br Materials and Methods br

Materials and Methods
Results
Discussion We sought to define properties of anti-Env Abs critical for mucosal HIV-1 protection, addressing two major issues in the vaccine field: the need to prioritize induction of IgA rather than IgG, and broadly-neutralizing activities rather than other antiviral functions. Given the dearth of female macaques and the extensive mAb panel available, we adapted our human vaginal explant model (Hladik et al., 2007, McElrath et al., 2010), containing HIV target and effector Z DEVD FMK Supplier in their proper tissue architecture (Hladik et al., 2007, McKinnon et al., 2014), to investigate Ab-mediated protection during the earliest events in vaginal infection, before seeding the viral reservoir. Only the bnAbs consistently protected against viral challenge; mAbs with known Fc-effector activities but lacking potent neutralizing function were not protective when tested alone or together. Of note, Cheeseman and colleagues found similar results to those reported here in ex vivo explants (Cheeseman et al., 2016b). We developed a secreted NanoLuc™ reporter virus system building upon methods for creating replication-competent HIV-1 IMCs expressing the Renilla Luc reporter (Edmonds et al., 2010, Seay et al., 2015). This system has the advantage of monitoring HIV-1 infectivity kinetics using a simple, two-step Luc assay using different Envs in an isogenic virus background. Thus, this ex vivo explant assay format is well-suited to assess Ab-mediated inhibition and may be tailored to screen clinically-infused bnAbs and vaccine-induced polyclonal Abs with selected virus panels. We determined relative tissue distributions of FcR-expressing leukocytes to define their accessibility in the ex vivo infectivity model to engage in Fc-binding Ab-mediated protection. We and others have demonstrated that CD4+ T cells are the first and predominant target cells for HIV-1 infection in the vaginal epithelium (Hladik et al., 2007), where few FcR-bearing effector cells other than DCs, particularly LCs, were found. Though well-positioned to engage Ab-bound virions or infected cells, the in vivo outcomes of such interactions are unclear due to the potential for HIV-1 to dysregulate DC function (Hladik and McElrath, 2008, Ahmed et al., 2015). By contrast, monocytes and macrophages expressing FcRs for IgG and IgA were relatively abundant, but were localized beneath the epithelium. Moreover, NK cells capable of mediating ADCC were rare throughout the tissue, in line with recent reports (Sips et al., 2016, Cheeseman et al., 2016a). Of note, DCs and monocytes and macrophages are also potential target cells and IgG nnAbs and nAbs have been shown in vitro to inhibit their infection by FcR-dependent (Holl et al., 2004, Holl et al., 2006a, Holl et al., 2006b, Peressin et al., 2011, Perez et al., 2009) and independent mechanisms (Lederle et al., 2014). Conceivably, these interactions could provide a second line of defense. Importantly, the relative frequency and distribution of monocytes, macrophages and NK cells in vaginal tissue may limit their timely recruitment to efficiently engage with Abs to prevent initial CD4+ T cell infection in this model. This may partially explain why even a cocktail of IgG nnAbs was ineffective, despite previously described synergy and enhanced cooperativity in HIV-1 inhibition by nnAbs (Moog et al., 2014, Pollara et al., 2014). By contrast, bnAbs can exert immediate inhibition, the potency of which corresponds to their in vitro neutralization potencies against the challenge virus. The balance of inhibitory and activating FcγRII, the most prevalent FcR in these tissues, and innate viral responses can also influence the net impact of HIV-1 Abs on early infection. These factors warrant further investigation but were beyond the scope of this study. We extended our investigations to a high-dose rectal challenge model as vaginal and rectal mucosae are anatomically and immunologically distinct compartments (McElrath et al., 2013, Mitchell et al., 2014, Cheeseman et al., 2016a, Sips et al., 2016), and the estimated risk of receptive anal intercourse is greater than that for receptive vaginal intercourse (Patel et al., 2014). Also, because neither the GalCer-blocking CH38 IgA2 nor ADCC-mediating CH54 IgG capture infectious virions, their greatest activity may be against cell-associated Env and better assessed in an in vivo challenge model. The mAb infusion schedules were designed to maintain mAb levels during the window that the first foci of infection would be established. Despite the potential for effector cell recruitment from the periphery, both nnAbs had no impact on SHIV acquisition, replication or on the number of transmitted founder viruses, unlike another C1-specific mAb (A32 IgG) (Santra et al., 2015). CH54 IgG and A32 IgG bind overlapping but distinct epitopes and have different ADCC breadth profiles (Bonsignori et al., 2012), which may explain their differing abilities to limit transmitted founder variants. By contrast, all CH31 bnAbs protected against rectal exposure, and the most potent neutralizing variant (CH31 IgG) was the most beneficial whether administered locally or systemically. Thus, the results from both the ex vivo and in vivo models were highly consistent and extend previous NHP findings indicating that bnAbs likely confer the most effective Ab-mediated protection (Burton et al., 2011, Dugast et al., 2014). However, a low dose intrarectal challenge model (Gautam et al., 2016) could be of interest to ascertain any protective activity of nnAbs individually or in combination.