An estimated 3.2 million children worldwide are living with HIV/AIDS . Consequently, child mortality rates have risen precipitously, especially in sub-Saharan Africa where roughly 8% of all deaths under 5 years of age are directly attributable to HIV infection . Mother-to-child transmission is overwhelmingly the most common route of HIV infection in children . Most mother-to-child transmission occurs shortly before or during delivery and is influenced by maternal viral load and immune status, the timing and route of delivery, viral subtype, and host genetic factors [4–6].
Receptors on leukocytes for the Fc domain of IgG antibodies form an important bridge between the humoral and cellular arms of the immune system . There are three main classes of Fcγ receptors, with class II (FcγRIIa) being the most widely distributed. A single nucleotide polymorphism in the gene for the Fcγ receptor subclass IIa is of particular interest. The histidine-131 variant leads to a receptor with high affinity for human IgG2, whereas the arginine allelic variant binds to IgG1 and IgG3 but poorly to IgG2 . Considerable differences in the distribution of the FcγRIIa polymorphism among ethnic groups have been reported, and this polymorphism has been found to play an important role in autoimmune diseases, especially systemic lupus erythematosus [9,10]. More recently, FcγRIIa polymorphism has been found to affect susceptibility to, or outcome of, infectious diseases. In infections with encapsulated bacteria, where IgG2 is central in host defense, those with the His131/His131 genotype have a lower incidence and intensity of disease, while heterozygotes have intermediate susceptibility and those with FcγRIIa Arg/Arg131 have enhanced susceptibility [11,12]. However, in parasitic diseases such as malaria, where IgG1 and IgG3 rather than IgG2 are of primary importance for protection, infants homozygous for the Arg131 gene are less likely to have a high-density infection . Therefore, depending on which IgG antibody subclasses are associated with protection, it may be advantageous to have one form of the FcγRIIa receptor over the other when encountering a specific pathogen.
For HIV-1, the biological relevance of Fcγ receptor polymorphism is uncertain. Fcγ receptors on HIV target cells have been implicated in in vitro mediation of viral entry and activation of HIV gene transcription via cross-linking with subneutralizing IgG antibody–virus immune complexes [14–16]. Further study has shown that IgG antibody subclasses differ in their ability to neutralize HIV-1 and inhibit HIV–cell fusion, with IgG1 and IgG3 being more efficient than IgG2 . Because of this poor ability of IgG2 to neutralize HIV and the role of subneutralizing levels of IgG in activation of HIV gene transcription, we hypothesized that the preferential IgG2-binding FcγRIIa His/His131 genotype may be associated with enhanced perinatal transmission of HIV-1 and mortality in HIV-infected children. Data have been analyzed from 448 HIV-infected women and their infants in a cohort study of perinatal mother-to-child transmission of HIV-1 conducted in western Kenya to look for evidence that infants homozygous for the FcγRIIa His131 genotype have increased susceptibility to perinatal HIV infection.
Study subjects and clinical procedures
From 1996 to 2001, pregnant women attending the antenatal clinic and delivery ward of the New Nyanza Provincial General Hospital in Kisumu, western Kenya, and their infants, were followed in a study designed to examine the effect of placental malaria on perinatal mother-to-child transmission of HIV-1 (VT study). Methods for this study have been described in detail elsewhere . Briefly, criteria for enrollment included a singleton uncomplicated pregnancy of ≥ 32 weeks’ gestation, no underlying chronic diseases (asymptomatic infection for HIV-positive women), and residence in the Kisumu area. The residents of this area are predominantly of the Luo ethnic group and HIV prevalence in pregnant women is approximately 26% [18–20].
Upon enrolment, a questionnaire was administered eliciting information on sociodemographic, behavioral, obstetric, and health status factors. Blood was drawn from mothers for HIV, hemoglobin, and malaria testing. At delivery, blood samples were taken from the mother, placenta, and cord to determine malaria infection and HIV viral load. At 1 month postpartum, additional blood was collected from the mother for CD4/CD8 cell counts, hemoglobin, and malaria assessment. Clinical information and blood samples were obtained monthly from infant participants to monitor incidence of malaria, anemia, and HIV for the first 2 years of life. The VT study originally enrolled 829 HIV-seropositive mothers and HIV transmission status was determined for 512 of the mother–infant pairs. For this genetics study, 448 mother–infant pairs were assessed, selected based on availability of DNA, information on maternal viral load, CD4 cell count and other clinical parameters.
Counseling was provided to all participants before and after HIV testing. At the time of the study (1996–2001), the Kenyan Ministry of Health recommended breastfeeding regardless of HIV status, and access to zidovudine or nevirapine was neither recommended by the Kenyan Ministry of Health nor available. Mothers gave their written informed consent to participate in the study and additional written consent was obtained upon enrollment of their child. Study methods were approved by the Institutional Review Board of the Centers for Disease Control and Prevention, Atlanta, USA and the Kenya Medical Research Institute Ethical Review Committee, Nairobi, Kenya.
Maternal HIV status was determined by a primary Serostrip HIV-1/2 (Saliva Diagnostic Systems, New York, USA) and a confirmatory Capillus HIV-1/HIV-2 test (Cambridge Diagnostics, Cambridge, UK) . Infant DNA was monitored for HIV infection by DNA polymerase chain reaction (PCR) using gpM-Z primers . Maternal CD4 cell counts were determined using fluorescent-activated cell sorting analysis (FACScan, Becton Dickinson, San Jose, California, USA). Maternal HIV-1 viral load at delivery and infant viral load at the visit corresponding to HIV conversion were measured using the Roche Amplicor HIV-1 monitor test versions 1.0 and 1.5, respectively (Roche Molecular Systems, Branchburg, New Jersey, USA).
FcγRIIa genotype determination
Infant and maternal DNA was extracted from peripheral blood mononuclear cells and a 366 base-pair fragment surrounding codon 131 of the FcγRIIa gene was amplified with gene-specific primers (9600 Geneamp PCR system, Applied Biosystems, Foster City, California, USA). The amplified product was then digested with the BstUI restriction enzyme (New England Biolabs, Beverley, Massachusetts, USA) and the length of digested fragments was analyzed by electrophoresis on an agarose gel to determine FcγRIIa genotype .
Infants were considered to be infected by HIV perinatally if they had two or more consecutive HIV-positive PCR tests, with the first positive PCR at or before 4 months of age . Mothers of perinatally infected infants were classified as ‘‘transmitters’’ and those of uninfected infants as ‘‘non-transmitters.’’ Infants who acquired HIV at or after 5 months of age were considered to have acquired HIV postnatally, and their mothers were included in the analysis as non-transmitters. Placental malaria was categorized into low (1–9999 × 106 parasites/l) or high (≥ 10 000 × 106 parasites/l) density according to a parallel epidemiologic study of the effect of placental malaria on perinatal HIV transmission in this population . CD4 cell counts were categorized as < 200, 200–499, and ≥ 500 × 106 cells/l . Gravidity was divided into primi- or secundigradvida versus multigravida, based on the fact that the immunological milieu of early pregnancies differs from that of later pregnancies [19,25]. All-cause mortality in HIV-positive children was recorded if the death occurred within the first 24 months of life and there was no more than 4 months between the last study visit and the death. No deaths occurring within the neonatal period could be included in the analysis because of the requirements for determining HIV status.
Univariate analyses were used to examine which covariates were associated with perinatal HIV-1 transmission. The effect of FcγRIIa genotypes on the odds of perinatal HIV-1 transmission was assessed in the 448 mother–infant pairs using a multivariate logistic regression model, controlling for confounding variables. Using Cox proportional-hazard regression with time-independent covariates, all HIV-positive children (n = 116), including both those who acquired HIV before and those who acquired it after the perinatal period, were included in a univariate and multivariate survival analysis. The FcγRIIa His/Arg131 genotype was used as a reference for the above analyses since this is the most prevalent genotype in human populations . The non-parametric Kruskal–Wallis test was performed to compare the median HIV-1 viral loads of children who acquired HIV perinatally (n = 100) and the ratio of mother to child's viral load by FcγRIIa genotype. Two-sided P values < 0.05 were considered statistically significant. Data analysis was performed using SAS (Release 8.02; SAS Institute, Cary, North Carolina, USA) and SPSS version 10.0 (SPSS, Chicago, Illinois, USA) software.
Characteristics of HIV transmission groups
There were no statistically significant differences in maternal parameters between those mothers included in this study of effect of FcγRIIa genotype on perinatal HIV transmission (n = 448) and those originally enrolled in the VT study but not included in the present investigation (n = 381). In this FcγRIIa study, perinatal HIV transmitters (n = 88) were more likely to have an episiotomy or perineal tear, a high HIV-1 viral load, and a low CD4 cell count than non-transmitters (n = 360). Consistent with the main findings of an epidemiological study in this population , these women were less likely to have placental malaria (particularly of low density) and more likely to be primi- or secundigravidae and deliver a low birth-weight baby (Table 1). Transmitting women were also more likely to have a longer duration of labor than non-transmitters (Table 1).
Distribution of FcγRIIa genotypes by HIV transmission status
There was no statistically significant association between maternal genotype and perinatal transmission of HIV (overall P = 0.56). However, among infants, Fcγ receptor polymorphism was significantly related to transmission (Table 1; overall P < 0.01). In a univariate logistic regression analysis, the odds of perinatal HIV-1 transmission of infants with the FcγRIIa His/His131 genotype were higher than the FcγRIIa His/Arg131 reference group [odds ratio (OR), 1.78; 95% confidence interval (CI), 1.05–3.03; Table 2]. Although failing to reach statistical significance, the FcγRIIa Arg/Arg131 group showed the opposite effect, with reduced transmission compared with the FcγRIIa His/Arg131 reference (OR, 0.56; 95% CI, 0.29–1.09).
Effect of infant FcγRIIa genotypes on perinatal HIV-1 transmission
Multivariate logistic regression was used to assess further the effect of infant FcγRIIa polymorphism on perinatal HIV-1 transmission. The analysis controlled for the confounding effect of maternal (viral load, placental malaria, gravidity, and duration of labor) and infant (birth weight) factors since these variables were found to be statistically significant in the univariate analysis. Because of high correlation with other variables considered, episiotomy/perineal tear and maternal CD4 cell count were not included in the multivariate model. The multivariate analysis demonstrated that infants with the FcγRIIa His/His131 genotype were more likely to become perinatally infected with HIV compared with infants with the FcγRIIa His/Arg131 genotype (adjusted OR, 2.22; 95% CI, 1.23–4.02; Table 2). A recent study on this cohort has shown that mother-to-child transmission rates varied by HIV-1 subtype . When the analysis controlled for HIV-1 subtype (primarily A or D in this region) in the subset of mothers with information available on this variable (n = 347), the association between infant FcγRIIa His/His131 and susceptibility to perinatal HIV-1 transmission remained (data not shown; P = 0.015).
Effect of FcγRIIa polymorphism on child mortality
All-cause mortality was used as a surrogate disease progression end-point. Out of 116 HIV-positive children, 50 documented deaths occurred during the 2-year study period. Thirty-eight of these deaths (76%) took place during the first year. In univariate analysis, both the child's age and the viral load at conversion to HIV-1-positive status were indicative of the rate of mortality. Infant FcγRIIa genotype and maternal educational level were borderline statistically associated with mortality risk (Table 3). These results remained similar when the analysis was limited to the first year of life (data not shown). In a Cox proportional hazard regression analysis controlling for the child's age and viral load, the risk of death in children with FcγRIIa His/His131 was higher, but not significantly so, than in the His/Arg131 group (adjusted hazard risk, 1.72; 95% CI, 0.92–3.20; P = 0.089).
The relationship between child FcγRIIa genotype and viral load at HIV conversion in perinatally infected children was also explored since viral load has been found to be a significant prognostic marker for HIV-1 disease progression in both adults and children [28,29]. Neither the median infant viral load at HIV conversion nor the ratio of maternal-to-infant viral load was associated with genotype (P = 0.304 and P = 0.588, respectively, data not shown).
To our knowledge, the current study is the first to explore the effect of polymorphism in the FcγIIa receptor for IgG on perinatal transmission of HIV-1. We found that infants with the FcγRIIa His/His131 genotype were significantly more likely to be infected by HIV-1 perinatally. Our results further suggest that there is a dose–response relationship for the effect of the His131 allele on the transmission, with His/His conferring the highest, Arg/Arg the lowest and His/Arg intermediary susceptibility to HIV infection. The fact that the FcγRIIa His131 is the only human Fcγ receptor to bind IgG2 efficiently suggests that the effect of this gene on susceptibility to HIV-1 infection may be related to the relative effect of IgG2 on HIV, in comparison with other IgG subclasses.
Previous in vitro studies have shown that neutralization of cell-free HIV and inhibition of fusion of HIV-1 to cells differs by IgG subclass, with IgG3 being the most and IgG2 the least effective . In addition, early in vitro studies demonstrated that, while high concentrations of IgG neutralize HIV-1 virions, antibodies at subneutralizing levels can actually enhance viral replication . At low levels of viral opsonization, it is believed that interaction with Fcγ receptors does not initiate endocytosis and intracellular degradation for initiation of an HIV-specific immune response, but it does enhances stability of the virus at the cell surface, allowing infection to proceed [16,30]. In newborns, most IgG is of maternal origin, as infant IgG production is still very low . While IgG1, IgG3, and IgG4 are efficiently transported across the placenta, such that infant levels approximate maternal levels at birth, IgG2 transport is poor . Taken together, the above studies suggest that both qualitative (IgG subclass) and quantitative (IgG level) factors are critical in the interaction of HIV with antibodies in relation to the Fcγ receptor.
In our study, maternal FcγRIIa genotype was not related to perinatal HIV transmission. A possible reason for this lack of effect is that most placental cell types that display class II Fcγ receptors are of fetal origin . Second, most HIV transmission occurs during delivery, as the protective environment of the placenta is compromised and the infant is exposed to maternal fluids, again making the infant rather than the maternal genotype of primary importance .
For logistic reasons (limited sample volume and cost), our assessment of the effect of FcγRIIa polymorphism on HIV disease progression in children was limited in that we were unable to follow CD4 cell count and viral load levels longitudinally. We also were unable to classify individual causes of death and, therefore, all-cause mortality was used as a surrogate for HIV-related mortality. Although mortality was marginally higher in HIV-infected infants with the FcγIIa His131 receptor (Table 3), this tendency remained statistically insignificant and was weakened upon control for other variables related to mortality. The fact that FcγRIIa genotype was unrelated to infant viral load upon HIV conversion, which has been shown to be predictive of disease progression [28,29], further suggests that there truly is no effect of FcγRIIa genotype on progression to AIDS in children. If so, this indicates that the neonatal setting, with its selective transfer of IgG subtypes, may provide a unique environment for FcγRIIa-influenced transmission, with later spread of the virus dominated by other factors.
In summary, we propose the model illustrated in Fig. 1, showing a possible mechanism of the effect of FcγRIIa polymorphism on perinatal HIV-1 transmission. When neonates come into contact with HIV-1 in the context of passively transferred maternal antibodies or are exposed to HIV-1/IgG2 complexes through membrane rupture during labor and delivery, low levels of IgG2 coupled with the inefficiency of IgG2 in HIV neutralization may place infants with the FcγIIa His131 receptor at risk of recognizing subneutralizing HIV-1–IgG2 immune complexes. Attachment of these complexes to the FcγIIa His131 receptor may lead to viral stabilization, resulting in infection followed by a non-specific immune stimulation, which creates an ideal environment for viral replication (Fig. 1). Macrophages expressing the FcγRIIa His131 receptor may then serve as reservoirs for viral dissemination.
In conclusion, our study demonstrates the importance of FcγRIIa polymorphism on susceptibility to perinatal HIV-1 transmission. There is no evidence, however, that FcγRIIa genotype is associated with HIV progression. These findings support the need to investigate further the association of FcγRIIa polymorphism with HIV-1 perinatal transmission and disease progression in different populations.
We are grateful for all of the mothers and children of the Kisumu area who participated in this study. We also thank the field and laboratory staff of the CDC/Kenya Medical Research Institute (KEMRI) who facilitated collection of data and processing of samples. We thank Dr Davy Koech, Director of KEMRI, for his support and his approval with regard to publication of this paper. We appreciate Mary Glenn Fowler, Marc Bulterys, Laurence Slutsker, and Mark Eberhard for their helpful comments on this manuscript.
Sponsorship: This study was supported by the Emerging Infections fund of the Genetics Working Group Project, NCID, CDC and by grant number AOT0483-PH1-2171, HRN-A-00-04-00010-02 from the United States Agency for International Development. K. C. Brouwer holds an Emerging Infectious Diseases Research Fellowship sponsored by the Association of Public Health Laboratories.
Disclaimer: Use of trade names and commercial sources is for identification only and does not imply endorsement by CDC or the US Department of Health and Human Services.
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