Identification of the critical attribute(s) of EBV gp350 antigen required for elicitation of a neutralizing antibody response in vivo

Highlights

• Identification of critical quality attributes for EBV gp350 vaccine development.

• Requirement of 72A1 epitope on recombinant gp350 for binding to CD21 on human B cells.

• Glycosylation of gp350 is essential for generation of neutralizing antibodies.

Abstract

Vaccine prophylaxis with EBV glycoprotein 350 (gp350) subunit plus adjuvant has been demonstrated clinically to protect individuals against infectious mononucleosis (IM), but the specifications of the antigen required to elicit this protection has remained largely theoretical. Previous studies have shown that antibodies to gp350 comprise the principle component of EBV-neutralizing sera. Further, a murine monoclonal antibody against gp350 (clone 72A1) is able to prevent infection by the virus both in vitro and in vivo. In the present study, we identify the 72A1 epitope on recombinant gp350 antigen as the site required for binding to CD21 on human B cells. We also identify the need for conformational-dependence of the antigen to generate EBV-neutralizing antibodies in vivo. Further, we have characterized the glycosylation status and antigenicity profiles of both native and denatured CHO-produced soluble gp350 as well as non-glycosylated protein produced in Escherichia coli. Collectively our in vitro and in vivo data demonstrate the requirement for a conformationally accessible 72A1 epitope on gp350 to elicit EBV-neutralizing responses, and establish this as a critical attribute of this vaccine antigen. These data provide direction for commercial vaccine development, as the absence of this epitope on either E. coli-expressed or denatured gp350, may limit production and purification options for the antigen.

Introduction

Epstein–Barr virus (EBV) is a prevalent gamma herpesvirus that infects human B-cells and epithelial cells and is the major cause of infectious mononucleosis (IM), a clinical syndrome characterized by fever, pharyngitis, and cervical lymphadenopathy primarily afflicting adolescents and young adults [1]. The virus is transmitted via saliva, and infects naive B cells through binding of the major viral surface glycoprotein, gp350, to CD21 on the B-cell surface. Following primary infection the virus establishes latency in B cells where it persists for life. This chronic B-cell reservoir undergoes recurrent lytic cycle reactivation, asymptomatic shedding of virus into the saliva, and spread to uninfected individuals, enabling a near complete permeation of EBV among humans. In addition to IM, EBV has been associated with a variety of proliferative diseases including Burkitt's and Hodgkin's lymphomas, nasopharyngeal carcinoma, and post-transplant lympoproliferative disease [2], [3], [4].

At present, no commercial vaccine is available for the prevention of EBV-associated diseases. Building on the observation that the majority of EBV-neutralizing capacity present in serum is directed against the major viral glycoprotein gp350 [5], [6], [7], [8], both adjuvanted gp350 subunit and vaccinia-vectored gp350 approaches have been evaluated in humans. The first clinical vaccine trial was performed in China using a vaccinia-vectored gp350 and demonstrated immunogenicity in vaccinated children [9]. This vectored approach generated strong anti-gp350 antibody responses and showed promise for protection against natural infection. In addition, trials performed using a CHO-derived soluble recombinant gp350 subunit antigen adjuvanted with 3-O-desacyl-4′-monophosphoryl A (AS04) [10], [11] successfully demonstrated safety, tolerability, and immunogenicity in young adults, where the vaccine induced strong antibody responses to gp350. More importantly, the vaccine had demonstrable efficacy (mean efficacy rate, 78.0% [95% confidence interval {CI}, 1.0–96.0%]) in preventing IM induced by EBV infection, but it had no efficacy in preventing asymptomatic EBV infection. The small proof-of-concept trial for adjuvanted gp350 subunit revealed a high level of efficacy at preventing acute IM, reaching 100% protection following the third dose [11].

Considerable analytical hurdles remain in using this recombinant antigen for product development. gp350 is one of the most heavily glycosylated proteins described where the polypeptide core represents only ∼90 kDa of its apparent 350 kDa molecular weight. The remaining ∼260 kDa results from post-translational modifications. The antigenicity profile of soluble gp350 antigen used in clinical trials has been described using a panel of epitope specific monoclonal antibodies (mAbs) [12], [13]. It has been shown that presence of the gp350 epitope reactive with the neutralizing murine mAb 72A1 (epitope group 1) mediates binding to CD21 (CR2) on B cells and hence would be an antigen characteristic crucial for effective vaccination [14]. However, a direct relationship between the conformational dependence of gp350 to bind this antibody and the antigen's capacity to bind natural CD21 receptor on human B cells has not been demonstrated.

In the present study, we examined the glycosylation status of CHO-produced soluble gp350 through mass spectrometry identification of N- and O-linked sites. Enzymatic deglycosylation of gp350 was performed to determine the contribution of these post-translational modifications on its size and thermal stability. Antigenicity profiles of native and denatured CHO-produced antigen, and non-glycosylated gp350 produced in Escherichia coli were characterized using a panel of epitope specific antibodies. Finally to evaluate the conformational requirement of gp350 for induction of neutralizing antibodies, the ability of native and denatured antigen to bind to gp350's natural receptor CD21, on human B cells, was determined as was their capacity to generate EBV-neutralizing antibody responses following immunization in vivo. Collectively our in vitro and in vivo data couple the conformational dependence for un-blocked 72A1 epitope on the gp350 antigen to elicit EBV-neutralizing responses and identify this epitope as the critical quality attribute for this vaccine antigen.