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.

Importance: This 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.

FIG 1

Cloning and expression of recombinant immunoglobulins from the 72A1 hybridoma that bind gp350 and neutralize EBV infection. (A) Simultaneous detection of murine kappa (IgK) and lambda (IgL) light-chain expression on 72A1 hybridoma cells by flow cytometry. (B) Testing recombinant antibodies for EBV gp350 binding by ELISA. 293 cells were transfected with the four different combinations of immunoglobulin expression plasmids [i.e., heavy-chain (H1 and H2) and light-chain (L1 and L2) variable regions of immunoglobulin mRNAs expressed in the 72A1 hybridoma cloned upstream of either the human IgG1 constant region or the human Ig(κ) constant region]. Cell supernatants were harvested 3 days after transfection, and reactivity to recombinant gp350 was measured by ELISA. Human sera from EBV-immune (Pos. Sera) or EBV-nonimmune (Neg. Sera) donors were used as controls. (C) EBV neutralization with recombinant H1L2 72A1. Human PBMC were stained with CFSE and exposed to medium, virus, or virus preincubated with recombinant antibody. Proliferation of human B cells was detected using flow cytometry by gating for live CD20⁺ cells and monitoring the reduction in CFSE intensity. The percentages of CFSE-low proliferating B cells relative to the percentages of CFSE-high nonproliferating B cells are indicated. The results from preincubation with H2L2 were similar to those with H1L1 and H2L1.

FIG 2

EBV, but not wild-type rhLCV, is sensitive to 72A1 neutralization. (A) CFSE-labeled human PBMC were exposed to medium, EBV, or EBV pretreated with 100, 50, or 25 ng/ml recombinant 72A1. Proliferation was detected using flow cytometry by gating live CD20⁺ cells and monitoring the reduction in CFSE intensity. The percentages of proliferating (CFSE-low) B cells are indicated. (B) CFSE-labeled rhesus macaque PBMC were similarly assayed following exposure to medium, rhLCV, or rhLCV pretreated with 100, 50, or 25 ng/ml recombinant 72A1.

FIG 3

Generation of chimeric rhLCV-hugp350. (A) Schematic representation of the 10-kb region of the WT-rhLCV BAC (top) containing the gp350 open reading frame and surrounding genes. The native rhgp350 of the WT-rhLCV BAC is shown in the middle structure, and the replacement with the EBV gp350 in the chimeric rhLCV-hugp350 BAC is shaded in gray in the bottom structure. BamHI restriction sites are indicated by a B, and the distance between the restriction sites is noted. DNA probes used for detection of rhgp350 or hugp350 fragments after Southern blotting are shown as an open or filled arrowhead, respectively. (B) Validation of rhgp350 replacement with hugp350 in the rhLCV BAC. BamHI digests of WT-rhLCV BAC and chimeric rhLCV-hugp350 BAC recovered from the rhLCV-hugp350 LCL were separated by agarose gel electrophoresis. The ethidium bromide (EtBr)-stained gel is shown on the left. After Southern blotting, the membrane was hybridized with rhgp350- or hugp350-specific probes, as shown on the right. (C) The rhLCV-hugp350 LCL was infected with the BAC-derived chimeric rhLCV-hugp350. DNA from LCLs stably transformed with EBV, WT-rhLCV, and the chimeric rhLCV-hugp350 was assayed for hugp350, huEBER, rhgp350, and rhEBNA2 genes by PCR. GAPDH was amplified as a control. (D) The rhLCV-hugp350 LCL expresses EBV gp350. WT-rhLCV and rhLCV-hugp350 LCLs were induced for lytic replication, and EBV gp350 was precipitated from the cell lysates by incubation with 72A1. Whole-cell lysates are shown on the left, and 72A1 immunoprecipitation (IP) is shown on the right. EBV-immune human serum was used for immunoblotting.

FIG 4

rhLCV-hugp350 immortalizes rhesus B cells similarly to the wild type and is sensitive to 72A1 neutralization. (A) The transformation efficiencies of WT-rhLCV and rhLCV-hugp350 were compared by calculating the DNA unit/TU ratio. Multiple large-scale virus preparations (lots 1 to 5) were assayed for B cell-transforming activity, and transforming units per milliliter were calculated as the dilution necessary for 50% outgrowth. The relative DNA content was assayed by real-time PCR, and all the samples were amplified in the same PCR run to allow comparison of DNA units. (B) rhLCV-hugp350 is as sensitive to 72A1 neutralization as EBV. Shown are CFSE neutralization assay results for rhLCV-hugp350 infection of rhesus PBMC (bottom) and EBV infection of human PBMC (top) when preincubated with 2-fold dilutions of recombinant 72A1. For both EBV and rhLCV infections, virus alone induced proliferation in approximately 25% of human and rhesus macaque B cells, respectively. Neutralization was calculated as follows: (percent CFSE-low cells with virus alone − percent CFSE-low cells of virus preincubated with antibody)/percent CFSE-low cells with virus alone.

FIG 5

Chimeric rhLCV-hugp350 infects rhesus macaques following oral inoculation. (A) Detection of rhLCV-hugp350 infection in circulating PBMC of experimentally infected macaques during acute (0 to 16 weeks) and persistent (>16 weeks) infection. Shown is a schematic representation of rhEBER reverse transcription (RT)-PCR-positive (filled circles) and -negative (open circles) PBMC aliquots for two macaques (Mm218-05 and Mm143-97) after oral inoculation with rhLCV-hugp350. (B) rhLCV-hugp350 establishes a persistent infection in rhesus macaques. The frequency of rhLCV-hugp350-infected B cells was determined by limiting dilution of PBMC, rhEBER RT-PCR, and Poisson distribution (24). The frequency of infected B cells during persistent infection in Mm218-05 is represented by triangles and that in Mm143-97 by circles.