If we assume that past smoking behavior causes HPV contamination, conditioning on contamination status at baseline to obtain our at risk population opens the possibility of selection bias through an unmeasured risk factor for HPV contamination (42, 43). and a non-mediated direct effect representing all other potential mechanisms of smoking. Results Compared to never smokers, current smokers had increased odds of HPV-16 contamination by the antibody-mediated indirect effect (odds ratio=1.29, 95% confidence interval: 1.11, 1.73); the estimated direct effect was very imprecise (OR=0.57, 95% CI: 0.26, 1.13). We observed a stronger estimated indirect effect among women who smoked at least half a pack of smokes daily (OR=1.61, 95% CI: 1.27, 2.15) than among women who smoked less than that threshold (OR = 1.09; 95% CI: 0.94, 1.44). Conclusions This is the first study to directly test the mechanism underlying smoking as an HPV cofactor. The results support current smoking as a risk factor earlier in the natural history of HPV, and are consistent with the hypothesis that smoking increases the risk of a subsequent contamination by reducing immunity. Keywords: Human Papillomavirus, HPV, Smoking, Antibodies, Mediation, Mechanism, Indirect Effect Introduction Human papillomavirus (HPV) infections are commonly acquired shortly after sexual initiation and are the necessary cause of cervical carcinogenesis (1). Most infections are cleared within 1-2 years, but some persist. Women with a persistent carcinogenic HPV contamination are at risk of developing precancerous lesions that may progress to cervical cancer (1, 2). Some infected women will develop antibodies, which could protect against a subsequent contamination by that HPV type (3-6). Risk factors are associated with acquiring antibodies from an HPV contamination. Greater sexual activity increases a woman’s exposure to HPV and thus her opportunity to develop antibodies (7, 8). Other factors, such as smoking, may impair a woman’s antibody response (9-11). For smoking, a diminished antibody response could be a JNJ-10397049 consequence of an impaired immune system. Smoking affects both the cellular and humoral immune response; it can reduce cytokines, natural killer cells, and immunoglobulins (12). Impairing the cellular response could lead to a persistent contamination by inadequate clearance of HPV; while impairing the humoral response could lead to a subsequent contamination by inadequate antibody protection. Despite smoking being associated with a lower natural antibody response, it is unclear whether it results in an increased risk of subsequent HPV contamination. To directly test that causal mechanistic research question in human subjects requires an analytic technique called mediation analysis. In a secondary analysis within a large clinical trial, we sought to evaluate the role of smoking and naturally acquired antibodies in subsequent HPV-16 infections. We posit a causal model of smoking, HPV antibodies, and HPV subsequent contamination to estimate two causal pathways using mediation analysis: 1) a smoking impaired antibody-mediated pathway; and 2) an alternative non-mediated pathway representing all other potential mechanisms of smoking. Methods Study populace The Atypical Squamous Cells of Undetermined Significance (ASCUS) Low-Grade Squamous JNJ-10397049 Intraepithelial Lesion (LSIL) Triage Study (ALTS) was a multicenter randomized controlled trial conducted by the National Malignancy Institute from 1996 to 1998 C before the development of the HPV vaccine C comparing clinical management strategies for Colec10 women with ASCUS or LSIL cytology results (13). The study enrolled 3,488 women with ASCUS and 1,572 with LSIL at four clinical centers in the United States and followed them for two years, during which the centers collected cervical specimens at six month intervals to test for HPV. In addition to completing a baseline questionnaire, 2,736 women also donated baseline blood samples allowing for the serological testing required for this analysis. We also required a populace that was at risk for a subsequent HPV-16 contamination during follow-up, meaning that each subject had been JNJ-10397049 previously exposed to HPV-16. Given the ALTS inclusion criteria of ASCUS or LSIL, we assumed all subjects had a past exposure to HPV-16, thus had the opportunity to develop antibodies; we address this assumption by a sensitivity analysis described in the statistical analyses. To create our at risk population from those who donated blood, we excluded women who were not HPV-16 unfavorable at baseline (n = 271); our results do not change if we include baseline HPV-16 positive women in the analytical study population JNJ-10397049 after a negative HPV test during follow-up. We additionally excluded women with cervical intraepithelial neoplasia grade 2 or more severe because they are further along the natural history (n = 487). Two subjects were missing smoking information and were excluded, leaving the final analytical cohort with 1,976 women of whom 131 tested positive for HPV-16 during follow-up. Our exposure, smoking status (never [referent], former, current) and our mediator, HPV antibody status (positive, unfavorable) were defined from the baseline questionnaire asking Do you currently.
