BACKGROUND:The critical role of antibody Fc-mediated effector functions in immune defense has been widely reported in various viral infections. These effector functions confer cellular responses through engagement with innate immune cells. The precise mechanism(s) by which immunoglobulin G (IgG) Fc domain and cognate receptors may afford protection are poorly understood, however, in the context of HIV/SHIV infections. Many different in vitro assays have been developed and utilized to measure effector functions, but the extent to which these assays capture distinct antibody activities has not been fully elucidated.RESULTS:In this study, six Fc-mediated effector function assays and two biophysical antibody profiling assays were performed on a common set of samples from HIV-1 infected and vaccinated subjects. Biophysical antibody profiles supported robust prediction of diverse IgG effector functions across distinct Fc-mediated effector function assays. While a number of assays showed correlated activities, supervised machine learning models indicated unique antibody features as primary contributing factors to the associated effector functions. Additional experiments established the mechanistic relevance of relationships discovered using this unbiased approach.CONCLUSIONS:In sum, this study provides better resolution on the diversity and complexity of effector function assays, offering a clearer perspective into this family of antibody mechanisms of action to inform future HIV-1 treatment and vaccination strategies.

Defining correlates of immunity by comprehensively interrogating the extensive biological diversity in naturally or experimentally protected subjects may provide insights critical for guiding the development of effective vaccines and antibody-based therapies. We report advances in a humoral immunoprofiling approach and its application to elucidate hallmarks of effective HIV-1 viral control. Systematic serological analysis for a cohort of HIV-infected subjects with varying viral control was conducted using both a high-resolution, high-throughput biophysical antibody profiling approach, providing unbiased dissection of the humoral response, along with functional antibody assays, characterizing antibody-directed effector functions such as complement fixation and phagocytosis that are central to protective immunity. Profiles of subjects with varying viral control were computationally analyzed and modeled in order to deconvolute relationships among IgG Fab properties, Fc characteristics, and effector functions and to identify humoral correlates of potent antiviral antibody-directed effector activity and effective viral suppression. The resulting models reveal multifaceted and coordinated contributions of polyclonal antibodies to diverse antiviral responses, and suggest key biophysical features predictive of viral control.

Antibodies are widely considered to be a frequent primary and often mechanistic correlate of protection of approved vaccines; thus evaluating the antibody response is of critical importance in attempting to understand and predict the efficacy of novel vaccine candidates. Historically, antibody responses have been analyzed by determining the titer of the humoral response using measurements such as an ELISA, neutralization, or agglutination assays. In the simplest case, sufficiently high titers of antibody against vaccine antigen(s) are sufficient to predict protection. However, antibody titer provides only a partial measure of antibody function, which is dependent on both the variable region (Fv) to bind the antigen target, and the constant region (Fc) to elicit an effector response from the innate arm of the immune system. In the case of some diseases, such as HIV, for which an effective vaccine has proven elusive, antibody effector function has been shown to be an important driver of monoclonal antibody therapy outcomes, of viral control in infected patients, and of vaccine-mediated protection in preclinical and clinical studies. We sought to establish a platform for the evaluation of the Fc domain characteristics of antigen-specific antibodies present in polyclonal samples in order to better develop insights into Fc receptor-mediated antibody effector activity, more fully understand how antibody responses may differ in association with disease progression and between subject groups, and differentiate protective from non-protective responses. To this end we have developed a high throughput biophysical platform capable of simultaneously evaluating many dimensions of the antibody effector response.

A recombinant vaccine containing Aventis Pasteur's canarypox vector (ALVAC)-HIV and gp120 alum decreased the risk of HIV acquisition in the RV144 vaccine trial. The substitution of alum with the more immunogenic MF59 adjuvant is under consideration for the next efficacy human trial. We found here that an ALVAC-simian immunodeficiency virus (SIV) and gp120 alum (ALVAC-SIV + gp120) equivalent vaccine, but not an ALVAC-SIV + gp120 MF59 vaccine, was efficacious in delaying the onset of SIVmac251 in rhesus macaques, despite the higher immunogenicity of the latter adjuvant. Vaccine efficacy was associated with alum-induced, but not with MF59-induced, envelope (Env)-dependent mucosal innate lymphoid cells (ILCs) that produce interleukin (IL)-17, as well as with mucosal IgG to the gp120 variable region 2 (V2) and the expression of 12 genes, ten of which are part of the RAS pathway. The association between RAS activation and vaccine efficacy was also observed in an independent efficacious SIV-vaccine approach. Whether RAS activation, mucosal ILCs and antibodies to V2 are also important hallmarks of HIV-vaccine efficacy in humans will require further studies.

Monica Vaccari, Shari N. Gordon, Slim Fourati, Luca Schifanella, Namal P.M. Liyanage, Mark Cameron, Brandon F. Keele, Xiaoying Shen, Georgia D. Tomaras, Erik Billings, Mangala Rao, Amy W. Chung, Karen G. Dowell, Chris Bailey-Kellogg, Eric P. Brown, Margaret E. Ackerman, Diego A. Vargas-Inchaustegui, Stephen Whitney, Melvin N. Doster, Nicolo Binello, Poonam Pegu, David C. Montefiori, Kathryn Foulds, David S. Quinn, Mitzi Donaldson, Frank Liang, Karin Loré, Mario Roederer, Richard A. Koup, Adrian McDermott, Zhong-Min Ma, Christopher J. Miller, Tran B. Phan, Donald N. Forthal, Matthew Blackburn, Francesca Caccuri, Massimiliano Bissa, Guido Ferrari, Vaniambadi Kalyanaraman, Maria G. Ferrari, DeVon Thompson, Marjorie Robert-Guroff, Silvia Ratto-Kim, Jerome H. Kim, Nelson L. Michael, Sanjay Phogat, Susan W. Barnett, Jim Tartaglia, David Venzon, Donald M. Stablein, Galit Alter, Rafick-Pierre Sekaly & Genoveffa Franchini.

Diverse Ab effector functions mediated by the Fc domain have been commonly associated with reduced risk of infection in a growing number of nonhuman primate and human clinical studies. This study evaluated the anti-HIVAb effector activities in polyclonal serum samples from HIV-infected donors, VAX004 vaccine recipients, and healthy HIV-negative subjects using a variety of primary and cell line-based assays, including Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cell-mediated viral inhibition, and Abdependent cellular phagocytosis. Additional assay characterization was performed with a panel of Fc-engineered variants of mAb b12. The goal of this study was to characterize different effector functions in the study samples and identify assays that might most comprehensively and dependably capture Fc-mediated Ab functions mediated by different effector cell types and against different viral targets. Deployment of such assays may facilitate assessment of functionally unique humoral responses and contribute to identification of correlates of protection with potential mechanistic significance in future HIV vaccine studies. Multivariate and correlative comparisons identified a set of Ab-dependent cell-mediated viral inhibition and phagocytosis assays that captured different Ab activities and were distinct from a group of ADCC assays that showed a more similar response profile across polyclonal serum samples. The activities of a panel of b12 monoclonal Fc variants further identified distinctions among the ADCC assays. These results reveal the natural diversity of Fc-mediated Ab effector responses among vaccine recipients in the VAX004 trial and in HIV-infected subjects, and they point to the potential importance of polyfunctional Ab responses.

Elite controllers (ECs) represent a unique model of a functional cure for HIV-1 infection as these individuals develop HIV-specific immunity able to persistently suppress viremia. Because accumulating evidence suggests that HIV controllers generate antibodies with enhanced capacity to drive antibody-dependent cellular cytotoxicity (ADCC) that may contribute to viral containment, we profiled an array of extra-neutralizing antibody effector functions across HIV-infected populations with varying degrees of viral control to define the characteristics of antibodies associated with spontaneous control. While neither the overall magnitude of antibody titer nor individual effector functions were increased in ECs, a more functionally coordinated innate immune-recruiting response was observed. Specifically, ECs demonstrated polyfunctional humoral immune responses able to coordinately recruit ADCC, other NK functions, monocyte and neutrophil phagocytosis, and complement. This functionally coordinated response was associated with qualitatively superior IgG3/IgG1 responses, whereas HIV-specific IgG2/IgG4 responses, prevalent among viremic subjects, were associated with poorer overall antibody activity. Rather than linking viral control to any single activity, this study highlights the critical nature of functionally coordinated antibodies in HIV control and associates this polyfunctionality with preferential induction of potent antibody subclasses, supporting coordinated antibody activity as a goal in strategies directed at an HIV-1 functional cure.

Nat. Med.; doi:10.1038/nm.4105; corrected online 16 June 2016 In the version of this article initially published online, an affiliation for Luca Schifanella was omitted and there was an error in the description of the phenotypic analyses of plasmablasts in the Online Methods. The error has been corrected for the print, PDF and HTML versions of this article.

AIDS Research and Human RetrovirusesVol. 30, No. S1 Correlates of Protection and ExposureFree AccessModulation of RAS Pathways as a Biomarker of Protection against HIV and as a Means to Improve Vaccine EfficacySlim Fourati, Monica Vaccari, Shari N. Gordon, Luca Schifanella, Mark Cameron, Brandon F. Keele, Xiaoying Shen, Georgia D. Tomoras, Erik Billings, Mangala Rao, Amy W. Chung, Karen Dowell, Chris Bailey-Kellogg, Eric Brown, Margaret E. Ackerman, Namal P.M. Liyanage, Diego A. Vargas-Inchaistegui, Stephen Whitney, Melvin N. Doster, Nicolo Binello, Poonam Pegu, David C. Montefiori, Kathryn Foulds, David S. Quinn, Mitzi Donaldson, Frank Liang, Karin Loré, Mario Roederer, Richard A Koup, Adrian McDermott, Zhong-Min Ma, Christopher J Miller, Tran B Phan, Donald N. Forthal, Matthew Blackburn, Francesca Caccuri, Guido Ferrari, Devon Thompson, Marjorie Robert-Guroff, Silvia Ratto-Kim, Jerome H. Kim, Nelson L. Michael, Sanjay Phogat, Susan W. Barnett, James Tartaglia, David Venzon, Donald M. Stablein, Galit Alter, Rafick-Pierre Sekaly, and Genoveffa FranchiniSlim FouratiVaccine & Gene Therapy Institute of Florida, Port Saint Lucie, FL, United StatesSearch for more papers by this author, Monica VaccariNational Cancer Institute, Animal Models and Vaccine Section, Bethesda, MD, United StatesSearch for more papers by this author, Shari N. GordonNational Cancer Institute, Animal Models and Vaccine Section, Bethesda, MD, United StatesSearch for more papers by this author, Luca SchifanellaNational Cancer Institute, Animal Models and Vaccine Section, Bethesda, MD, United StatesSearch for more papers by this author, Mark CameronVaccine & Gene Therapy Institute of Florida, Port Saint Lucie, FL, United StatesSearch for more papers by this author, Brandon F. KeeleNational Cancer Institute, AIDS and Cancer Virus Program, Frederick, MD, United StatesSearch for more papers by this author, Xiaoying ShenDuke Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, Georgia D. TomorasDuke Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, Erik BillingsU.S. Military HIV Research Program (MHRP), Walter Reed Army Institute of Research, Silver Spring, MD, United StatesSearch for more papers by this author, Mangala RaoU.S. Military HIV Research Program (MHRP), Walter Reed Army Institute of Research, Silver Spring, MD, United StatesSearch for more papers by this author, Amy W. ChungRagon Institute of MGH, MIT and Harvard, Boston, MA, United StatesSearch for more papers by this author, Karen DowellDartmouth College, Computer Science, Hanover, NH, United StatesSearch for more papers by this author, Chris Bailey-KelloggDartmouth College, Computer Science, Hanover, NH, United StatesSearch for more papers by this author, Eric BrownDartmouth College, Thayer School of Engineering, Hanover, NH, United StatesSearch for more papers by this author, Margaret E. AckermanDartmouth College, Thayer School of Engineering, Hanover, NH, United StatesSearch for more papers by this author, Namal P.M. LiyanageNational Cancer Institute, Animal Models and Vaccine Section, Bethesda, MD, United StatesSearch for more papers by this author, Diego A. Vargas-InchaisteguiNational Cancer Institute, Immune Biology of Retroviral Infection Section, Bethesda, MD, United StatesSearch for more papers by this author, Stephen WhitneyAdvanced BioScience Laboratories, Inc., Rockville, MD, United StatesSearch for more papers by this author, Melvin N. DosterNational Cancer Institute, Animal Models and Vaccine Section, Bethesda, MD, United StatesSearch for more papers by this author, Nicolo BinelloNational Cancer Institute, Animal Models and Vaccine Section, Bethesda, MD, United StatesSearch for more papers by this author, Poonam PeguNational Cancer Institute, Animal Models and Vaccine Section, Bethesda, MD, United StatesSearch for more papers by this author, David C. MontefioriDuke University Medical Center, Durham, NC, United StatesSearch for more papers by this author, Kathryn FouldsNational Institutes of Health, Vaccine Research Center, Bethesda, MD, United StatesSearch for more papers by this author, David S. QuinnKarolinska Institutet, Stockholm, SwedenSearch for more papers by this author, Mitzi DonaldsonKarolinska Institutet, Stockholm, SwedenSearch for more papers by this author, Frank LiangNational Institutes of Health, Vaccine Research Center, Bethesda, MD, United StatesSearch for more papers by this author, Karin LoréNational Institutes of Health, Vaccine Research Center, Bethesda, MD, United StatesSearch for more papers by this author, Mario RoedererNational Institutes of Health, Vaccine Research Center, Bethesda, MD, United StatesSearch for more papers by this author, Richard A KoupNational Institutes of Health, Vaccine Research Center, Bethesda, MD, United StatesSearch for more papers by this author, Adrian McDermottNational Institutes of Health, Vaccine Research Center, Bethesda, MD, United StatesSearch for more papers by this author, Zhong-Min MaUniversity of California, California National Primate Research Center, Davis, CA, United StatesSearch for more papers by this author, Christopher J MillerUniversity of California, California National Primate Research Center, Davis, CA, United StatesSearch for more papers by this author, Tran B PhanUniversity of California, Irvine School of Medicine, Irvine, CA, United StatesSearch for more papers by this author, Donald N. ForthalUniversity of California, Irvine School of Medicine, Irvine, CA, United StatesSearch for more papers by this author, Matthew BlackburnNational Cancer Institute, Animal Models and Vaccine Section, Bethesda, MD, United StatesSearch for more papers by this author, Francesca CaccuriNational Cancer Institute, Animal Models and Vaccine Section, Bethesda, MD, United StatesSearch for more papers by this author, Guido FerrariDuke University Medical Center, Durham, NC, United StatesSearch for more papers by this author, Devon ThompsonAdvanced BioScience Laboratories, Inc, Rockville, MD, United StatesSearch for more papers by this author, Marjorie Robert-GuroffNational Cancer Institute, Immune Biology of Retroviral Infection Section, Bethesda, MD, United StatesSearch for more papers by this author, Silvia Ratto-KimU.S. Military HIV Research Program (MHRP), Walter Reed Army Institute of Research, Silver Spring, MD, United StatesSearch for more papers by this author, Jerome H. KimU.S. Military HIV Research Program (MHRP), Walter Reed Army Institute of Research, Silver Spring, MD, United StatesSearch for more papers by this author, Nelson L. MichaelU.S. Military HIV Research Program (MHRP), Walter Reed Army Institute of Research, Silver Spring, MD, United StatesSearch for more papers by this author, Sanjay PhogatSanofi Pasteur, Swiftwater, PA, United StatesSearch for more papers by this author, Susan W. BarnettNovartis Vaccines and Diagnostics Inc., Cambridge, MA, United StatesSearch for more papers by this author, James TartagliaSanofi Pasteur, Swiftwater, PA, United StatesSearch for more papers by this author, David VenzonNational Cancer Institute, Biostatistics and Data Management Section, Bethesda, MD, United StatesSearch for more papers by this author, Donald M. StableinThe EMMES Corporation, Rockville, MD, United StatesSearch for more papers by this author, Galit AlterRagon Institute of MGH, MIT and Harvard, Boston, MA, United StatesSearch for more papers by this author, Rafick-Pierre SekalyCase Western Reserve University, Pathology, Cleveland, OH, United StatesSearch for more papers by this author, and Genoveffa FranchiniNational Cancer Institute, Animal Models and Vaccine Section, Bethesda, MD, United StatesSearch for more papers by this authorPublished Online:30 Oct 2014https://doi.org/10.1089/aid.2014.5182b.abstractAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail P03.04 LBBackground: Adjuvants modulate the immune response and can improve the immunogenicity of vaccines. Immunogenicity studies in humans suggest that MF59 is a more efficient adjuvant than alum as it triggers enhanced B and T cell immune responses.Methods: We performed a study (P162MRV144) in macaques using repeated challenges with SIVmac251 powered to benchmark the results of RV144 vaccine that showed limited efficacy in a phase III clinical trial in Thailand. We compared the efficacy of ALVAC-SIV/gp120 vaccine administered with alum or MF59 adjuvants to placebo in the SIV mac251 model. Transcriptional profiling of blood from 54 immunized macaques before vaccination (pre-vax), after immunization with ALVAC-SIV alone (post-1st) and after immunization with ALVAC-SIV combined with the protein/adjuvant MF59 or Alum immunization (post-3rd) was performed. Differential expression analysis and pathway enrichment analysis was used to identify genes and pathways associated with protection. A naïve Bayes classifier was build to predict vaccine protection.Results: We found that alum protected macaques from SIVmac251 acquisition (log-rank test: p = 0.0205), confirming the main results of the RV144 study. However MF59 did not protect macaques from SIVmac251 acquisition (log-rank test: p = 0.562), despite MF59's ability to elicit higher systemic T-cell and antibodies responses. Association between the changes in transcriptional profiles and risk of SIVmac251 acquisition resulted in the identification of 12-gene expression signature able to predict prior to vaccination protection by ALVAC + alum (ROC: Accuracy = 65.2%, p ≤ 0.05). Seven of the twelve genes of the signature were related to Ras signaling.Conclusions: System biology revealed that RAS, a signal transducer that facilitates cross talk among B-cells, T-cells and antigen presenting cells, as a biomarker of vaccine efficacy in the ALVAC + alum treated animals. These data suggest that activation of RAS may constitute a novel approach to improve vaccine efficacy against HIV.FiguresReferencesRelatedDetailsCited bySystems serology for evaluation of HIV vaccine trials30 January 2017 | Immunological Reviews, Vol. 275, No. 1Microscale purification of antigen-specific antibodiesJournal of Immunological Methods, Vol. 425 Volume 30Issue S1Oct 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Slim Fourati, Monica Vaccari, Shari N. Gordon, Luca Schifanella, Mark Cameron, Brandon F. Keele, Xiaoying Shen, Georgia D. Tomoras, Erik Billings, Mangala Rao, Amy W. Chung, Karen Dowell, Chris Bailey-Kellogg, Eric Brown, Margaret E. Ackerman, Namal P.M. Liyanage, Diego A. Vargas-Inchaistegui, Stephen Whitney, Melvin N. Doster, Nicolo Binello, Poonam Pegu, David C. Montefiori, Kathryn Foulds, David S. Quinn, Mitzi Donaldson, Frank Liang, Karin Loré, Mario Roederer, Richard A Koup, Adrian McDermott, Zhong-Min Ma, Christopher J Miller, Tran B Phan, Donald N. Forthal, Matthew Blackburn, Francesca Caccuri, Guido Ferrari, Devon Thompson, Marjorie Robert-Guroff, Silvia Ratto-Kim, Jerome H. Kim, Nelson L. Michael, Sanjay Phogat, Susan W. Barnett, James Tartaglia, David Venzon, Donald M. Stablein, Galit Alter, Rafick-Pierre Sekaly, and Genoveffa Franchini.Modulation of RAS Pathways as a Biomarker of Protection against HIV and as a Means to Improve Vaccine Efficacy.AIDS Research and Human Retroviruses.Oct 2014.A99-A99.http://doi.org/10.1089/aid.2014.5182b.abstractPublished in Volume: 30 Issue S1: October 30, 2014PDF download

Embryonic stem cells (ESCs), characterized by their ability to both self‐renew and differentiate into multiple cell lineages, are a powerful model for biomedical research and developmental biology. Human and mouse ESCs share many features, yet have distinctive aspects, including fundamental differences in the signaling pathways and cell cycle controls that support self‐renewal. Here, we explore the molecular basis of human ESC self‐renewal using Bayesian network machine learning to integrate cell‐type‐specific, high‐throughput data for gene function discovery. We integrated high‐throughput ESC data from 83 human studies (∼1.8 million data points collected under 1,100 conditions) and 62 mouse studies (∼2.4 million data points collected under 1,085 conditions) into separate human and mouse predictive networks focused on ESC self‐renewal to analyze shared and distinct functional relationships among protein‐coding gene orthologs. Computational evaluations show that these networks are highly accurate, literature validation confirms their biological relevance, and reverse transcriptase polymerase chain reaction (RT‐PCR) validation supports our predictions. Our results reflect the importance of key regulatory genes known to be strongly associated with self‐renewal and pluripotency in both species (e.g., POU5F1, SOX2, and NANOG), identify metabolic differences between species (e.g., threonine metabolism), clarify differences between human and mouse ESC developmental signaling pathways (e.g., leukemia inhibitory factor (LIF)‐activated JAK/STAT in mouse; NODAL/ACTIVIN‐A‐activated fibroblast growth factor in human), and reveal many novel genes and pathways predicted to be functionally associated with self‐renewal in each species. These interactive networks are available online at www.StemSight.org for stem cell researchers to develop new hypotheses, discover potential mechanisms involving sparsely annotated genes, and prioritize genes of interest for experimental validation. Stem Cells 2014;32:1161–1172

Self-renewal, the ability of a stem cell to divide repeatedly while maintaining an undifferentiated state, is a defining characteristic of all stem cells. Here, we clarify the molecular foundations of mouse embryonic stem cell (mESC) self-renewal by applying a proven Bayesian network machine learning approach to integrate high-throughput data for protein function discovery. By focusing on a single stem-cell system, at a specific developmental stage, within the context of well-defined biological processes known to be active in that cell type, we produce a consensus predictive network that reflects biological reality more closely than those made by prior efforts using more generalized, context-independent methods. In addition, we show how machine learning efforts may be misled if the tissue specific role of mammalian proteins is not defined in the training set and circumscribed in the evidential data. For this study, we assembled an extensive compendium of mESC data: ∼2.2 million data points, collected from 60 different studies, under 992 conditions. We then integrated these data into a consensus mESC functional relationship network focused on biological processes associated with embryonic stem cell self-renewal and cell fate determination. Computational evaluations, literature validation, and analyses of predicted functional linkages show that our results are highly accurate and biologically relevant. Our mESC network predicts many novel players involved in self-renewal and serves as the foundation for future pluripotent stem cell studies. This network can be used by stem cell researchers (at http://StemSight.org) to explore hypotheses about gene function in the context of self-renewal and to prioritize genes of interest for experimental validation.