Epstein-Barr Virus gp350 Can Functionally Replace the Rhesus Lymphocryptovirus Major Membrane Glycoprotein and Does Not Restrict Infection of Rhesus Macaques

Abstract

Primary Epstein-Barr virus (EBV) infection is the most common cause of infectious mononucleosis, and persistent infection is associated with multiple cancers. EBV vaccine development has focused on the major membrane glycoprotein, gp350, since it is the major target for antibodies that neutralize infection of B cells. However, EBV has tropism for both B cells and epithelial cells, and it is unknown whether serum neutralizing antibodies against B cell infection will provide sufficient protection against virus infection initiated at the oral mucosa. This could be stringently tested by passive antibody transfer and oral virus challenge in the rhesus macaque model for EBV infection. However, only neutralizing monoclonal antibodies (MAbs) against EBV are available, and EBV is unable to infect rhesus macaques because of a host range restriction with an unknown mechanism. We cloned the prototypic EBV-neutralizing antibody, 72A1, and found that recombinant 72A1 did not neutralize rhesus lymphocryptovirus (rhLCV) infection of macaque B cells. Therefore, we constructed a chimeric rhLCV in which the native major membrane glycoprotein was replaced with EBV gp350. This chimeric rhLCV became sensitive to neutralization by the 72A1 MAb, efficiently immortalized macaque B cells in vitro, and successfully established acute and persistent infection after oral inoculation of rhesus macaques. Thus, EBV gp350 can functionally replace rhLCV gp350 and does not restrict rhLCV infection in vitro or in vivo. The chimeric rhLCV enables direct use of an EBV-specific MAb to investigate the effects of serum neutralizing antibodies against B cell infection on oral viral challenge in rhesus macaques. IMPORTANCEThis study asked whether the EBV major membrane glycoprotein could functionally replace the rhLCV major membrane glycoprotein. We found that an rhLCV humanized with EBV gp350 is capable of efficiently immortalizing monkey B cells in vitro and reproduces acute and persistent infection after oral inoculation of macaques. These results advance our understanding of why EBV cannot infect rhesus macaques by proving that viral attachment through gp350 is not the mechanism for EBV host range restriction. Humanization of rhLCV with EBV gp350 also confers susceptibility to a potent EBV-neutralizing MAb and provides a novel and significant enhancement to the rhesus macaque animal model where both the clinical utility and biological role of neutralizing MAbs against B cell or epithelial cell infection can now be directly tested in the most accurate animal model for EBV infection. P rimary Epstein-Barr virus (EBV) infection is the most common cause of infectious mononucleosis (IM), and persistentEBV infection is associated with cancers of B cell origin, e.g., Burkitt lymphoma and Hodgkin lymphoma, as well as epithelial cell origin, e.g., nasopharyngeal carcinoma and gastric carcinoma. A vaccine that prevents EBV-associated diseases would provide significant public health benefits (1).The major membrane glycoprotein gp350 is considered an attr...

An Antibody Targeting the Fusion Machinery Neutralizes Dual-Tropic Infection and Defines a Site of Vulnerability on Epstein-Barr Virus

“…Alternatively, the virus may initially infect infiltrating B cells, which would lead to subsequent targeting of epithelial cells. Neither scenario is mutually exclusive; thus a combination of epithelial and B cell neutralizing antibodies may be required to most effectively block incoming virus at the oral mucosa (Herrman et al, 2015).…”

“…pCAGGS expression plasmids for gH, gL, gB, and pT7EMCLuc (which carries a luciferase-containing reporter plasmid under the control of the T7 promoter) were kindly provided by Dr. R. Longnecker (Haan et al, 2001; Okuma et al, 1999; Plate et al, 2011). Plasmids for the expression of humanized, recombinant 72A1 were provided by Dr. F. Wang (Herrman et al, 2015). p509, an expression plasmid encoding BZLF1, was provided by Dr. W. Hammerschmidt (Delecluse et al, 1998).…”

Neutralizing Antibodies Protect against Oral Transmission of Lymphocryptovirus

“… 53 The 72A1 mAb, which showed protection in mice, does not cross-react with the rhesus gp350 protein. 54 A chimeric rhLCV virus expressing gp350 from EBV is infectious in macaques and susceptible to neutralization by 72A1, 54 but passive transfer studies of neutralizing mAbs against rhLCV have not been reported to date.…”

Construction and Characterization of a Humanized Anti-Epstein-Barr Virus gp350 Antibody with Neutralizing Activity in Cell Culture

“…Prior to undertaking the humanization of mouse (m)72a1, we re-examined the reported differences in anti-gp350 reactivity observed with the use of antibody constructs based on two separate sets of m72a1 variable region (VR) cDNAs [ 33 , 34 ]. An examination of archived chimeric antibody samples generated in our laboratory prior to our earlier publication showed that the strongest anti-gp350 immunoreactivity was found in a chimeric antibody based on GenBank cDNA sequences KT211017.1 and KT211018.1 ( Figure S1 ) [ 34 ].…”

“…The murine 72a1 HC partial coding sequence (GenBank KT211017.1) [ 34 ] was modified by addition of the Bam HI restriction enzyme recognition site upstream of the HC VR ATG start sequence and the introduction of a Sca I site at nucleotide position 409 ( DNA sequence S1 ). The murine 72a1 lambda LC partial coding sequence (GenBank: KT211018.1) [ 34 ] was modified to remove an internal Sca I restriction enzyme site at nucleotide position 133 and restriction enzymes Hind III and Hinc II introduced upstream of the LC ATG start sequence and at nucleotide 372, respectively ( DNA sequence S2 ). Nucleotide modification to introduce new restriction enzyme sites was not expected to change the 72a1 protein sequences.…”