Dr. Wine graduated from Tel Aviv University in the Department of Molecular Microbiology and Biotechnology at the Faculty of Life Sciences under the supervision of Prof. Amihay Freeman and Prof Felix Frolow, where using structural biology, protein chemistry, computerized modeling and molecular biology tools, he studied various aspects of protein-protein interactions for the design of novel composite materials within the nano-biotechnology field. During his postdoctoral training at the laboratory of Prof. George Georgiou (UT, Austin), Dr. Wine focused on developing a novel approach for the in-depth analysis of the humoral response following vaccine or disease. Following his postdoc training he established his research group at Tel Aviv University. As a group leader, Dr. Wine utilizes his recently developed approach combined with earlier acquired tools to study: i) maternal-infant immunity by profiling the molecular composition, dynamics and attributes of maternal, trans-placental and breastmilk vaccine-specific antibodies; ii) Anti-drug antibodies following treatment with Biologics and ; iii) anti-bacterial antibodies to be used as next generation antibiotics.
Collectively, Dr. Wine’s research group aims to address basic immunological questions as well as application-focused research for vaccine evaluation, development and design, immunodiagnostic discovery, and monoclonal antibody engineering.
Research Group Activity
Monoclonal antibody against a novel target in antibiotic-resistance bacteria.
This project aims to provide a novel therapeutic drug to combat drug-resistant pathogenic bacteria. The main goals will be to progress our novel monoclonal antibody MAb-B7 that targets enteropathogenic bacteria to development and clinical trials. This goal will be achieved by two main objectives. The first is to establish the MAb’s molecular mechanism of action (MoA) in an ex-vivo model system, thus laying the foundation for in-vivo experiments. The second is to progress towards clinical trials by i) evaluating the appropriate bactrial strain and animal model for conducting efficacy studies; ii) designing an effective dosing strategy; and iii) evaluating efficacy in monotherapy for treatment of enteropathogenic antibiotic-resistant bacteria and synergy in combination therapy with traditional antibiotics.
Personnel: Postdoc - Yaron Hillman, M.Sc. – Dan Lustiger
Antibody mediated control of cytomegalovirus at the materno-fetal interface
Human cytomegalovirus (HCMV) is the most common viral infection in newborns worldwide and the leading cause of congenital neurosensorial disease of infectious origin. The development of a vaccine and of antibody therapies against congenital HCMV infection is limited by our poor understanding of the mechanisms involved in the vertical transmission of the virus. The IgG response to HCMV envelope glycoproteins is probably a key determinant of viral control but could also participate in immunopathological responses at the placental level. The objectives of the project are to 1) determine the molecular and functional characteristics of the IgG response to HCMV envelope glycoproteins during primary infection in pregnancy; 2) identify serological correlates of HCMV transmission using multi-parametric analyses and systems serology; 3) characterize the interactions between IgG, HCMV and innate immune cells at the placental level. The identification of the mechanisms underlying viral control and tissue damage at the materno-fetal interface will have a considerable impact on the development of new vaccines and antibody-based therapies against congenital HCMV infection and potentially also against other pathogens transmitted to the fetus such as the Zika virus.
Personnel: PhD. – Shai Rosenstain, M.Sc. – Aya Kigel
In-depth molecular analysis of vaccine-specific maternal and transplacental antibody repertoires by deep sequencing and shotgun proteomics
Many vaccine-preventable diseases, like influenza, pertussis, and tetanus cause substantial morbidity and mortality in pregnant women, newborns and infants. Immunization during pregnancy has the potential to provide protection to the newborn and infant by the transplacental transfer of vaccine specific maternal antibodies. However, the immunobiology underlying immunization during pregnancy, that leads to the protection of the newborn are not well understood. Current vaccine formulations were designed for and tested in non-pregnant populations; yet substantial immune modulations take place during different stages of pregnancy and potentially can impact the humoral response following maternal immunzation. We thus have insufficient data on quantity and quality of the immune response during pregnancy and how this relates to immunity provided from the mother to the newborn. First, we hypothesize that the nature and breadth of the humoral immune response following vaccination differs in pregnant and non-pregnant vaccinees. Next, we hypothesize that the vaccine-specific antibodies that cross the plaecenta, comprise distinct repertoire features thus, the placenta functions as a differential barrier for antibody transfer. To test these hypotheses, we will use proteomic and genomic/transcriptomic measurements of antibody repertoires in the maternal and cord blood compartments. The measurements will be based on antibody clonal diversity/frequency, V(D)J germline usage and SHM, where we expect to find changes in i) vaccine-specific B cell frequency, antibody clonal diversity, germline usage and SHM in pregnant women and ii) distinct repertoire features in the transplacental vaccine-specific antibody compartment compared the maternal compartment. We will utilize deep sequencing and proteomic technologies to provide, for the first time, insight into the immunobiology of a promising intervention aimed to prevent early life infectious morbidity and mortality and establish new research avenues for vaccine research in vulnerable populations.
Personnel: PhD. – Limor Levy, M.Sc. Rita Zovonoriov
The molecular landscape of Anti-Drug Antibodies following treatment with therapeutic Biologics
Therapeutic proteins have revolutionized modern medicine by providing advanced medical treatments that were completely impossible prior to biotechnological advancements in recent decades. However, immune responses to therapeutic proteins commonly arise and can interfere with treatment regimens. In particular, anti-drug immune responses result in a loss of drug efficacy, and in severe cases, clinical adverse-events that threaten patient safety. A key component of anti-drug immune responses is the generation of anti-drug-antibodies (ADAs) that target and often neutralize the therapeutic protein. While some mechanisms of ADA generation are known, there is very little information regarding the molecular composition of antibodies elicited in response to protein therapies. Here we will apply recently-developed approaches to elucidate the molecular composition of ADAs, using therapeutic anti-TNFα MAb as a tractable clinical model system. TNF-α antagonist are broadly administered to combat a range of autoimmune diseases and serve as an ideal model for studying ADA formation in humans.
To elucidate the impact of ADAs on the loss of drug-efficacy, we will perform high-throughput sequencing of ADA-encoding B-cells combined with proteomic analysis of ADAs in patients’ serum enabling a new functional analysis of ADAs in blood. The proposed investigation will serve as a platform approach for analysis of ADAs in immune responses to protein therapeutics and provide the first repertoire-based, molecular-scale study of ADAs. This work will generate new insights regarding ADA formation and define the molecular biases for immune interference with protein therapeutics, thereby accelerating approaches to improve protein drug development and treatment in the coming years.
Personnel: PhD. – Anna Vaisman-Mentesh
Within the CYMAF project, Dr. Wine will lead working package 2 (WP2).
The overall aim of WP2 is WP2 will test the hypothesis that envelope glycoprotein-specific IgG have reduced effector functions as compared to tegument protein-specific IgG during primary HCMV infection. The structural basis of IgG effector functions will be explored by measuring the glycosylation profile of their Fc fragment. The role of CD4 helper T cells will be evaluated by measuring TFH cell responses. These analyses will be conducted using a bank of serum and PBMC samples collected by partner 2 (IT) under approval of the institutional ethics committee. Samples obtained from 30 pregnant women during the first 2 months of primary HCMV infection and 30 gestational age-matched pregnant women with chronic infection will be analyzed. This cohort size allowed the identification of serological signatures of viral control and vaccine responses as demonstrated in previous studies (14,15). The evolution of IgG characteristics over the first 6 months of primary HCMV infection will be analyzed in a sub-cohort of 10 pregnant women.
A pilot study will be conducted in parallel to explore the antibody repertoire of envelope glycoprotein and tegument protein-specific IgG during primary HCMV infection. In order to process them fresh, the samples required for this pilot study will be collected by partner 3 (IL) following approval by the institutional ethics committee.
The role of the Wine group within WP2 will include 2 tasks:
1/ IgG repertoire analyses will be performed as previously described on 4 pregnant women with primary HCMV infection prospectively recruited by partner 3 (IL). Memory B cells and plasmablasts will be purified by cell sorting and will be submitted to V gene Next Generation Sequencing (NGS). Antigen-specific IgG will be purified by affinity chromatography and will be submitted to LC-MS/MS shotgun proteomics.
2/ Synthetic genes encoding the high-frequency, proteomically identified VH sequences will be paired
with the VL repertoire from ag-specific plasmablasts, expressed as Fabs in Escherichia coli, and
screened for ag-specificity by ELISA. VH/VL pairs exhibiting high ELISA signal will be cloned and
purified as IgG from HEK 293F cells. For the purpose of WP4, anti-HCMV envelope
glycoprotein-specific human mAb will be cloned from public sequences into pcDNA3.4 expression
vector and purified as IgG from HEK 293F cells.
Wine, Y., Cohen-Hadar, N., Freeman, A. & Frolow, F. Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X-ray structure analysis. Biotechnology and Bioengineering 98, 711-718. (2007).
Saggy, I., Wine, Y., Shefet-Carasso, L., Nahary, L., Georgiou, G. & Benhar, I. Antibody isolation from immunized animals: comparison of phage display and antibody discovery via V gene repertoire mining. Protein Engineering, Design and Selection 25, 539-549. (2012).
DeKosky, B.J., Ippolito, G.C., Deschner, R.P., Lavinder, J.J., Wine, Y., Rawlings, B.M., Varadarajan, N., Giesecke, C., Dorner, T., Andrews, S.F., Wilson, P.C., Hunicke-Smith, S.P., Willson, C.G., Ellington, A.D. & Georgiou, G. High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire. Nature Biotechnology 31, 166-169. (2013).
Cotham, V.C., Wine, Y., Brodbelt, J.S. Selective 351 nm Photodissociation of Cysteine-Containing Peptides for Discrimination of Antigen-Binding Regions of IgG Fragments in Bottom-Up Liquid Chromatography - Tandem Mass Spectrometry Workflows. Analytical Chemistry 85, 5577-5585. (2013).
*Wine, Y. (co-first author), *Boutz, D.R., *Lavinder, J.J., Miklos, A.E., Hughes, R.A., Hoi, K.H., Jung, S.T., Horton, A.P., Murrin, E.M., Ellington, A.D., Marcotte, E.M. & Georgiou, G. Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response. Proceedings of the National Academy of Sciences of the United States of America 110, 2993-2998. (2013).
*Lavinder, J.J., *Wine, Y. (co-first author), Giesecke, C., Ippolito, G.C., Horton, A.P, Lungu, O.I., Hon Hoi, K., DeKosky, B.J., Murrin, E.M., Wirth, M.M., Ellington, A.D., Dörner, T., Marcotte, E.M., Boutz, D.R., Georgiou, G. Identification, Dynamics, and Functionality of the Constituent Antibodies within Human Serum after Vaccination. Proceedings of the National Academy of Sciences of the United States of America. 111, 2259-2264. (2014).
*Boutz, D.R., *Horton, A.P., *Wine, Y. (co-first author), Lavinder, J.J, Georgiou, G., Marcotte, E.M. Proteomic Identification of Monoclonal Antibodies from Serum. Analytical Chemistry. 86, 4758-4766. (2014).
Lavinder, J.J., Hon Hoi, K., Reddy, S.T., Wine, Y., Georgiou, G. Systematic Characterization and Comparative Analysis of the Rabbit Immunoglobulin Repertoire. PloS one. 9, e101322. (2014).
Wine ,Y., Horton. A.P., Ippolito, G.C., Georgiou, G. Serology in the 21st century: the molecular-level analysis of the serum antibody repertoire. Current opinion in immunology. 35, 89-97. (2015).
Breden, E.T. Luning Prak, B. Peters, F. Rubelt, C.A. Schramm, C.E. Busse, J.A. Vander Heiden, S. Christley, S.A.C. Bukhari, A. Thorogood, F.A. Matsen IV, Y. Wine et al.. Reproducibility and Reuse of Adaptive Immune Receptor Repertoire Data. Frontiers in Immunology, 8 1817. (2017).
Vaisman-Mentesh, A. and Wine, Y. Monitoring phage biopanning by Next-Generation Sequencing, Method in Molecular Biology. Methods in Molecular Biology, Phage Display-Material and Methods, Humana Press, NY, 463-473. (2018).
O. Avram et al., ASAP - A Webserver for Immunoglobulin-Sequencing Analysis Pipeline. Frontiers in Immunology 9, (2018).