of disease progression, no significant differences were observed between HIV-1-infected children with and without CCR5Δ32 (P = 0.52). Thus when the 65 HIV-1-infected children ≥8 years of age were analyzed, 38 were progressors (4 of 5 heterozygotes and 34 of 60 wild type), 18 slow progressors (1 of 5 heterozygotes and 17 of 60 wild type) and 9 long term resistant hosts (all wild type). Discussion. We showed that CCR5Δ32 in one allele does not confer absolute protection against vertical transmission of HIV-1 infection and the allele frequencies in children do not differ according to maternal risk factor for HIV-1 infection; moreover among vertically HIV-1-infected children with heterozygous and wild type conditions, we observed no differences in predictive biologic indicators and disease progression. Regarding HIV-1 transmission previous studies in heterozygous adults are discordant. Samson et al.2 and Michael et al.12 observed the existence of a protective effect of the CCR5Δ32 allele given that they reported a frequency of the heterozygous state that was lower in those who were infected than in the general population; whereas Huang et al.,3 Dean et al.4 and Balotta et al.5 found no protective effect of heterozygosity. Discordant results have been obtained also in perinatally HIV-1-exposed infected and uninfected children. In agreement with our results some reports suggested that heterozygosity for the CCR5Δ32 does not appear to offer resistance in mother-to-infant transmission of HIV-1.6-8 However, in previous reports of HIV-1-exposed children a dilution effect of CCR5Δ32 allele caused by multiracial genetic heritage of the patients enrolled was possible. We avoided potential selection biases because both parents of all our patients were of Caucasian origin. In contrast to our data Shearer et al.10 as well as Mandl et al.11 showed a trend suggesting protection against HIV-1 vertical transmission in infants with CCR5Δ32 heterozygosity; however, because of the low frequency of this genotype in their study population, in both cases this phenomenon could not be completely clarified. It is possible that maternal viral loads correlate with an increased incidence of HIV-1 infection in the heterozygotes for CCR5Δ32. The fact that a maternal risk factor for HIV-1 infection does not play a role in determining the frequency of heterozygosity in HIV-1-infected children confirms previous data in which it was observed that in adults heterozygosity does not vary in subjects infected through sexual or parenteral route.5 Discordant data are reported on the influence of genetic variations in the CCR5 on disease progression. Dean et al.4 described a clearly slower disease progression among heterozygotes. Huang et al.3 reported a positive impact on biologic indicators (viral load and CD4+ cell count), but no difference was observed between morbidity and mortality. A beneficial effect on progression was not confirmed by Smith et al.16 Moreover Michael et al.12 observed a lower rate of progression for heterozygous patients in whom the virus was classified as macrophage-tropic. In vertically HIV-1-infected children the study of the predictive role of the CCR5Δ32 allele is more clear-cut than in adults because the date of infection is known. However, once again the results are not concordant. Our findings are in agreement with those of Rousseau et al.6 who did not find a significant association between a single copy of CCR5Δ32 and disease progression among perinatally HIV-1-infected children. On the contrary Misrahi et al.9 suggested that CCR5Δ32 heterozygous genotype slows development of HIV-1-related disease. Differences in antiretroviral treatment are unlikely to provide an explanation for these contrasting results, because in all these studies patients were followed up in periods when zidovudine monotherapy was the most common regimen before AIDS onset. There are no data available about the tropism of the virus initially infecting our children. If the viral strain is macrophage-tropic, it is possible that CCR5Δ32 is not the sole genetic determinant of HIV-1 disease prognosis, but still unknown genetic polymorphisms may alter the risk of disease progression. Alternatively if the viral strain is T cell-tropic, disease progression may be not related to the CCR5 gene. In conclusion our data support the concept that CCR5 is not essential as a coreceptor for vertically transmitted HIV-1 infection and that infection may advance independently of the presence of a functional CCR5 coreceptor. More accurate analyses on the interaction between the virus and its cellular receptors are needed better to define the factors that play the main role in perinatal HIV-1 transmission and disease progression. Susanna Esposito, M.D. Gianguglielmo Zehender, Ph.D. Gian Vincenzo Zuccotti, M.D. Chiara Vegni, M.D. Luisa Galli, M.D. Vico Vecchi, M.D. Carlo Agostoni, M.D. Laura Rancilio, M.D. Chiara De Maddalena, B.S. Mario Clerici, M.D. Nicola Principi, M.D. Paediatric Department IV (SE, NP), Institute of Infectious Diseases (GZ, CDM), Paediatric Department V (GVZ, CA); Neonatology Department (CV), Paediatric; Department I (LR) and Immunology; Department (MC); University of Milan; Milan Paediatric Department III; University of Florence (LG); Florence Paediatric Department; Ospedale degli Infermi; Rimini (VV) Italy