A Novel Insertion in the Hepatitis B Virus Surface Protein Leading to Hyperglycosylation Causes Diagnostic and Immune Escape

Abstract

Chronic hepatitis B virus (HBV) infection is a global health threat. Mutations in the surface antigen of HBV (HBsAg) may alter its antigenicity, infectivity, and transmissibility. A patient positive for HBV DNA and detectable but low-level HBsAg in parallel with anti-HBs suggested the presence of immune and/or diagnostic escape variants. To support this hypothesis, serum-derived HBs gene sequences were amplified and cloned for sequencing, which revealed infection with exclusively non-wildtype HBV subgenotype (sgt) D3. Three distinct mutations in the antigenic loop of HBsAg that caused additional N-glycosylation were found in the variant sequences, including a previously undescribed six-nucleotide insertion. Cellular and secreted HBsAg was analyzed for N-glycosylation in Western blot after expression in human hepatoma cells. Secreted HBsAg was also subjected to four widely used, state-of-the-art diagnostic assays, which all failed to detect the hyperglycosylated insertion variant. Additionally, the recognition of mutant HBsAg by vaccine- and natural infection-induced anti-HBs antibodies was severely impaired. Taken together, these data suggest that the novel six-nucleotide insertion as well as two other previously described mutations causing hyperglycosylation in combination with immune escape mutations have a critical impact on in vitro diagnostics and likely increase the risk of breakthrough infection by evasion of vaccine-induced immunity.

Keywords: N-linked glycosylation; diagnostic escape; hepatitis B virus; immune escape.

Figure 1

The major hydrophilic region (MHR) of mutated viral variants contains diagnostic escape mutations and additional glycosylation motifs. (A) The putative topology of SHBs. SHBs consists of four transmembrane domains. The MHR between transmembrane domains II and III (aa100-163) is the major target of neutralizing antibodies and is commonly associated with mutations. The N-linked glycosylation site at N146 is highly conserved. (B) MHR of serum-derived SHBs variants. Cluster 1 represents a subgenotype (sgt) D3 isolate (similar to the sgtD3 reference, serotype ayw3) but with an unusual ayw4 serotype, caused by mutation T127L. In the remaining clusters, several mutations were found: three previously described diagnostic escape mutations (cluster 2: T131I; cluster 3: G130R/K141I; yellow), a six-nucleotide insertion (cluster 4; orange), and substitutions introducing N-glycosylation motifs (clusters 2–4; boxed). The conserved N-glycosylation site (N146) is shown in green. Black arrows denote additional N-glycosylation sites. Grey arrows denote HBsAg subtype w/r determinant defining amino acids.

Figure 2

Ectopic sequons of the variant SHBs serve as N-glycosylation acceptor sites and cause hyperglycosylation of SHBs. (A) Construction of SHBs expression plasmids. Expression of N-terminally 6xHis- and FLAG-tagged SHBs is controlled by the CMV immediate/early promoter present in pcDNA3.1(+). The sequence encoding amino acids 100–226 (encompassing the MHR (aa100-163)) of the reference subgenotype (sgt) D3 strain was exchanged with the corresponding sequences of the viral variants. Mutations causing additional glycosylation are underlined. Representative Western blot of (B) intracellular and (C) extracellular SHBs expression pattern. HepG2 cells were transiently transfected with 6xHis-FLAG-SHBs-expression plasmids. Four days post-transfection, supernatants were collected and cells were lysed. Cell lysates/supernatants were subjected to SDS-PAGE and blotted. Membranes were probed with an anti-FLAG antibody to visualize SHBs. (D) SHBs in the supernatants was treated with PNGase F and subjected to Western blot as described above. Bands of deglycosylated SHBs of three independent experiments were quantified with Image Studio Lite. MHR = major hydrophilic region; NC = negative control (empty pcDNA3.1(+) was transfected); R = reference strain of sgtD3.

Figure 3

Detectability of SHBs is severely impaired by the combination of hyperglycosylation and immune escape variants. (A) Top panel: HepG2 cells were transiently transfected with SHBs-expressing plasmids and supernatants were collected four days after transfection. SHBs in the supernatants was adjusted to equal amounts (as quantified by immunoblot) and measured in the quantitative HBsAg assay (Abbott ARCHITECT). Bottom panel: supernatants containing equal amounts of SHBs (as quantified by immunoblot) were tested in the HBsAg confirmatory test (Abbott ARCHITECT). (B) Top panel: supernatants containing equal amounts of SHBs (as quantified by immunoblot) were measured in the qualitative HBsAg assay (DiaSorin Liaison). Bottom panel: supernatants containing equal amounts of SHBs (as quantified by immunoblot) were tested in the HBsAg confirmatory test (DiaSorin Liaison). All panels show the results of three independent experiments. R = SHBs of subgenotype D3 reference strain; 1/2/3/4 = SHBs containing aa1-99 of the reference strain and aa100-226 of a patient-derived sequence of cluster 1/2/3/4, respectively (also refer to Figure 2A). NC = negative control (supernatant of empty pcDNA3.1(+)-transfected cells). NR = nonreactive. ns = not significant, ** = p < 0.01, **** = p < 0.0001 (unpaired t-test).

Figure 4

Hyperglycosylation of variant HBsAg masks epitopes recognized by anti-HBs which was induced in six human donors each by vaccination or HBV infection. SHBs within cell culture supernatants was diluted to equal amounts (corresponding to 10 IU/mL WT HBsAg) and preincubated with human anti-HBs-positive sera, containing 50 IU/L anti-HBs. After incubation, quantitative unbound anti-HBs levels were measured with the ARCHITECT anti-HBs-test (Abbott ARCHITECT). Data are shown as relative reactivity of vaccinee (A), convalescent (B), and naïve (C) human sera with variant HBsAg. Original data used for calculation of relative reactivities are presented in Table S1. R = SHBs of subgenotype D3 reference strain; 1/2/3/4 = SHBs containing aa1-99 of the reference strain and aa100-226 of the patient-derived sequences of cluster 1/2/3/4, respectively (also refer to Figure 2A). NC = negative control (supernatant of empty pcDNA3.1(+)-transfected cells). ns = not significant, * = p < 0.05, *** = p < 0.001, **** = p < 0.0001 (unpaired t-test).