Penn Veterinary Medicine | University of Pennsylvania

Description of Research Expertise

Research Interests
Herpes simplex virus entry mechanisms; vaccinia virus proteins involved in protection.

Key words: herpes, HSV, virology, glycoproteins, virus entry.

Description of Research
The research in my laboratory is conducted in collaboration with Dr. Gary H. Cohen of the School of Dental Medicine, UPenn. Our longterm goal is to understand molecular events that mediate virus entry into susceptible cells and promote the pathogenesis of the virus in its human host.

Research on herpes simplex virus (HSV):
Four viral glycoproteins, gB, gD, gH and gL are essential for virus entry and spread. HSV entry is mediated by one of several different receptors. including HVEM (HveA) a TNF receptor, and nectin-1, a cell adhesion molecule that is a member of the Ig superfamily. We showed that purified gD interacts directly with purified HVEM and nectin-1 using such techniques as co-precipitation, ELISA and biosensor. A major accomplishment was to solve the three-dimensional structure of gD with HVEM. The structure of the complex enabled us to carry out structure-based mutagenesis of both proteins. gD mutants with altered receptor usage are being tested in mouse models of HSV pathogenesis. Our future direction is to understand entry events that are downstream of the gD/receptor interaction and involve gB, gH/gL and possibly other cell proteins. Our goal is to carry out structure-function studies similar to those for gD, including solution of their 3-D structures. gB has been crystallized and its structure will soon be known. We recently found that gB partitions into lipid rafts at the time of virus attachment, implicating a raft-specific cellular molecule in entry. In addition, the conformation of gB changes when virions bind nectin-1 at temperatures permissive for entry. Thus, the gD/receptor interaction triggers the downstream events that lead to fusion. This is an active area of ongoing research. In addition, we found that after infection, gD alters the distribution of nectin-1 in cells and also dissociates nectin-1 from its intracellular ligand afadin. Moreover, others and we found that the virus can use both direct fusion and at least two different endocytic pathways of entry, leading us into interesting areas of cell biology.

Poxvirus Research:
We are using similar techniques to those above to study the envelope proteins of vaccinia virus (VV). Live VV is currently the only vaccine available for protection against smallpox (and now monkeypox), a “class A” biological agent according to the CDC. Poxviruses are large complex viruses that are wrapped in two distinct viral envelopes. The inner envelope, called IMV contains at least 5 virion glycoproteins. The outer envelope (called EEV) contains an additional 5 or 6 proteins. Some of these stimulate neutralizing and/or protective antibody responses in animals. Our first goal has been to develop an antibody cocktail that will serve as a replacement for vaccinia immune globulin (VIG), currently offered to smallpox vaccinees with adverse reactions to the vaccine. The second goal is to develop a subunit vaccine against smallpox using VV proteins. A third goal is to understand more about the structure and function of the envelope glycoproteins of this complex virus. Our approach is to express VV proteins in a baculovirus expression system and to purify the recombinant proteins (5 so far) on a scale that is sufficient to develop immunologic reagents (goal 1) and to test the proteins using in-vivo models for efficacy (goal 2). The proteins and antibodies have also been used to address the third goal. The proteins have been evaluated alone and in combination in both mouse and monkey models in a collaboration with investigators at the NIH. A combination vaccine protected mice against a lethal VV challenge and ameliorated the effects of monkeypox in monkeys. These reagents will be further tested against the smallpox virus. Panels of monoclonal antibodies have been developed and are being studied to gain clues about the function of the VV glycoproteins and to test for efficacy in mouse challenge models.

Rotation Projects for 2006-2007
1. A number of mutated forms of herpes glycoproteins gD, gB and gH have been created and tested in mammlian expression systems. However, many of these have yet to be expressed in large quantities for more careful protein analysis. As a rotation project, the student will subclone the ectodomain of one of these mutant forms into a baculovirus expression system. The student will become skilled in this technology and will learn how to purify the proteins and to analyze their properties. Purified receptors will be used in binding assays (Westerns, ELISA, biacore) and as immunogens to prepare antisera (the latter done commercially). Understanding the characteristics of these proteins as HSV receptors is critical for understanding the phenotypes of virus mutants carrying mutations in gD that alter receptor tropism. The student will learn a variety of molecular biological and virological techniques and will become skilled in the use of the baculovirus expression system as well as protein purification and characterization.

2. To clone and express vaccinia virus proteins that are important components of the host immune response using the baculovirus system. Similar strategies and techniques to those described above will be employed. To carry out bioassays, the student will collaborate with others in the lab who are permitted to work with vaccinia virus so that the student is not exposed to the virus.

3. Creation of mutations in the genes for one of the herpes or VV glycoproteins under investigation in our lab. Such mutants will will enhance our understanding of these proteins and the choice of the particular mutant will depend on the current research in the lab. This project will entail the use of the Quick Change mutagenesis and will focus on making key mutations that we help the student select, and characterization of the mutant protein using mammalian expression.

4. We have acquired the technology to use live cell confocal microscopy and a number of people in the lab are familiar with this exciting new technology. We have also prepared a number of FP tagged cells and viruses and the student will use the materials at hand to master the technique and apply it to new questions concerning virus entry, receptor down regulation and events that focus on glycoproteins during viral replication.

Lab personnel:
Eric Lazear, B.S., CAMB graduate student (3rd year)
Brian Hannah, B.S., CAMB graduate student (3rd year)
Katie Stiles, B.S., CAMB graduate student (2nd year)
Chwang Hong Foo, B.S. CAMB graduate student (2nd year)
Claude Krummenacher, Ph.D., Research Associate
Tina Cairns, Ph.D., Postdoctoral Scientist
Florent Bender, Ph.D., Postdoctoral Scientist
J. Charles Whitbeck, Ph.D., Senior Research Scientist
Yi Zuo, DMD, Research Scientist
Huan Lou, BS, Research Specialist
Manuel Ponce de Leon, MS, Research Specialist

Former graduate students:
J.T. Matthews, Ph.D. Senior Scientist, Aventis Pharmaceuticals
C. Seidel-Dugan, Ph.D. Senior Scientist, Elixis Pharmaceuticals
D. L. Sodora, , Ph.D., Associate Professor, University of Texas
Shan-Ling Hung, Ph.D. May, 1992; Professor of Oral Biology, National Yang-Ming University, Taipei, Taiwan R.O.C.
Deborah Long,, Ph.D. Aug., 1992. Group Leader, Virology program, Wyeth, Inc
Hsien-Yuan Chiang, Ph.D. Professor of Microbiology, National Defense Medical Center Taipei, Taiwan
Ruth Tal-Singer, Ph.D., Section Chief, Smith-Kline Beecham
Christopher Handler, Ph.D. CRA, Quintiles, Rockville, MD
Anthony Nicola, Ph.D., Assistant Professor, Virginia Commenwealth University
Tao Peng, Ph.D. (Biology Grad. Group) Staff Scientist, Immusol
Sarah Connolly, Ph.D.. Post-doctoral fellow, Dr. Robert Lamb, Northwestern University.

Selected Publications

Atanasiu D, Whitbeck JC, Cairns TM, Reilly B, Cohen GH and Eisenberg RJ.: Bimolecular complementation reveals that glycoproteins gB and gH/gL of herpes simplex virus interact with each other during cell fusion. Proc. Nat’l Acad Sci USA 2007.

Aldaz-Carroll L, Xiao Y, Whitbeck JC, Ponce de Leon M, Lou H, Kim M, Yu J, Reinherz EL, Isaacs SN, Eisenberg RJ and Cohen GH. : Major neutralizing sites on vaccinia virus glycoprotein B5 are expressed differently on variola ortholog B6. J. Virol. 81: 8131-8139, 2007.

Chen Z, Earl P, Americo J, Damon, I, Smith S, Yu F, Sebrell A, Suzanne Emerson, S, Cohen GH, Eisenberg RJ, Gorshkova I , Schuck P, Satterfield W, Moss B and Purcell R.: Characterization of Chimpanzee/Human monoclonal antibodies to vaccinia virus A33 protein and its variola homolog in vitro and in a vaccinia virus mouse protection model. J. Virol. 81: 8989-8995, 2007.

Jing L, Chong TM, Byrd B, McClurkan CL, Huan J, Story BT, Dunkley KM, Aldaz-Carroll L, Eisenberg RJ, Cohen GH, Kwok WW, Sette A, and Koelle D. : Dominance and diversity in the primary human CD4 T cell response to replication-competent vaccinia virus. J. Immunol. 178: 6374-6386, 2007.

Hannah BP, Heldwein EE, Bender FC, Cohen GH and Eisenberg RJ.: Mutational evidence of internal fusion loops in HSV glycoprotein B . J. Virol. 81: 4858-4865, 2007.

Bender, FC, Samanta M, Heldwein EE, Ponce de Leon, M, Bilman E, Lou H, Eisenberg RJ and Cohen GH. : Antigenic and mutational analyses of herpes simplex virus glycoprotein B reveal four functional regions. J. Virol 81: 3827-3841, 2007.

Tengvall S, Lundqvist A, Eisenberg RJ, Cohen GH, Harandi AM.: Mucosal administration of CpG oligodeoxynucleotide elicits strong CC and CXC chemokine responses in the vagina and serves as a potent Th1-tilting adjuvant for recombinant gD2 protein vaccination against genetal herpes. J. Virol 80: 5283-5291, 2006.

Chen Z, Earl P, Americo J, Damon I, Smith SK, Zhou Y-H, Yu F, Sebrell A, Emerson S, Cohen G, Eisenberg RJ, Svitel J, Schuck P, Satterfield W, Moss B, and Purcell R. : Chimpanzee/human mAbs to vaccinia virus B5 protein neutralize vaccinia and smallpox viruses and protect mice against vaccinia virus. Proc. Nat’l Acad Sci USA. 103: 1882-1887, 2006.

Aldaz-Carroll, Lydia. Xiao, Yuhong. Whitbeck, J Charles. de Leon, Manuel Ponce. Lou, Huan. Kim, Mikyung. Yu, Jessica. Reinherz, Ellis L. Isaacs, Stuart N. Eisenberg, Roselyn J. Cohen, Gary H.: Major neutralizing sites on vaccinia virus glycoprotein B5 are exposed differently on variola virus ortholog B6. Journal of Virology 81(15): 8131-9, Aug 2007.

Lawrence, Steven J. Lottenbach, Kathleen R. Newman, Frances K. Buller, R Mark L. Bellone, Clifford J. Chen, John J. Cohen, Gary H. Eisenberg, Roselyn J. Belshe, Robert B. Stanley, Samuel L Jr. Frey, Sharon E.: Antibody responses to vaccinia membrane proteins after smallpox vaccination. Journal of Infectious Diseases 196(2): 220-9, Jul 15 2007.

Jing, Lichen. Chong, Tiana M. Byrd, Benjamin. McClurkan, Christopher L. Huang, Jay. Story, Brian T. Dunkley, Karissa M. Aldaz-Carroll, Lydia. Eisenberg, Roselyn J. Cohen, Gary H. Kwok, William W. Sette, Allesandro. Koelle, David M.: Dominance and diversity in the primary human CD4 T cell response to replication-competent vaccinia virus. Journal of Immunology 178(10): 6374-86, May 15 2007.

Cairns, Tina M. Friedman, Lisa S. Lou, Huan. Whitbeck, J Charles. Shaner, Marie S. Cohen, Gary H. Eisenberg, Roselyn J.: N-terminal mutants of herpes simplex virus type 2 gH are transported without gL but require gL for function. Journal of Virology 81(10): 5102-11, May 2007.

Xiao, Yuhong. Aldaz-Carroll, Lydia. Ortiz, Alexandra M. Whitbeck, J Charles. Alexander, Edward. Lou, Huan. Davis, Heather L. Braciale, Thomas J. Eisenberg, Roselyn J. Cohen, Gary H. Isaacs, Stuart N.: A protein-based smallpox vaccine protects mice from vaccinia and ectromelia virus challenges when given as a prime and single boost. Vaccine 25(7): 1214-24, Jan 26 2007.