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Research Letter

Toll-like receptor 9 polymorphism is associated with increased Epstein–Barr virus and Cytomegalovirus acquisition in HIV-exposed infants

Beima-Sofie, Kristina; Wamalwa, Daltonb; Maleche-Obimbo, Elizabethb; Lingappa, Jairam R.a,c,d; Mackelprang, Romela; Gantt, Sorene; John-Stewart, Gracea,c,d,f; Casper, Coreya,c,g; Slyker, Jennifer A.a,f

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doi: 10.1097/QAD.0000000000001680
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Toll-like receptors (TLRs) recognize conserved antigenic motifs, enabling broad nonspecific pathogen recognition. Single nucleotide polymorphisms (SNPs) in TLR genes may impact the natural history of viral infections, including HIV [1–8] and human herpesviruses [9]. The TLR9 1635 (rs352140) locus is of interest because having one or more copies of 1635A is associated with increased risk of HIV acquisition in discordant couples [4] and infants [1]. Paradoxically, 1635AA was associated with lower HIV viral loads or slower CD4+ decline/HIV progression compared with 1635GG in some studies [2,6], including the infant cohort in which it was found to be associated with increased HIV acquisition risk [1]. However, other studies found 1635AA to be associated with lower CD4+ cell count or higher HIV viral loads, suggesting differences in genetically distinct populations [5,7].

Cytomegalovirus (CMV) and Epstein–Barr virus (EBV) are often acquired in infancy [10–12], establish persistent infection with prolonged shedding, and are opportunistic infections in HIV [10,13–17]. To date, there are few studies of TLR polymorphisms in herpesvirus infections. Mutations in TLR3 increase susceptibility to herpes simplex virus encephalitis in children after primary infection [18]. SNPs in TLR2, TLR4, and TLR9 have been examined in the context of congenital CMV; TLR9 1635A was associated with risk in Polish [19–21], but not Japanese infants [22]. We are aware of no published studies examining TLR9 polymorphisms and primary EBV infection.

In a Kenyan infant cohort, having one or more copies of the TLR9 1635A allele was found to be associated with increased HIV acquisition risk and lower HIV viral load [1]. In this secondary analysis, we evaluate whether 1635A is also associated with EBV and CMV risk or viremia. HIV-infected mother–infant pairs participated in an observational study in Nairobi, Kenya from 1999 to 2003 [23,24]. HIV, CMV, and EBV DNA levels were assessed from birth to 1 year in HIV-exposed uninfected (HEU) infants and HIV-infected (HIV+) infants, with sampling at birth, 1, 3, 6, 9, and 12 months of age; HIV+ children were followed for an additional year at quarterly intervals. Follow-up [23,25] and CMV [10] and EBV [11] assays are detailed elsewhere. For host genetic studies, infant DNA was extracted from cryopreserved blood and genotyped for TLR9 1635 (rs352140) and a set of genetic ancestry informative markers (AIMS) using an Illumina Custom Oligo Pooled Assay microarray platform (San Diego, California, USA) [1].

All analyses were conducted using StataSE 14.0 (College Station, Texas, USA), using two-tailed tests with alpha = 0.05. Based on previous associations between TLR9 and HIV, initial analyses were conducted assuming a dominant model of inheritance; we confirmed this was also the most appropriate inheritance model for CMV and EBV. The Mann–Whitney U test was used to compare the baseline (first detectable) viral DNA level in plasma between genotype groups, because this time-point had the most data and was the peak level for most infants. The analysis for viral acquisition differed for EBV and CMV because the pattern of acquisition differed by virus and HIV status. As EBV transmissions were dispersed over the first 2 years [11], Cox proportional hazards regression was performed to assess the risk of EBV infection, Kaplan–Meier was used to estimate probabilities of infection at 12 months, and the z test was used to compare probabilities between genotypes. As nearly all infants were CMV positive by 3 months of age, there were not adequate time-points for a time-to-event analysis [10]; Pearson's Chi-squared test was used to compare the proportion of infants with CMV detected by 1 month between TLR genotypes. AIMS were homogenous in this population and did not affect point estimates of association; given the small sample size, these were excluded from final analyses (data not shown).

The study cohort of 368 infants with host genetic data has been described elsewhere [1]. Prevalence of the 1635A allele was 49% (180/368) in the overall cohort, with 188 GG (51%), 136 GA (37%), and 44 AA (12%).

CMV and TLR9 genotype data were available in a subset 37 HIV+ infants and 19 HEU. At 1 month of age, 42% of HIV+ infants with one or more copies of the 1635A allele had acquired CMV, compared with 11% in infants with the 1635GG genotype (P = 0.03; Table 1). We did not detect a difference in baseline CMV viral load by TRL9 genotype in either the HIV+ or HEU infants.

Table 1
Table 1:
Transmission and viral containment in HIV infected and exposed uninfected infants by Toll-like receptor 9 allele polymorphism.

EBV and TLR9 genotype data were available in 62 HIV+ and 42 HEU infants. The probability of EBV infection by 12 months of age was 0.82 in HIV+ infants with one or more copies of 1635A, and 0.67 in infants with two copies of the 1635G allele (P = 0.2). The 1635A variant was not associated with EBV risk, univariately [hazard ratio (HR) = 1.5, 95% confidence interval (CI) = 0.74–2.9; P = 0.3], or adjusting for maternal CD4+ percentage at 32 weeks gestation [adjusted (a)HR = 1.4, 95% CI = 0.71–2.7; P = 0.3]. However, infants with one or more copies of the 1635A allele had a median baseline EBV viral load that was 0.4 log10 copies/ml lower than infants with two copies of the 1635G allele (P = 0.02).

In HEU infants, having one or more copies of the 1635A allele was associated with a 4.2-fold increased risk of EBV acquisition univariately (HR = 4.2, 95% CI = 1.2–15; P = 0.02), and adjusting for maternal CD4+ percentage (aHR = 4.2, 95% CI = 1.2–15; P = 0.03). The probability of EBV infection by 12 months of age was 0.67 in infants with one or more copies of 1635A and 0.21 in infants with two copies of 1635G (P = 0.003).

This small study suggests polymorphisms in TLR9 1635 may alter defenses against herpesvirus infections during infancy. Importantly, these effects appear to be modified by HIV infection, which has global and profound impacts on host immunity. Immunosuppression or unmeasured maternal factors may explain some of the differences observed between HIV+ and HEU infants. The A/G substitution is synonymous and occurs in exon 2 of the TLR9 gene, and it is unknown whether this SNP affects TLR9 expression or reflects changes in another gene in linkage disequilibrium. Studies to determine the effect of 1635A on TLR9 expression and function are needed to understand its impact on acquisition and viral control.


We would like to acknowledge the CTL study research personnel, laboratory staff, and data management teams in Nairobi, Kenya, and Seattle, Washington; the Department of Paediatrics and Child Health at Kenyatta National Hospital for providing facilities for laboratory and data analysis; Julie Overbaugh and Sandy Emery for provision of HIV diagnostics and viral studies; Meei-Li Huang for the EBV assays, and the University of Washington Northwest Genomics Center and the Center for Clinical Genomics at the University of Washington for technical assistance. We thank the Kizazi Working Group (UW Global WACh) for comments and insights provided during article development. Most of all, we thank the women and children who participated in the study.

The publication was made possible with support from the National Institutes of Health (NIH) awards K01AI087369 (NIAID, PI J.A.S.), R21AI073115 (NIAID, PI J.R.L.), R01HD023412 (G.J.-S.), U54 CA190146 (C.C.), and K24HD054314 (NICHD, PI G.J.-S.). K.B.-S. was supported by the University of Washington (UW) Institute of Translational Health Sciences Multidisciplinary Clinical Research Training Program (TL1 TR 000422) and the Fogarty International Clinical Research Scholars Program (grant number 5 R24 TW007988) from National Institutes of Health. This research was also supported by the UW Center for AIDS Research (CFAR) (new investigator and HIV-associated malignancy awards), an NIH funded program (P30AI027757) and the UW Global Center for Integrated Health of Women, Adolescents and Children (Global WACh) and the Canadian Institutes for Health Research (grant no. 136825; S.G.). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the funders.

Role of the funding source: The funding sources were not involved in the analyses or interpretation of data.

Conference presentation: The work was presented in part at the 2016 Conference on Retroviruses and Opportunistic Infections (CROI), poster no. 780.

Authors’ contributions: K.B.-S., R.M., and J.R.L. conducted the genomics studies and analysis, J.A.S. conducted the herpesvirus studies and analyses, G.J.-S./D.W. and E.M.-O. designed the cohort study and provided the clinical data and specimens, S.G. and C.C. provided expertise in the interpretation of the herpes data and manuscript preparation, J.A.S. and K.B.-S. wrote the article together.

Conflicts of interest

There are no conflicts of interest.


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