BASIC SCIENCE Influence of the Toll-Like Receptor 9 1635A/G Polymorphism on the CD4 Count, HIV Viral Load, and Clinical Progression Natalia Soriano-Sarabia, PhD,* Alejandro Vallejo, PhD,† Reposo Ramı ´rez-Lorca, PhD,‡ Marı ´a del Mar Rodrı ´guez,* Ana Salinas,‡ Ildefonso Pulido, MD,§ Marı ´a E. Sa ´ez, PhD,‡ and Manuel Leal, MD, PhD* Objective: To analyze the influence of single-nucleotide poly- morphisms (SNPs) in TLR2 (1892A/C and 2258G/A), TLR4 (896A/G and 1196C/T), and TLR9 (1635A/G) genes on CD4 count, HIV viral load, and clinical progression in a cohort of naive HIV-infected patients. Methods: TLR2, TLR4, and TLR9 SNPs were analyzed in 369 naive HIV-infected patients by real-time polymerase chain reaction and melting curve technology. TLR2 1892C/A and TLR9 1635A/G SNPs were also analyzed in a non–HIV-infected population. Multivariate multiple regression analysis and Cox regression analyses were performed to assess the potential association between the SNPs and the end points. Results: TLR2 and TLR4 SNPs were not associated with the end points of the study. Regarding TLR9 1635A/G SNP, patients with the AA genotype showed statistically lower CD4 count (P = 0.003) and higher HIV viral load (P = 0.0018) compared with AG+GG genotypes at cohort entry. The multivariate analysis showed a sig- nificant association between the 1635AA genotype and both end points. Cox regression analysis showed that HIV clinical progression to clinical stage C and death due to AIDS-related events under anti- retroviral therapy was earlier in patients with the 1635AA genotype (P = 0.035, P = 0.017, respectively). Conclusions: TLR9 1635A/G SNP might have a role in HIV clinical disease progression. Key Words: toll-like receptors, polymorphisms, HIV clinical progression (J Acquir Immune Defic Syndr 2008;00:000–000) INTRODUCTION Toll-like receptors (TLRs) belong to the family of pattern recognition receptors that recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immune responses. 1,2 To date, 10 TLRs and their ligands have been described in humans and are expressed by different cells of the immune system, either at the cell membrane (TLR1, 2, 6, 4, 5, 10) or in endosomes (TLR3, 7, 8, 9). These receptors are structurally characterized by the presence of a Toll/IL-1 receptor motif in the intra- cellular domain. TLR ligation triggers signalling pathways that activate transcription factors, initiating adaptive immune responses. 3,4 Growing data supporting associations between single- nucleotide polymorphisms (SNPs) in TLRs and the increased risk of bacteria and viral infections are being reported. These include TLR2, that recognizes the widest repertoire of pathogen-associated molecular patterns due to its ability to recognize ligands as a heterodimer with TLR1 and TLR6, 5 and TLR4, that is highly specific for lypopolysaccharide, the main component of the gram-negative bacteria cell wall 6 and respiratory syncytial virus. 7 Hence, the association between common SNPs in TLR2 and TLR4 and several infectious diseases has been deeply studied demonstrating that TLR2 Arg753Gln (2258G/A) and Pro631His (1892C/A) and TLR4 Asp299Gly (896A/G) and Thr399Ile (1196C/T) might be involved in the susceptibility to bacterial and/or viral infections. 7–12 In the context of HIV infection, the role of SNPs in TLRs may provide relevant information on HIV pathogenesis because the in vitro activation and signalling through TLR4, 13 TLR2, and TLR9 14 might enhance HIV replication. Further- more, HIV may impair the in vitro immune response after TLR4 15,16 and TLR2 16 stimulation. Only 2 recent studies have analyzed the role of SNPs in TLRs in the clinical context of HIV infection. One of them reported a potential association between the TLR4 Asp299Gly SNP and a higher susceptibility to develop active tuberculosis in HIV-infected patients in Tanzania. 17 The other study reported an association between Received for publication February 6, 2008; accepted June 23, 2008. From the *Laboratories of Immunovirology; and †Molecular Virology, Seville Biomedicine Institute (IBIS), Service of Infectious Diseases, Virgen del Rocı ´o Hospital, Seville, Spain; ‡Department of Structural Genomics, Neocodex SL, Charles Darwin s/n, Isla de la Cartuja, Seville, Spain; and §Department of Internal Medicine, Virgen del Rocı ´o Hospital, Seville, Spain. Supported by Fondo de Investigaciones Sanitarias (PI 060915), Red de Investigacio ´n en SIDA, Instituto de Salud Carlos III (RIS RD06/006/0021), and Servicio Andaluz de Salud (0241/2005). Presented at the 11th European AIDS Conference/EACS, October 24–27, 2007, Madrid, Spain. Correspondence to: Manuel Leal, MD, PhD, Service of Infectious Diseases, Virgen del Rocı ´o Hospital, 410013, Seville, Spain (e-mail: mleal@cica.es). Copyright Ó 2008 by Lippincott Williams & Wilkins J Acquir Immune Defic Syndr  Volume 00, Number 0, October 1, 2008 1