Identification of multiple potent neutralizing and non-neutralizing antibodies against Epstein-Barr virus gp350 protein with potential for clinical application and as reagents for mapping immunodominant epitopes

Abstract

Prevention of Epstein-Barr virus (EBV) infection has focused on generating neutralizing antibodies (nAbs) targeting the major envelope glycoprotein gp350/220 (gp350). In this study, we generated 23 hybridomas producing gp350-specific antibodies. We compared the candidate gp350-specific antibodies to the well-characterized nAb 72A1 by: (1) testing their ability to detect gp350 using enzyme-linked immunosorbent assay, flow cytometry, and immunoblot; (2) sequencing their heavy and light chain complementarity-determining regions (CDRs); (3) measuring the ability of each monoclonal antibody (mAb) to neutralize EBV infection in vitro; and (4) mapping the gp350 amino acids bound by the mAbs using competitive cell and linear peptide binding assays. We performed sequence analysis to identify 15 mAbs with CDR regions unique from those of murine 72A1 (m72A1). We observed antigen binding competition between biotinylated m72A1, serially diluted unlabeled gp350 nAbs (HB1, HB5, HB11, HB20), and our recently humanized 72A1, but not gp350 non-nAb (HB17) or anti-KSHV gH/gL antibody.

Keywords: Complementarity-determining region; Epitope; Epstein-Barr virus; Immunodominant; Immunosuppression; Infection; Neutralizing antibodies; gp350.

Figure 1.. Specificity of anti-gp350 antibodies.

(A) SDS-PAGE analysis of anti-EBV gp350 antibodies purified from indicated hybridoma (HB) supernatants. (B) ELISA screening of HB supernatants for anti-gp350-specific antibodies. Soluble EBV gp350 protein was used as the target antigen at 0.5 μg/ml. m72A1 at 10 μg/ml and KSHV anti-gH/gl (54A1) were used as positive and negative (not shown) controls, respectively. Bound antibodies were detected using HRP-conjugated anti-mouse IgG (1:2,000). Twenty-three HB clones with ELISA signals two times greater than those of PBS control were considered to be positive/reactive to gp350. (C) Immunoblot analysis with gp350-transfected stable CHO lysate to determine specificity of anti-gp350-producing HB supernatants. (D) Flow cytometric analysis of surface expression of gp350 protein on gp350-expressing CHO cells. Cells were stained with indicated anti-gp350 mAbs (1:250), followed by secondary goat anti-mouse conjugated to AF488.

Figure 1.. Specificity of anti-gp350 antibodies.

(A) SDS-PAGE analysis of anti-EBV gp350 antibodies purified from indicated hybridoma (HB) supernatants. (B) ELISA screening of HB supernatants for anti-gp350-specific antibodies. Soluble EBV gp350 protein was used as the target antigen at 0.5 μg/ml. m72A1 at 10 μg/ml and KSHV anti-gH/gl (54A1) were used as positive and negative (not shown) controls, respectively. Bound antibodies were detected using HRP-conjugated anti-mouse IgG (1:2,000). Twenty-three HB clones with ELISA signals two times greater than those of PBS control were considered to be positive/reactive to gp350. (C) Immunoblot analysis with gp350-transfected stable CHO lysate to determine specificity of anti-gp350-producing HB supernatants. (D) Flow cytometric analysis of surface expression of gp350 protein on gp350-expressing CHO cells. Cells were stained with indicated anti-gp350 mAbs (1:250), followed by secondary goat anti-mouse conjugated to AF488.

Figure 2.. Determination of novel anti-gp350 antibody sequences.

(A) Agarose gel analysis of PCR products of heavy chain of select novel anti-gp350 antibodies (HB1, HB4, HB7, HB13, and HB15) and m72A1 was used as a positive control. (B) Amino acid sequencing of the heavy (V_H) and light (V_L) chain variable region complementarity-determining regions (CDR) 1–3 of the new gp350 mAbs and mouse (m72A1) and humanized 72A1 (h72A1). (C) IMGT/V-QUEST analysis to determine the germline gene families for V_H and V_L of the 15 new mAbs and m72A1.

Figure 3.. Comparison of murine 72A1 (m72A1) and humanized 72A1 (h72A1).

(A) Sequence comparison of murine (m72A1) and humanized (h72A1) 72A1. ClustalW alignment of heavy chain (i) and light chain (ii) variable region amino acid sequences. Regions of identical sequence are represented by *. Regions of similarity are represented by:. (B) ELISA comparison screening of m72A1 and h72A1 for anti-gp350-specificity. Soluble EBV gp350 protein was used as the target antigen at 0.5 μg/ml. m72A1 and h72A1 were serially diluted (5–0.062 μg/ml) and 1x phosphate buffered saline (PBS) was used as a negative control (data not shown). Bound h72A1 and m72A1 antibodies were detected using HRP-conjugated anti-mouse IgG and anti-human IgG (1:2,000) as relevant. (C) ELISA determining the reactivity of humanized 72A1 to murine IgG. Soluble EBV gp350 protein was used as the target antigen at 0.5 μg/ml. Plates were incubated with 10 μg/ml of m72A1 and h72A1, followed by three washes. Bound antibodies were detected using HRP-conjugated anti-mouse IgG or anti-human IgG (1:2,000). (D) Flow cytometric analysis of m72A1 and h72A1 gp350 specificity. CHO wild-type cells and gp350-expressing CHO cells were stained with m72A1 and h72A1, followed by secondary goat anti-mouse or anti-human conjugated to AF488. Unstained cells and cells stained with secondary goat anti-mouse or anti-human conjugated to AF488 alone were used as negative controls. 2° represents secondary antibody.

Figure 4:. Neutralization activity of novel anti-gp350 mAbs against EBV-eGFP in Raji cells.

(A) EBV-eGFP titration in Raji cells to determine optimal dose of infection. (B) EBV-eGFP was pre-incubated with 15 indicated serial diluted (12.5–100 μg/ml), maxispin column-purified anti-gp350 mAbs, followed by incubation with 10⁵ Raji cells for 48 h. EBV-eGFP+ cells were enumerated using flow cytometry. Anti-gp350 (m72A1) nAb served as positive control and non-neutralizing anti-gp350 (2L10) mAb and anti-KSHV gH/gL mAb (54A1) served as negative controls. (C) EBV-eGFP was pre-incubated with 7 indicated serially diluted (12.5–100 μg/ml) protein G affinity chromatography- and size-exclusion chromatography-purified anti-gp350 mAbs, followed by incubation with 10⁵ Raji cells for 48 h. EBV-eGFP+ cells were enumerated using flow cytometry. Anti-gp350 (m72A1 and h72A1) nAbs served as positive controls and anti-KSHV gH/gL mAb (54A1) served as negative control.

Figure 5.. Analysis of anti-gp350 antibodies linear epitope binding.

(A) Schematic diagram of EBV gp350 protein, illustrating the ectodomain and the splice site (aa 501–699) for making gp220, the transmembrane domain, TM (aa 841–897) and the cytoplasmic domain, CT (aa 898–907). To analyze and classify binding of anti-gp350 mAbs to linear epitopes on the protein, EBV gp350 was separated into 9 regions of ~100 aa. (B) Summarized analysis of anti-gp350 mAb linear epitope binding to various regions of gp350. Three major immunodominant regions were identified, region 1 (aa 1–100), 2 (aa 101–202) and 5 (aa 401–502).