To test this hypothesis, plasma samples from 12 superinfected women each assigned to three singly infected women were tested against a panel of eight viruses representing four different HIV-1 subtypes at matched time points post-superinfection (5 years post-initial infection). NAb responses post-superinfection when compared to singly infected individuals (RR = 1.68, CI: 1.232.30, p = 0.001). This was true even after controlling for NAb breadth developed prior to superinfection, contemporaneous CD4+ T cell count and viral load. Similarly, both unadjusted and adjusted analyses showed significantly higher potency in superinfected instances compared to settings. Notably, Resiquimod two superinfected individuals were able to neutralize variants from four different subtypes at plasma dilutions >1300, suggesting that their NAbs show elite activity. Cross-subtype breadth was recognized within a yr of superinfection in both of these individuals, which was within 1.5 years of their initial infection. These data suggest that sequential infections lead to augmentation of the NAb response, a process that may provide insight into potential mechanisms that contribute to the development of antibody breadth. Consequently, a successful vaccination strategy that mimics superinfection may lead to the development of broad NAbs in immunized individuals. == Author Summary == A broad and potent antibody response is considered essential for an effective HIV-1 vaccine that may protect against varied circulating strains. As a result, there is fantastic interest in both the sponsor and viral factors that impact the development of the neutralizing antibody (NAb) response in natural HIV-1 infections. HIV-infected individuals who become superinfected with a second disease from a different resource partner represent unique cases for studying the antibody response, as superinfection displays exposure to different HIV-1 antigenic variants, and hence may provide insight into the development of broadly NAbs. In support of this model, we display here that superinfected individuals develop broader and more potent NAb reactions than singly infected individuals, a result that is likely due to the improved antigenic activation from two viruses compared to one. Our findings remained unchanged after controlling for other factors that have been shown to influence the NAbs response, such as CD4+ T cell count and viral weight. This Resiquimod study demonstrates that superinfection yields antibodies that have the capacity to recognize varied circulating HIV-1 variants. Consequently, further characterization of these superinfected individuals’ NAb reactions could lead to novel insights into pathways that elicit broadly NAbs. == Intro == Multiple studies have shown the potential of HIV-specific neutralizing antibodies (NAbs) FACC to protect against illness using nonhuman primate models[1],[2]. However, it remains unclear how to elicit a NAb response of adequate breadth and potency to protect humans against varied circulating HIV-1 variants, which can differ by several orders of magnitude in neutralization level of sensitivity[1],[2]. Consequently, investigating naturally-occurring antibody reactions that can neutralize viruses across the major viral subtypes remains a major focus of study[3]. In the past few years, multiple HIV-specific broadly neutralizing monoclonal antibodies have been isolated from HIV-infected individuals with elite neutralizing activity[4][8]. This subset of individuals comprises about 1% of chronically-infected individuals and are regarded as elite neutralizers based on their ability Resiquimod to potently neutralize viruses from multiple subtypes[9]. The collection of broad monoclonal antibodies recognized to date, which were isolated more than a decade after initial HIV-1 illness in some cases, have undergone considerable somatic hypermutation, a process that would be hard to mimic having a HIV-1 vaccine[2],[10]. Also, these monoclonal antibodies have been isolated from individuals who were presumably infected with a single HIV-1 strain, although in most cases, the possibility of superinfection (SI) was not addressed. Within singly infected populations, NAb breadth has been positively associated with viral diversity[11]. Consequently, individuals infected with multiple HIV-1 strains as a result of SI by a second resource partner may generate broadly NAbs in response to activation from both viruses. Initially, it was hypothesized that SI resulted from a fragile NAb response that was unable to protect the individual from reinfection. A small study of three SI instances and three viral strains offered some support for this model[12]. However, in a larger study using a panel of 16 viruses from a number of different subtypes, Blish et al. showed no significant variations in the NAb breadth or potency Resiquimod in six superinfected instances Resiquimod immediately before acquisition of the second virus compared to 18 singly infected settings at matched time points[13]. In this study, where the focus was on correlates of safety from SI, the NAb repertoire and breadth developed in the years following SI were not examined. In the past year, two studies have provided evidence of a broadening of the NAb response after SI. Inside a South African individual that became superinfected 1315.