Project Summary Biological sex impacts the immune system, as evidenced by differences between men and women in vaccine- induced humoral responses. There is a considerable gap in knowledge, however, surrounding the immunological responsiveness of transgender people, a population at considerably higher risk for HIV and other STIs. To address this gap, we propose to develop an animal model of feminizing hormone therapy to study the effects of estrogen/anti-testosterone therapy on HIV vaccine-induced immune responses. Cross-sex hormone therapy (XHT) is the most common approach for transition-related care, but immunological studies of transgender individuals are generally limited to case studies. Animal models of gonadectomy and/or supranormal hormone treatment have implicated sex hormones as potent modulators of the immune system, with estrogens generally appearing to enhance immune responses and testosterone inhibiting them. However, ambiguous (and sometimes paradoxical) observations have obscured an overarching consensus for sex hormone effects on immunity. In recent years, mouse models of XHT have provided important insights into effects of prolonged hormone therapy on bone physiology, reproductive function etc., but information on the effects of sex hormone therapy on immune responsiveness is scarce. For this project, we will develop a mouse model of XHT that recapitulates clinical hormone therapy for male-to-female transition in humans. We hypothesize that mice and humans share sets of immune-related genes that are impacted by a feminizing hormone regimen, and that mice undergoing XHT will exhibit altered immune responses to a HIV vaccine compared to control male mice. To test these hypotheses, we will define the relevance of this mouse model to human transition-related care through a single-cell transcriptomics approach to define differentially expressed genes in control and XHT mice, and then compare these cell-type specific sets of differentially expressed genes to those obtained from the same analyses of people undergoing feminizing hormone therapy (pre and two years post-initiation of hormone therapy) (Specific Aim 1). To test the effects of hormone therapy on in vivo vaccine responses, we will immunize control male mice and XHT mice with a HIV vaccine regimen that is highly sensitive to sex-dependent factors (Specific Aim 2). Should this study reveal an immunoregulatory effect of feminizing hormone therapy on HIV vaccine responsiveness in mice, it will provide a new platform for assessing the effects of sex hormones on vaccine responses. Moreover, this animal model could then be used to test various vaccination parameters (adjuvant, dose, interval, etc.) for sex hormone-dependent effects, with the ultimate goal of designing an HIV vaccine that maximizes efficacy but minimizes adverse outcomes.