Purified hexameric Epstein-Barr virus-encoded BARF1 protein for measuring anti-BARF1 antibody responses in nasopharyngeal carcinoma patients

Abstract

WHO type III nasopharyngeal carcinoma (NPC) is highly prevalent in Indonesia and 100% associated with Epstein-Barr virus (EBV). NPC tumor cells express viral proteins, including BARF1, which is secreted and is considered to have oncogenic and immune-modulating properties. Recently, we found conserved mutations in the BARF1 gene in NPC isolates. This study describes the expression and purification of NPC-derived BARF1 and analyzes humoral immune responses against prototype BARF1 (B95-8) and purified native hexameric BARF1 in sera of Indonesian NPC patients (n = 155) compared to healthy EBV-positive (n = 56) and EBV-negative (n = 16) individuals. BARF1 (B95-8) expressed in Escherichia coli and baculovirus, as well as BARF1-derived peptides, did not react with IgG or IgA antibodies in NPC. Purified native hexameric BARF1 protein isolated from culture medium was used in enzyme-linked immunosorbent assay (ELISA) and revealed relatively weak IgG and IgA responses in human sera, although it had strong antibody responses to other EBV proteins. Higher IgG reactivity was found in NPC patients (P = 0.015) than in regional Indonesian controls or EBV-negative individuals (P < 0.001). IgA responses to native BARF1 were marginal. NPC sera with the highest IgG responses to hexameric BARF1 in ELISA showed detectable reactivity with denatured BARF1 by immunoblotting. In conclusion, BARF1 has low immunogenicity for humoral responses and requires native conformation for antibody binding. The presence of antibodies against native BARF1 in the blood of NPC patients provides evidence that the protein is expressed and secreted as a hexameric protein in NPC patients.

FIG. 1.

BARF1 (peptide) sequence and specific animal antibody reactivity against B95-8-derived recombinant BARF1 protein and antigenic peptides. (A) BARF1 sequence containing 2 common amino acid substitutions, V29A and H130R, found in Indonesian isolates (boldface). Antigenic peptides designed by computer analysis, named N peptide (aa 40 to 79), M peptide (aa 151 to 188), and C peptide (aa 187 to 221), are underlined. (B) Antibody reactivity to peptides measured in ELISA. Guinea pig antibodies against C peptide, M peptide, N peptide, and mouse monoclonal anti-C peptide (4A6) specifically react to their target peptides. The rabbit polyclonal antibody anti-BARF1-His (K150.3) binds to both C and M peptides. Rabbit polyclonal antibody raised against the purified native sBARF1 reacts to the C peptide. (C) Antibody reactivity of healthy individuals and NPC patients to the BARF1 C peptide measured in IgG ELISA, showing slightly elevated levels in NPC patients. (D) IgG immunoblot analysis of 3 representative NPC sera with BARF1-His expressed by the Sf9 insect cell expression system and BARF1-GST expressed by E. coli. HH514-derived viral capsid antigens were used as a positive control for EBV antibody presence in patient sera. Guinea pig anti-C peptide antibody (+) showed a clear band on the blot strips, but no BARF1 reactivity was detected in the patient sera despite the presence of diverse antibody responses to other EBV proteins, as visualized on the HH514 strips.

FIG. 2.

BARF1 is secreted as a hexameric glycosylated protein from human HEK293 cells. (A) BARF1 is secreted as a hexamer in the culture supernatant of stably transfected 293HEK-BARF1 cells. Ten microliters of medium was mixed 1:1 with either reduced (R) or nonreduced (NR) loading buffer and separated by SDS-PAGE and Western blotting. A 29-kDa and a 160-kDa band can be seen, representing the monomeric and the hexameric structures of sBARF1, respectively. (B) The glycosylation status of BARF1 in serum-free culture supernatant was determined by direct treatment with several glycosidases. PNGase F cleaves high-mannose N-linked sugar groups. Neuraminidase removes sialic acid groups, and O-glycosidase cleaves O-linked sugar chains. The digested protein was loaded on an SDS-PAGE Western blot and detected using 4A6 anti-BARF1 antibody. (C) Manipulation of BARF1 secretion from HEK293 cells. Under normal growth conditions, hardly any BARF1 can be detected in 293HEK cell lysates; BARF1 is completely secreted in the culture supernatant. Treatment of cells with the transport blockers monensin (Mon) and brefeldin A (BFA) retains sBARF1 in the cell lysate and almost completely blocks secretion into the culture medium. (D) Schematic overview of intercellular transport blockage by brefeldin A and monensin. (E) Native-sBARF1 was purified from culture medium using ConA affinity chromatography followed by size exclusion chromatography and visualized by Coomassie staining as hexameric sBARF1 (NR) and monomeric sBARF1 (R). Purity of ∼98% was routinely reached in repeated experiments.

FIG. 3.

Low immune responses against native BARF1 in human sera. (A) IgG antibodies against native human sBARF1 detected by antibody capture ELISA. Data are shown as OD₄₅₀ values for EBV-negative healthy donors (n = 16), EBV-positive healthy donors (n = 56), and NPC patients (n = 155). (B) IgA antibodies against native human sBARF1 were detected by ELISA using EBV-negative healthy donors (n = 15), EBV-positive healthy donors (n = 57), and NPC patients (n = 155). (C) Scatter plot of IgG anti-BARF1 versus IgG anti-EBNA responses. (D) Scatter plot of IgG anti-BARF1 versus IgG anti-VCAp18 responses. Filled circles, NPC patients; open circles, EBV-positive healthy donors; stars, EBV-negative healthy donors.

FIG. 4.

Immunoblot confirmation of specific antibody responses to native sBARF1. Native sBARF1 was loaded on a reduced SDS-PAGE gel, blotted to nitrocellulose, and cut into strips. Sera from the highest responders in the NPC patient panel and control sera were analyzed at 1:100 dilution. 4A6 anti-BARF1 antibody was used as a positive control (C). A band at the correct height was detected with most of the selected NPC patient sera only upon considerable overexposure (minutes) when using ECL visualization. The 4A6 signal was revealed in a few seconds.