The human phase 2B RV144 ALVAC-HIV vCP1521/AIDSVAX B/E vaccine trial, held in Thailand, resulted in an estimated 31.2% efficacy against HIV infection. By contrast, vaccination with VAX003 (consisting of only AIDSVAX B/E) was not protective. Because protection within RV144 was observed in the absence of neutralizing antibody activity or cytotoxic T cell responses, we speculated that the specificity or qualitative differences in Fc-effector profiles of nonneutralizing antibodies may have accounted for the efficacy differences observed between the two trials. We show that the RV144 regimen elicited nonneutralizing antibodies with highly coordinated Fc-mediated effector responses through the selective induction of highly functional immunoglobulin G3 (IgG3). By contrast, VAX003 elicited monofunctional antibody responses influenced by IgG4 selection, which was promoted by repeated AIDSVAX B/E protein boosts. Moreover, only RV144 induced IgG1 and IgG3 antibodies targeting the crown of the HIV envelope V2 loop, albeit with limited coverage of breakthrough viral sequences. These data suggest that subclass selection differences associated with coordinated humoral functional responses targeting strain-specific protective V2 loop epitopes may underlie differences in vaccine efficacy observed between these two vaccine trials.

Phagocytosis can be induced via the engagement of Fcγ receptors by antibody-opsonized material. Furthermore, the efficiency of antibody-induced effector functions has been shown to be dramatically modulated by changes in antibody glycosylation. Because infection can modulate antibody glycans, which in turn modulate antibody functions, assays capable of determining the induction of effector functions rather than neutralization or titer provide a valuable opportunity to more fully characterize the quality of the adaptive immune response. Here we describe a robust and high-throughput flow cytometric assay to define the phagocytic activity of antigen-specific antibodies from clinical samples. This assay employs a monocytic cell line that expresses numerous Fc receptors: including inhibitory and activating, and high and low affinity receptorsallowing complex phenotypes to be studied. We demonstrate the adaptability of this highthroughput, flow-based assay to measure antigen-specific antibody-mediated phagocytosis against an array of viruses, including influenza, HIV, and dengue. The phagocytosis assay format further allows for simultaneous analysis of cytokine release, as well as determination of the role of specific Fcγ-receptor subtypes, making it a highly useful system for parsing differences in the ability of clinical and vaccine induced antibody samples to recruit this critical effector function.

SUMMARY The global diversity of HIV-1 represents a critical challenge facing HIV-1 vaccine development. HIV-1 mosaic antigens are bioinformatically optimized immunogens designed for improved coverage of HIV-1 diversity. However, the protective efficacy of global HIV-1 vaccine antigens has not previously been evaluated. Here we demonstrate the capacity of bivalent HIV-1 mosaic antigens to protect rhesus monkeys against acquisition of heterologous challenges with the difficult-to-neutralize simian-human immunodeficiency virus SHIV-SF162P3. Adenovirus/poxvirus and adenovirus/adenovirus vector-based vaccines expressing HIV-1 mosaic Env, Gag, and Pol afforded a significant reduction in the per-exposure acquisition risk following repetitive, intrarectal SHIV-SF162P3 challenges. Protection against acquisition of infection was correlated with vaccine-elicited binding, neutralizing, and functional non-neutralizing antibodies. These data demonstrate the protective efficacy of HIV-1 mosaic antigens and suggest a potential strategy towards the development of a global HIV-1 vaccine. Moreover, our findings suggest that the coordinated activity of multiple antibody functions may contribute to protection against difficult-to-neutralize viruses.

The immune tolerance to rat kidney allografts induced by a perioperative treatment with anti-CD28 Abs is associated with a severe unresponsiveness of peripheral blood cells to donor Ags. In this model, we identified an accumulation in the blood of CD3−class II−CD11b+CD80/86+ plastic-adherent cells that additionally expressed CD172a as well as other myeloid markers. These cells were able to inhibit proliferation, but not activation, of effector T cells and to induce apoptosis in a contact-dependent manner. Their suppressive action was found to be under the control of inducible NO synthase, an enzyme also up-regulated in tolerated allografts. Based on these features, these cells can be defined as myeloid-derived suppressor cells (MDSC). Interestingly, CD4+CD25highFoxP3+ regulatory T cells were insensitive in vitro to MDSC-mediated suppression. Although the adoptive transfer of MDSC failed to induce kidney allograft tolerance in recently transplanted recipients, the maintenance of tolerance after administration of anti-CD28 Abs was found to be dependent on the action of inducible NO synthase. These results suggest that increased numbers of MDSC can inhibit alloreactive T cell proliferation in vivo and that these cells may participate in the NO-dependent maintenance phase of tolerance.

In vivo, the activity of antibodies relies critically on properties of both the variable domain, responsible for antigen recognition, and the constant domain, responsible for innate immune recognition. Here, we describe a flexible, microsphere-based array format for capturing information about both functional ends of disease-specific antibodies from complex, polyclonal clinical serum samples. Using minimal serum, we demonstrate IgG subclass profiling of multiple antibody specificities. We further capture and determine the subclass of epitope-specific antibodies. The data generated in this array provides a profile of the humoral immune response with multi-dimensional metrics regarding properties of both variable and constant IgG domains. Significantly, these properties are assessed simultaneously, and therefore information about the relationship between variable and constant domain characteristics is captured, and can be used to predict functions such as antibody effector activity.

Eliciting neutralizing antibodies is thought to be a key activity of a vaccine against human immunodeficiency virus (HIV). However, a number of studies have suggested that in addition to neutralization, interaction of IgG with Fc gamma receptors (Fc␥R) may play an important role in antibody-mediated protection. We have previously obtained evidence that the protective activity of the broadly neutralizing human IgG1 anti-HIV monoclonal antibody (MAb) b12 in macaques is diminished in the absence of Fc␥R binding capacity. To investigate antibody-dependent cellular cytotoxicity (ADCC) as a contributor to Fc␥R-associated protection, we developed a nonfucosylated variant of b12 (NFb12). We showed that, compared to fully fucosylated (referred to as wild-type in the text) b12, NFb12 had higher affinity for human and rhesus macaque Fc␥RIIIa and was more efficient in inhibiting viral replication and more effective in killing HIV-infected cells in an ADCC assay. Despite these more potent in vitro antiviral activities, NFb12 did not enhance protection in vivo against repeated low-dose vaginal challenge in the simian-human immunodeficiency virus (SHIV)/macaque model compared to wild-type b12. No difference in protection, viral load, or infection susceptibility was observed between animals given NFb12 and those given fully fucosylated b12, indicating that Fc␥R-mediated activities distinct from Fc␥RIIIa-mediated ADCC may be important in the observed protection against SHIV challenge.

While development of an HIV vaccine that can induce neutralizing antibodies remains a priority, decades of research have proven that this is a daunting task. However, accumulating evidence suggests that antibodies with the capacity to harness innate immunity may provide some protection. While significant research has focused on the cytolytic properties of antibodies in acquisition and control, less is known about the role of additional effector functions. In this study, we investigated antibody-dependent phagocytosis of HIV immune complexes, and we observed significant differences in the ability of antibodies from infected subjects to mediate this critical effector function. We observed both quantitative differences in the capacity of antibodies to drive phagocytosis and qualitative differences in their Fc␥R usage profile. We demonstrate that antibodies from controllers and untreated progressors exhibit increased phagocytic activity, altered Fc domain glycosylation, and skewed interactions with Fc␥R2a and Fc␥R2b in both bulk plasma and HIV-specific IgG. While increased phagocytic activity may directly influence immune activation via clearance of inflammatory immune complexes, it is also plausible that Fc receptor usage patterns may regulate the immune response by modulating downstream signals following phagocytosis-driving passive degradation of internalized virus, release of immune modulating cytokines and chemokines, or priming of a more effective adaptive immune response.

While the development of a protective HIV vaccine is the ultimate goal of HIV research, to date only one HIV vaccine trial, the RV144, has successfully induced a protective response. The 31% protection from infection achieved in the RV144 trial was linked to the induction of non-neutralizing antibodies, able to mediate ADCC, suggestive of an important role of Fc-mediated functions in protection. Likewise, Fc-mediated antiviral activity was recently shown to play a critical role in actively suppressing the viral reservoir, however, the Fc-effector mechanisms within tissues that provide protection from or after infection are largely unknown. Here we aimed to define the landscape of effector cells and Fc-receptors present within vulnerable tissues. We found negligible Fc-receptor expressing NK cells in the female reproductive and gastrointestinal mucosa. Conversely, Fc-receptor expressing macrophages were highly enriched in most tissues, but neutrophils mediated superior antibody-mediated phagocytosis. Modifications in Fc domain of VRC01 antibody increased phagocytic responses in both phagocytes. These data suggest that non-ADCC mediated mechanisms, such as phagocytosis and neutrophil activation, are more likely to play a role in preventative vaccine or reservoir-eliminating therapeutic approaches.