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The role of killer cell immunoglobulin-like receptors (KIRs) in the transmission of HIV-1 has not been extensively studied. Here, we investigated the association of KIR gene content polymorphisms with perinatal HIV-1 transmission. The KIR gene family comprising 16 genes was genotyped in 313 HIV-1 positive Kenyan mothers paired with their infants. Gene content polymorphisms were presented as presence of individual KIR genes, haplotypes, genotypes and KIR gene concordance. The genetic data were analyzed for associations with perinatal transmission of HIV. There was no association of infant KIR genes with perinatal HIV-1 transmission. After adjustment for gravidity, viral load, and CD4 cell count, there was evidence of an association between reduction in perinatal HIV-1 transmission and the maternal individual KIR genes KIR2DL2 (adjusted OR = 0.50; 95% CI: 0.24–1.02, P = 0.06), KIR2DL5 (adjusted OR = 0.47; 95% CI: 0.23–0.95, P = 0.04) and KIR2DS5 (adjusted OR = 0.39; 95% CI: 0.18–0.80, P = 0.01). Furthermore, these maternal KIR genes were only significantly associated with reduction in perinatal HIV transmission in women with CD4 cell count 350 cells/ μl and viral load <10000 copies/ml. Concordance analysis showed that when both mother and child had KIR2DS2, there was less likelihood of perinatal HIV-1 transmission (adjusted OR = 0.44; 95% CI: 0.20–0.96, P = 0.039). In conclusion, the maternal KIR genes KIR2DL2, KIR2DL5, KIR2DS5, and KIR2DS2 were associated with reduction of HIV-1 transmission from mother to child. Furthermore, maternal immune status is an important factor in the association of KIR with perinatal HIV transmission.
The present study used samples from mothers and their infants from an epidemiological investigation of the relationship between placental malaria (PM) and perinatal transmission of HIV-1 [vertical transmission (VT study)] that was carried out in western Kenya between 1996 and 2001 [32, 35]. The Luo is the dominant ethnic group in the study area. During the VT study period, the prevalence of infections in pregnant women attending antenatal clinic was about 25% for HIV-1 and 20% for P. falciparum, respectively [36]. In the VT project, women were enrolled if they had singleton uncomplicated pregnancies of at least 32 weeks gestation and no known underlying chronic illnesses [32]. Information on reproductive history, socio-demographics, heath/clinical status, and malaria treatment was collected at enrollment and delivery. Blood samples were collected from mothers at enrollment, delivery, and one month postpartum for HIV diagnosis, HIV viral load, CD4 count, malaria diagnosis, and hemoglobin level determination. In addition, infants were followed up and blood samples collected monthly were used for HIV diagnosis. In the VT epidemiological study, HIV-positive women were given enrollment priority; while among HIV-negative women, women with PM were given enrollment priority. In total, 269 HIV-negative and 829 HIV-positive pregnant women were originally enrolled in the VT epidemiological study. For this host genetics study, we analyzed blood samples from 313 HIV-positive mothers paired with their infants for KIR gene content polymorphisms, based on availability of mother-infant paired samples. Counseling was provided to all women before and after HIV testing. At the time of the VT study (1996–2001), the Kenyan Ministry of Health recommended breastfeeding regardless of HIV status, and access to zidovudine or nevirapine was by then neither recommended by the Kenyan MOH nor available [37]. Written informed consent for participation in the study was obtained from the mothers for themselves and their infants. Study methods of the VT project, including the host genetics tests and analysis described here, were approved by the Kenya Medical Research Institute Ethical Review Committee, Nairobi, Kenya and the Institutional Review Board of the Centers for Disease Control and Prevention, Atlanta, USA. Maternal HIV status was determined based on a combination of initial testing with Sero Strip HIV-1/2 (Saliva Diagnostic Systems, New York, USA) and confirmation with Capillus HIV-1/HIV-2 test (Cambridge Diagnostics, Cambridge, UK). Infant HIV status was monitored monthly by DNA polymerase chain reaction (PCR) using gpM-Z primers. Maternal CD4 cell count was determined using fluorescent-activated cell sorting analysis (FACScan, Becton Dickinson, San Jose, California, USA) based on manufacturer instructions. Maternal HIV-1 viral load at delivery was measured using the Roche Amplicor HIV-1 monitor test versions 1.0 and 1.5, respectively (Roche Molecular Systems, Branchburg, New Jersey, USA)[32]. Thick smears made from placental and peripheral blood of mothers were stained with Giemsa and examined by microscopy. The number of asexual parasites/300 leukocytes was counted. Parasite density was estimated assuming 8000 leukocytes/μl. Peripheral blood hemoglobin concentrations (g/dl) were quantified using the HemoCue system (HemoCue, Brea, California) [32]. The KIR genotyping method used in this study has been described previously [38]. Briefly, DNA was extracted from blood samples from 313 mother-infant pairs using the QIAamp DNA blood mini kit (Qiagen, Valencia, California, USA). KIR genotyping for 16 KIR genes was carried out using KIRSSO genotyping test (One Lambda Inc., Canoga Park, California, USA) based on the manufacturer instructions. The results were read on a Luminex 200 IS (Luminex Corp., Austin, Texas, USA). The presence of individual KIR genes was determined using HLA Fusion Beta software (One Lambda Inc., Canoga Park, California, USA). Positive control DNA samples with different profiles of KIR gene content from the International Histocompatibility Working Group (IHWG) were used in all experiments. Infants were considered to be perinatally infected with HIV if they had two or more consecutive HIV-positive PCR tests, with the first positive PCR at or before 4 months of age[32]. Mothers of perinatally infected infants were classified as “transmitters” and those of uninfected infants as “non-transmitters.” Mothers of infants who acquired HIV at or after 5 months of age (considered postnatally acquired HIV) were also included in the analysis as non-transmitters [32]. Placental malaria was categorized into low (1–9999 parasites/μl) or high (≥ 10 000 parasites/μl) density per the parallel VT epidemiological study [32]. CD4 cell count was grouped as ≤ 200, 200–499, and ≥ 500cells/μl and viral load as <1000, 1000–9999 and ≥ 10000 copies/ml. Gravidity was divided into primi- or secundigradvida versus multigravida to allow assessment of possible differences in immunological environment between early and later pregnancies [35, 39]. KIR gene content polymorphisms were assessed in various ways: (1) the presence of 16 individual KIR genes [40]; (2) KIR haplotype A (presence of KIR3DL3, KIR2DL3, KIR2DL1, KIR2DP1, KIR3DP1, KIR2DL4, KIR3DL1, KIR2DS4 and KIR3DL2 only) and haplotype B (presence of KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5, KIR2DS5, KIR2DP1, KIR3DL2, KIR3DL3, KIR3DS1 and KIR3DP1) [41–43]; and (3) KIR genotypes AA, AB and BB. Genotype AA includes individuals with KIR3DL3, KIR2DL3, KIR2DL1, KIR2DP1, KIR3DP1, KIR2DL4, KIR3DL1, KIR2DS4, and KIR3DL2 while genotype BB comprises individuals without KIR2DL1, KIR2DL3, KIR3DL1 and KIR2DS4 [38]. Individuals not classified as either genotype AA or BB were regarded as genotype AB [44]. KIR concordance was defined as mothers and their infants having the same KIR gene content for single genes, haplotypes or genotypes. Statistical analyses compared HIV-transmitting mothers to those who did not transmit HIV in terms of characteristics of the mothers, characteristics of the babies, and KIR gene content of both mothers and babies. To determine whether characteristics of mothers or babies affected HIV transmission in unadjusted analyses, exact Chi-square tests were used for categorical characteristics, and exact Wilcoxon tests were used for other characteristics. For all other comparisons, logistic regression models were used. Due to sparse data issues, the Firth likelihood penalty was used for logistic regression models where possible [45]. If the model using Firth penalty did not converge, exact logistic regression was used. Both univariable and multivariable models were used to assess the relationship between HIV transmission status and KIR gene content of mothers or infants, where KIR gene content included single genes, genotype and haplotype. Based on the number of HIV transmitters, it was determined that multivariable models should control for no more than three variables to reduce the possibility of over fitting the data. Gravidity (<3 vs. ≥3), viral load (<10,000 copies/ml vs. ≥10,000 copies/ml), and CD4 count (<350 cells/ul vs. ≥350 cells /ul) were controlled for in the multivariable models as these were known predictors of MTCT of HIV [5, 46, 47]. Concordance models assessed whether a mother and child having the same single genes, haplotype, or genotype, affected HIV transmission. Again, both univariable and multivariable models were fit, and multivariable models controlled for gravidity, CD4 count, and viral load, which were categorized as mentioned above. For several single genes that appeared related to HIV transmission from mother to child, models with interaction terms were used to assess whether the relationship between KIR gene content of mothers and MTCT differed for different levels of CD4 count or viral load, where CD4 count and viral load were dichotomized as mentioned above. Models controlled for gravidity and were fit for CD4 count and viral load separately. The false discovery rate (FDR) was used to determine significance, controlling for multiple comparisons within each of the comparison groups separately, where comparison groups were defined by gene content type (single genes or not), whether the genes considered were from infants, mothers, or concordance between the two, and if the models used were adjusted. Two additional groups were defined for the interaction models, one group with interaction with CD4 and the other group with viral load interaction. Since there are two gene content types, three subject types, and two model types, plus two additional interaction model groups, there were 14 comparison groups for which FDR was used to determine significance individually.
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