We propose a two-pronged approach to explore basic aspects of the entry/fusion process of herpes simplex virus (HSV) that enables virus-cell and cell-cell fusion (Aim 1) and to use that knowledge to understand how the entry glycoproteins stimulate humoral responses important in virus neutralization and protection (Aim 2). These fundamental problems apply to all herpesviruses and to other enveloped viruses. We have amassed a rich armamentarium of purified glycoprotein constructs, monoclonal antibodies (Mabs) and expression plasmids to address significant questions about the fusion pathway as well as the humoral responses in a variety of populations. The coordinated entry process requires four glycoproteins, gD, gB, a heterodimer of gH/gL, and either of two cell receptors, herpesvirus entry mediator (HVEM) and nectin-1. Each step is blocked by epitope specific virus-neutralizing Mabs.
In Aim 1, we hypothesize that conformational changes to each glycoprotein convert it into a functional form. Receptor binding on one face of gD induces a conformational change to a different face that enables it to interact with gH/gL. The conformation of gH/gL is altered and its second face interacts with and activates the gB trimer to carry out fusion. We propose to solve the structures of Fab/glycoprotein complexes that define each step. We will also carry out cryo-EM studies of gB to gain insight into its prefusion form. A major biochemical approach will be to lock flexible regions involved in conformational changes using reversible disulfide bonds. We show that engineering such bonds in gD freezes conformations in place that normally have to change. Reduction of the bond restores the ability of gD to function as measured kinetically in living cells using a split luciferase assay.
In Aim 2, we will use our Mabs to dissect the polyclonal responses to protein-based vaccines and in naturally HSV infected people. Some Mabs act in a synergistic fashion with others to enhance neutralization. We will discern the correlates of the humoral responses to individual epitopes of each glycoprotein that contribute to virus neutralization. To do this on larger cohorts, we have miniaturized techniques to evaluate epitope profiles and to isolate antibodies to specific proteins. These include competition analysis using the Biacore and the Octet biosensor (see letter from Dr. J. Rucker), a magnetic bead assay to capture Abs to individual glycoproteins from sera and the high-throughput Alphascreen technology that directly tests sera for their capacity to interfere with gD/receptor interactions. We have collected sera from human vaccine trials and from a large cohort of naturally infected humans. We will obtain sera from guinea pigs immunized with different combinations of the glycoproteins. Finally, our Mabs will be used in passive immunization experiments. We are collaborating with Drs. F. Rey (crystallization), A. Steven (cryo-EM), D. Bernstein and R. Cardin (guinea pig studies), A. Wald (naturally infected humans) and J. Cohen (passive immunization studies). Our proposal thus explores the basic biology of HSV entry/fusion and uses this knowledge for translational studies.

Public Health Relevance

Herpes simplex virus (HSV) causes human diseases including cold sores, serious eye infections, acute encephalitis, and ulcerative genital lesions. The outer surface of the virus contains proteins needed to gain entry into host cells to cause disease. I use molecular biological approaches to understand how these proteins work. They are also critical targets of the host's immune response and my research focuses on uncovering correlates between the response to these proteins and protection from HSV disease and will have a major impact on vaccine design.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Virology - B Study Section (VIRB)
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Natarajan, Ramya
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University of Pennsylvania
Schools of Dentistry/Oral Hygn
United States
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Atanasiu, Doina; Saw, Wan Ting; Lazear, Eric et al. (2018) Using Antibodies and Mutants To Localize the Presumptive gH/gL Binding Site on Herpes Simplex Virus gD. J Virol 92:
Hook, Lauren M; Cairns, Tina M; Awasthi, Sita et al. (2018) Vaccine-induced antibodies to herpes simplex virus glycoprotein D epitopes involved in virus entry and cell-to-cell spread correlate with protection against genital disease in guinea pigs. PLoS Pathog 14:e1007095
Hensel, Michael T; Marshall, Jason D; Dorwart, Michael R et al. (2017) Prophylactic Herpes Simplex Virus 2 (HSV-2) Vaccines Adjuvanted with Stable Emulsion and Toll-Like Receptor 9 Agonist Induce a Robust HSV-2-Specific Cell-Mediated Immune Response, Protect against Symptomatic Disease, and Reduce the Latent Viral Reservoir. J Virol 91:
Cairns, Tina M; Ditto, Noah T; Lou, Huan et al. (2017) Global sensing of the antigenic structure of herpes simplex virus gD using high-throughput array-based SPR imaging. PLoS Pathog 13:e1006430
Fontana, Juan; Atanasiu, Doina; Saw, Wan Ting et al. (2017) The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane. MBio 8:
Atanasiu, Doina; Saw, Wan Ting; Eisenberg, Roselyn J et al. (2016) Regulation of HSV glycoprotein induced cascade of events governing cell-cell fusion. J Virol :
Persson, Josefine; Zhang, Yuan; Olafsdottir, Thorunn A et al. (2016) Nasal Immunization Confers High Avidity Neutralizing Antibody Response and Immunity to Primary and Recurrent Genital Herpes in Guinea Pigs. Front Immunol 7:640
Cairns, Tina M; Huang, Zhen-Yu; Gallagher, John R et al. (2015) Patient-Specific Neutralizing Antibody Responses to Herpes Simplex Virus Are Attributed to Epitopes on gD, gB, or Both and Can Be Type Specific. J Virol 89:9213-31
Saw, Wan Ting; Matsuda, Zene; Eisenberg, Roselyn J et al. (2015) Using a split luciferase assay (SLA) to measure the kinetics of cell-cell fusion mediated by herpes simplex virus glycoproteins. Methods 90:68-75
Cairns, Tina M; Huang, Zhen-Yu; Whitbeck, J Charles et al. (2014) Dissection of the antibody response against herpes simplex virus glycoproteins in naturally infected humans. J Virol 88:12612-22

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