The Journal of Immunology Low-Level Exposure to HIV Induces Virus-Specific T Cell Responses and Immune Activation in Exposed HIV-Seronegative Individuals Clara Restrepo,* Norma I. Rallo ´n,* Jorge del Romero, † Carmen Rodrı ´guez, † Victoria Hernando, † Mariola Lo ´pez,* Alejandra Peris,* Sara Lozano,* Jose ´ M. Sempere-Ortells, ‡ Vincent Soriano,* and Jose ´ M. Benito* HIV-specific T cells response and T cell activation are frequently seen in exposed seronegative individuals (ESN). In this study, we report HIV-specific response and level of T cell activation in ESN partners of HIV-infected patients presenting low or undetectable levels of HIV-RNA. We evaluated 24 HIV-serodiscordant couples. ESN were classified into three categories of exposure to HIV (very low, low, and moderate-high), considering levels of HIV-RNA in their infected partner and frequency of sexual high-risk practices within the last 12 mo. HIV-specific T cell responses and activation levels in T cell subsets were evaluated by flow cytometry. We reported that 54% of ESN had detectable HIV-specific T cells response, being the highest prevalence seen in the low exposure group (64%). Several T cell subsets were significantly increased in ESN when compared with controls: CD4 + CD38 + ( p = 0.006), CD4 + HLA-DR 2 CD38 + ( p = 0.02), CD4 + CD45RA + CD27 + HLA-DR 2 CD38 + ( p = 0.002), CD8 + CD45RA + CD27 + CD38 2 HLA-DR + ( p = 0.02), and CD8 + CD45RA + CD27 2 CD38 + HLA-DR + ( p = 0.03). Activation of CD8 + T cells was increased in ESN with detectable HIV T cell responses compared with ESN lacking these responses ( p = 0.04). Taken together, these results suggest that persistent but low sexual HIV exposure is able to induce virus-specific T cells response and immune activation in a high proportion of ESN, suggesting that virus exposure may occur even in conditions of maximal viral suppression in the HIV-infected partner. The Journal of Immunology, 2010, 185: 982–989. S ome individuals remain HIV-seronegative despite repeated unprotected exposures to the virus (1). These exposed but seronegative (ESN) individuals include commercial sex workers (2), men having sex with men (3), injection drug users (4), hemophiliacs who received contaminated blood preparations (5), health care workers with accidental percutaneous exposure to infected blood (6), infants born to HIV-infected mothers (7), and individuals with HIV-infected heterosexual partners (8–11). The mechanisms of protection from infection in ESN individuals are largely undefined and most likely are multifactorial (1, 12). Host factors, including coreceptor susceptibility (13) and adaptive im- munity (14), as well as viral factors, including replication level (15), have all been involved in this phenomenon. The role of HIV-specific adaptive immunity in resistance to in- fection in ESN has attracted much attention. High expression levels of b-chemokines, as well as HIV-specific IFN-g or IL-2– secreting T cells have been recognized in different cohorts of ESN individuals (16–21). However, others have not confirmed these findings acknowledging a lack of HIV-specific immune responses in ESN individuals (3). Detection of HIV-specific cyto- toxic T lymphocytes in these individuals may simply be the con- sequence of exposure to the virus and not necessarily be associated with resistance to infection (20, 21). On the other hand, the recog- nition of significant mucosal and systemic cellular activation in ESN individuals clearly supports that viral exposure has occurred (22, 23). Most studies conducted in ESN individuals have examined het- erogeneous populations, given the difficulties to recruit such indi- viduals (2–6), have focused on HIV-specific immune responses, and have used cross-sectional designs. Thus, the interpretation of results in terms of a protective role of immune responses on resistance to HIV infection is often difficult. In contrast, most studies examining ESN individuals with stable sexual HIV-infected partners have se- lected only couples with an HIV-partner presenting high-level vire- mia (8, 9). In this regard, and given the cross-sectional design of these studies, the demonstration of HIV-specific immune responses cannot be considered as a proof of resistance to infection as it can only reflect exposure to the virus. Capture of viral particles or pro- teins by certain specialized cells rather than infection is necessary to elicit cell-mediated immune responses (24). To our knowledge, there are no studies examining the prevalence of detectable HIV-specific T cells response and systemic immune activation in ESN individuals split out according to the level of viral exposure. Materials and Methods Study population Participants were recruited at Centro Sandoval, a sexually transmitted dis- ease (STD) clinic located in Madrid, Spain. The study population consisted of ESN persons in stable relationship with their HIV-infected partner on reg- ular follow-up. Criteria for inclusion were sexual partnership with an HIV- infected individual during at least 12 mo prior to the inclusion. The HIV *Infectious Diseases Department, Hospital Carlos III; † Centro Sanitario Sandoval, Madrid; and ‡ Departamento de Bitotecnologı ´a, Universidad de Alicante, Alicante, Spain Received for publication January 25, 2010. Accepted for publication April 30, 2010. This work was supported by grants from Fundacio ´n para la Investigacio ´n y la Pre- vencio ´n del SIDA en Espan ˜a, Fundacio ´n Investigacio ´n y Educacio ´n en SIDA, and Fondo de Investigacio ´n Sanitaria (grants ISCIII-REMC RD06/006 and PI050283). Address correspondence and reprint requests to Dr. Clara Restrepo, Hospital Carlos III, Servicio de Enfermedades Infecciosas, Sinesio Delgado 10, 28029, Madrid, Spain. E-mail address: clararestrepog@gmail.com Abbreviations used in this paper: A, always; ESN, exposed seronegative individuals; F, frequently; HC, healthy control; IQR, interquartile range; N, never; NA, not avail- able; S, sporadic; STD, sexually transmitted disease. Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000221