ADCC mediating antibodies target a wide range of epitopes

ADCC-mediating FLAG tag target a wide range of epitopes on the envelope (Env) protein, as well as epitopes on Gag (Grunow et al., 1995), Pol (Isitman et al., 2012), Nef (Yamada et al., 2004) and Vpu (Wren et al., 2013; Tiemessen et al., 2009) proteins, though the significance of ADCC activity directed against proteins other than Env remains unclear. Within Env, ADCC-mediating antibodies target epitopes similar to neutralizing antibodies, including the membrane proximal external region (MPER), CD4 binding site (CD4bs) and V1-V2 and V3 regions as well as unique epitopes on the gp120 inner domain naturally occluded by gp41 and exposed following CD4 binding (CD4-induced [CD4i] epitopes) (reviewed in (Pollara et al., 2013; Gach et al., 2011)). A detailed study by Guan et al. (2013) finely mapped three unique clusters (A, B and C) of CD4i-specific antibodies using a competition-based ELISA approach (Guan et al., 2013). Cluster A epitopes become exposed following the conformational change resulting from Env binding to cellular CD4 that occurs during viral entry or following infection and subsequent cell–cell spread when CD4 and the viral envelope are co-expressed on the surface of the same cell (Finnegan et al., 2001; Acharya et al., 2014; Veillette et al., 2014). Prototypic Cluster A mAbs A32 and C11 bind to similar epitopes on gp120 but only weakly cross-compete with each other, suggesting they have unique specificities (Guan et al., 2013). Fab inhibition experiments have demonstrated that A32-like antibodies frequently constitute the majority of the ADCC activity observed in chronically-infected (Ferrari et al., 2011) and RV144-vaccinated (Bonsignori et al., 2012) individuals. The Cluster B epitope is defined by only one antibody, N12-i15, and targets a conformational epitope on gp120 that requires CD4 binding and involves the V1–V2 loop. Cluster C antibodies recognizing the co-receptor binding sites (CoRBS) are the only CD4i antibodies capable of neutralizing cell-free virus and are further sub-categorized into 4 groups as specified by competition with either 17b or 19e mAbs. Among these ADCC-mediating antibodies, the A32 and C11 mAbs from cluster A are the most potent (Guan et al., 2013), and these CD4i mAbs have non-overlapping specificities. Few studies have examined ADCC mAb breadth or the ability to recognize diverse envelopes from different HIV-1 clades. Recently, two studies identified epitope breadth (Wren et al., 2013) and increased cross-clade ADCC activity (Madhavi et al., 2014) as characteristics associated with superior ADCC-mediating antibody responses. Despite this work, we still know very little about the features defining a protective ADCC-mediating immune response or the optimal methods required to dissect this response at the monoclonal level. Further, few studies have attempted to define the contribution of epitope-specific mAbs other than A32 to the overall ADCC response or to determine the kinetics of ADCC Ab development following infection. While considerable progress in identifying HIV-specific bnAbs has been made using high-throughput methods such as protein-specific screens and microneutralization assays, no high-throughput, functional methods for isolating ADCC-mediating antibodies have been described. In this report, we screened peripheral blood mononuclear cells (PBMCs) approximately 2.5years post infection (PI) from an individual who demonstrated cross-clade ADCC and neutralizing Ab responses (Piantadosi et al., 2009; Bosch et al., 2010). Memory B cells that expressed B cell receptors (BCRs) that bound HIV envelope protein were isolated using HIV virus-like particles (VLPs). Using this approach, we identified three ADCC-mediating antibodies with cross-clade activity that targeted two distinct epitopes: the C11-like epitope, and the V3 region.
Methods
Results
Discussion A detailed understanding of the epitope specificity of ADCC-mediating antibodies is a pre-requisite for developing effective immunization strategies that optimize protection by these antibodies. In comparison to the wealth of data characterizing bnAbs, surprisingly little is known about the characteristics defining a broad and potent ADCC-mediating immune response. In this study, we describe a method to identify memory B cells producing ADCC-mediating antibodies from HIV-infected individuals. We applied this method to isolate mAbs from an individual with a cross-clade ADCC and neutralizing Ab response (Bosch et al., 2010). The three ADCC antibodies identified here mediate potent ADCC activity, and competition studies suggest that two target a CD4-induced epitope that resembles the epitope bound by the C11 antibody. Subsequent analysis determined that these two mAbs, QA255.157 and QA255.253, demonstrated impressive cross-clade breadth, mediating activity against 10 of 11 envelopes tested from clades A, B and C. When expressed as LALA variants, Abs QA255.157 and QA255.253 inhibited QA255 plasma IgG activity consistently beyond 189dpi. Thus, these data provide evidence that CD4i antibodies that target an epitope also bound by mAb C11 constitute a substantial ADCC response and that these antibodies contribute to ADCC activity within the first six months of infection.