CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

at days 30, 90, 180, 270 and 360 following infection. Over 100 biophysical, functional and clinical parameters including glycosylation, subclass distribution, phagocytosis, NK degranulation, cytokine release and viral set point were included in the analysis. Results: Significant changes were observed in both the functional and biophysical characteristics of the evolution of humoral immune response following acute HIV infection. Antibody glycosylation matured dramatically during infection, transitioning from a highly functional (highly galactosylated) profile, to a less functional (highly agalactosylated) profile, typically associated with increaded inflammation and characteristic of chronic HIV infection. Fc functionality increased over infection, with increases in antibody-mediated phagocytosis, NK degranulation and complement in a highly antigen-specific manner. p24-specific functional activity evolved more modestly over time compared to gp120- and gp41-specific response. Both antibody subclass changes and glycosylation drove changes in the evolution of antibody effector activity, highlighting the natural modifications in the humoral immune response that enable the directed recruitment of the innate immune system to target and control HIV. Conclusion: Antibody functionality evolves rapidly following acute HIV infection in an antigen-specific manner, pointing to specific antibody effector functions in the early control of HIV infection. 298 GP41−SPECIFIC ANTIBODIES MEDIATE POTENT ANTIBODY−DEPENDENT CELLULAR CYTOTOXICITY Megan M. Stumpf 1 , Katherine L. Williams 1 , Nicole Naiman 1 , Meghan Garrett 1 , Nitya Ramadoss 2 , Peter Kim 2 , Julie Overbaugh 1 1 Fred Hutchinson Cancer Research Center, Seattle, WA, USA, 2 Stanford University, Stanford, CA, USA Background: HIV-specific antibodies (Abs) are capable of limiting viral infection by neutralizing cell-free virus and/or targeting infected cells for destruction. The latter process is referred to as antibody-dependent cellular cytotoxicity (ADCC). ADCC-mediating Abs have been identified as a correlate of protection from HIV in the RV144 Phase III HIV vaccine trial as well as in experimental vaccine studies in non-human primates. High levels of passively acquired ADCC-mediating activity has also been correlated with improved clinical outcome in HIV-infected infants. Due to requirements for fusion and entry, certain regions of gp41 are highly conserved. However, little is known about antibodies that target these conserved regions and whether they mediate FcR-dependent effector activity, like ADCC. Methods: HIV-specific B cells were sorted from PBMC samples obtained from a subtype-A infected Kenyan woman 914 days post-infection. Following sequence amplification and protein expression, the Abs were tested for HIV binding by ELISA and in a rapid and fluorometric ADCC (RF-ADCC) assay. We used the following four coating antigens: a clade B gp41 protein, a clade C gp41 ectodomain protein, a clade A gp140 protein which includes the extracellular domain of gp41, and a six-helical mimetic gp41 protein which consists of the conserved HR1 and HR2 regions in a continuous trimer. Competition ELISAs were performed to further characterize the individual epitopes. Results: We identified four gp41-specific ADCC-mediating Abs from unique B cell lineages that target two distinct regions of the gp41 ectodomain. Abs QA255.006 and QA255.016 bind the heptad-repeat regions whereas QA255.067 and QA255.072 target the C-C loop of gp41. QA255.006 and QA255.016 both mediated strong ADCC activity against all four gp41 antigens tested in the RF-ADCC assay, including the gp41 mimetic. In contrast, QA255.067 and QA255.072 maintained strong activity against the other three gp41 antigens but did not demonstrate any measurable ADCC activity against the mimetic antigen. Conclusion: Here we describe four new ADCC-mediating antibodies, isolated from one individual that are derived from distinct B cell lineages, that target gp41. Two of these antibodies recognize the trimeric post-fusion conformation mimetic containing the conserved HR1 and HR2 regions. Further characterization of these novel functional antibodies may inform the design of a broad and effective HIV vaccine. 299 DISSECTING THE PHENOTYPE AND TRANSCRIPTOME OF IGG3+ B CELLS Saheli Sadanand , Wen-Han Yu, Dario Garcia-Dominguez, Galit Alter Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Background: HIV-specific IgG3 antibodies (Abs) have been established as a key signature associated with robust humoral immune activity in the context of both natural HIV infection and the efficacious RV144 vaccination trial.

While highly functionally potent, IgG3 Abs are short-lived and not abundant. An improved understanding of the mechanism(s) by which IgG3+ B cells are actively induced and preserved could suggest strategies for eliciting highly potent and stable IgG3 Abs. It is unclear whether intrinsic factors are important for selection and maintenance of IgG3-secreting B cells. We hypothesized that IgG3+ B cells have a distinct phenotype and transcriptome that preserves IgG3 subclass expression. Methods: Peripheral B cells from HIV negative donors and HIV positive donors (elite controllers, viremic controllers and treated chronic progressors) were phenotypically profiled using multicolor flow cytometry. For RNA-sequencing, peripheral non-naïve bulk isotype-specific B cells were sorted into lysis buffer and whole transcriptome amplification was performed using a modified SMART-Seq2 protocol. For in vitro stimulations, resting memory B cells were stimulated with anti-IgG or anti-IgM along with CpG and soluble CD40 ligand for four days. Results: IgG3+ peripheral B cell frequencies are similar in HIV negative and HIV positive donors. We evaluated expression of markers associated with B cell-T cell interactions and homing to the germinal center. IgG3+ B cells have a unique and intermediate phenotype that is independent of HIV disease status (Figure). Transcriptional analyses of peripheral non-naive IgM+, IgG3+ an IgG1+ B cells further revealed that IgG3+ B cells were more similar to IgM+ B cells than to IgG1+ B cells. Notably, both IgM+ and IgG3+ B cells had higher expression of genes involved in class-switch recombination. Finally, preliminary in vitro data suggest that upon stimulation IgG3+ B cells significantly modulate expression of over 600 genes, including some involved in DNA repair and BCR signaling. Conclusion: Collectively, the phenotypic and transcriptional profile of IgG3+ B cells likely promotes germinal center reentry as opposed to plasmablast differentiation. Our data suggest B cell-T cell interactions and genes that may be useful targets for blocking further class-switch recombination, thereby skewing activated IgG3+ B cells towards plasmablast differentiation and leading to more production of highly potent IgG3 Abs. 300 DEFINING CORRELATES OF ANTIBODY-POLYFUNCTIONALITY IN RV144 VACCINEES Stephanie Fischinger 1 , Todd J. Suscovich 1 , Hendrik Streeck 2 , Galit Alter 1 1 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA, 2 Institute for HIV Research, Essen, Germany Background: Results from the RV144 trial pointed to a potentially important role for non-neutralizing but functional antibodies in protection against HIV acquisition. Importantly, while ADCC activity was primarily associated with protection in the case-control study, RV144 vaccine induced antibodies were able to recruit a broader range of antiviral functions including antibody- dependent cellular phagocytosis (ADCP), neutrophil phagocytosis (ADNP), activation of natural killer cells (NK), and the recruitment of complement. However, the specific antibody modifications that permitted the induction of this polyfunctional response are incompletely understood but may provide critical insights to enhance protective immunity. Methods: Systems serology profiling was therefore performed in plasma samples from 300 RV144 vaccinees including both functional and biophysical profiling of the humoral immune response against both the priming (MN- cladeB) and boosting (A244-cladeAE) HIV envelope immunogens. Results: Heterogeneous responses for antibody mediated monocyte phagocytosis, antibody mediated neutrophil phagocytic, activation of antibody-dependent complement deposition (ADCD) and NK cell-activating activity were observed across the vaccinees and across the two antigens. The functional responses to prime- and boost-antigen were highly correlated in the overall cohort. Levels of IgG3 against either antigen, which was shown to be a correlate of protection was strongly correlated with ADCP, ADNP, ADCD and IFN-y release by NK cells. Polyfunctionality was associated with IgG3 levels, but more strongly associated with antigen-specific IgG1 levels, pointing to a potentially critical role for altered IgG1 glycosylation in driving antibody polyfunctionality within this vaccine trial. Conclusion: The data argue for a synergistic role for both IgG3 and particularly modified IgG1 antibodies in the induction of polyfunctional antibody effector profiles, pointing to novel correlates of antiviral immunity that may be leveraged to drive enhanced antibody effector function in future vaccine design.

Poster Abstracts

CROI 2018 105