Identification and Applications of Llama-Derived Single Domain Antibodies Binding to Glycoprotein D of Herpes Simplex Virus 2
Geoghegan, Eileen Michelle
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There are currently no protective vaccines or microbicides that can prevent the transmission of herpes simplex virus 2 (HSV-2). The purpose of this thesis was to develop a microbicide based on a llama-derived single domain antibody (VHH) binding to glycoprotein D (gD2) of HSV-2. After immunizing two llamas with gD2 and creating a phage library displaying the VHH repertoire of the immunized llamas, ten unique VHH sequences were identified that bind to gD2. Although many of the VHHs were able to bind gD2 when expressed and purified from E. coli, none of the VHHs were able to neutralize the virus in vitro or in vivo. Only when a gD2-binding VHH called R33 was expressed as a pentamer did it exhibit virus neutralization activity against HSV-2. When R33 was expressed as a fusion protein with the HSV-2 antimicrobial peptide TATC, the in vitro antiviral activity of the R33-TATC was increased compared to a non-gD2 binding VHH expressed with TATC and to the TATC peptide alone. Creation of a single domain antibody immunotoxin by expression of R33 with the active domain of exotoxin A from Pseudomonas aeruginosa resulted in specific and potent killing of HSV-2 infected cells in vitro. Although the isolated VHH that bind to gD2 are unable to neutralize HSV-2 on their own in a monomeric form, they can be used to specifically deliver other effector proteins resulting in potent antiviral activity against HSV-2.