These data are consistent with a recent report showing that anti-Ad5 neutralizing antibody is not associated with risk of HIV infection in the absence of vaccination [14]. the Step trial volunteers was analyzed using chimeric rAd5/35 vectors to characterize their specificity for Ad5 fiber and non-fiber external (capsid) proteins. Immune responses and HIV seropositivity were correlated with the specificity of Ad5-neutralizing antibodies. Neutralizing antibodies induced by the vaccine in Ad5 seronegative subjects were directed preferentially to Ad5 capsid proteins, although some fiber-neutralizing antibodies could be detected. Pre-vaccination Ad5 serostatus did not affect the capsid-directed response after three vaccinations. In contrast, anti-fiber antibody titers were significantly higher in volunteers who were Ad5 seropositive prior to vaccination. Those Ad5 seropositive subjects who generated anti-capsid responses showed a marked reduction in vaccine-induced CD8 responses. Unexpectedly, anti-vector immunity differed qualitatively in Ad5 seropositive participants who became HIV-1 infected compared to uninfected case controls; Ad5 seropositive participants who later acquired HIV had lower neutralizing antibodies to capsid. Moreover, Ad35 seropositivity was decreased in HIV-infected subjects compared with uninfected case controls, while seroprevalence Itga10 for other serotypes including Ad14, Ad28 and Ad41 was comparable in both groups. Conclusions Together, these findings suggest that the case subjects were less immunologically responsive prior to contamination. Subjects infected during the Step trial had qualitative differences in immunity that increased their risk of HIV-1 contamination impartial of ZINC13466751 vaccination. Introduction The Step ZINC13466751 study was a phase IIB clinical trial designed to test a recombinant adenovirus 5 (rAd5) based HIV vaccine in 3000 participants who were seronegative or seropositive for pre-existing Ad5 neutralizing antibodies. An interim analysis of Step study participants revealed no beneficial effect of vaccination on HIV viral load or acquisition of contamination in vaccinated individuals vs. placebo controls and also showed a pattern towards increased HIV contamination after vaccination in uncircumcised men with pre-existing Ad5 neutralizing antibodies [1], [2]. Thus, understanding the nature and immune effects of Ad5 seropositivity in humans is important for the development of rAd-based vaccines against AIDS. Ad5 viruses are non-enveloped virions composed of fiber and two major capsid proteins, hexon and penton base (penton), all of which are uncovered around the virion surface. Neutralizing antibodies to these proteins mediate viral inactivation. Specifically, anti-fiber antibodies can prevent viral entry, and antibodies to other capsid proteins can also interfere with viral uptake and viral endosomal escape during cell ZINC13466751 contamination [3]. These antibodies also synergize with each other to achieve maximum viral neutralization [3], [4]. Prior research on pre-existing neutralizing antibodies to Ad5 in participants of this trial established the titer of neutralizing antibodies to the whole Ad5 virion [1], [2]. We have previously developed chimeric rAd vectors that can be used to analyze the role of specific neutralizing antibodies against individual viral proteins [5]. The panel of rAd reporters included two chimeric vectors and the parental, unmodified rAd5 and rAd35 vectors. The rAd35 vector was used as the control backbone because human sera rarely neutralize this serotype at high titers and participants seropositive for this control backbone confound the analysis and must be excluded from the subsequent immune analysis. The two chimeric vectors are rAd5 Fiber (F)35 and rAd35 F5 vectors, with the fiber of Ad35 grafted onto rAd5 or with the fiber of Ad5 grafted onto rAd35, respectively. rAd5 F35 vectors were used to detect neutralizing antibodies to Ad5 capsid and rAd35 F5 vectors were used to detect anti-Ad5 fiber neutralizing antibodies. The properties of these reporter vectors reflect those found on the native Ad serotypes and therefore can be used to ZINC13466751 detect the specificity and function of the respective viral components and analyze anti-Ad immunity ZINC13466751 in humans [5]..