Linear epitopes on the capsid protein of norovirus commonly elicit high antibody response among past-infected individuals

Abstract

Background: Human norovirus (HuNoV) is the leading cause of acute nonbacterial gastroenteritis globally, and its infection is usually self-limited, so most people become past Norovirus (NoV)-infected individuals. It is known that some antibody responses may play a critical role in preventing viral infection and alleviating disease; however, the characteristics and functions of particular antibody responses in persons with previous infections are not fully understood. Capsid proteins, including VP1 and VP2, are crucial antigenic components of NoV and may regulate antibody immune responses, while epitope-specific antibody responses to capsid proteins have not been comprehensively characterized.

Methods: We prepared purified VP1 and VP2 proteins by ion exchange chromatography and measured serum antigen-specific IgG levels in 398 individuals by ELISA. Overlapping 18-mer peptides covering the full length of VP1 and VP2 were synthesized, and then we identified linear antigenic epitopes from 20 subjects with strong IgG positivity. Subsequently, specific antibody responses to these epitopes were validated in 185 past infected individuals, and the conservation of epitopes was analyzed. Finally, we obtained epitope-specific antiserum by immunizing mice and expressed virus-like particles (VLPs) in an insect expression system for a blockade antibody assay to evaluate the receptor-blocking ability of epitope-specific antibodies.

Results: The IgG responses of VP1 were significantly stronger than those of VP2, both of which had high positive rates of over 80%. The overall positive rate of VP1-IgG and/or VP2-IgG was approximately 94%, which may be past NoV-infected individuals. Four linear antigenic B-cell epitopes of capsid proteins were identified, namely, VP1_199-216, VP1_469-492, VP2_97-120, and VP2_241-264, all of which were conserved. The IgG response rates of the above epitopes in past NoV-infected individuals were 38.92%, 22.16%, 8.11% and 28.11%, respectively. In addition, VP1_199-216- and VP1_469-492-specific antibodies can partially block the binding of VLPs to the receptor histo-blood group antigen (HBGA).

Conclusion: This is the first study to describe specific antibody responses of VP2 and to identify its B-cell epitopes. Our findings offer data for a more thorough understanding of norovirus capsid protein-specific IgG responses and could provide useful information for designing and developing vaccines.

Keywords: Epitopes; IgG; Norovirus; VP1; VP2.

Fig. 1

VP1- and VP2-specific IgG responses in non-current NoV-infected individuals. A The magnitude B and positive rate of VP1- and VP2-specific IgG responses. Serum samples were collected from 398 individuals who were not currently NoV-infected. All OD₄₅₀ values were subtracted from blank control values. With irrelevant protein BSA as the negative control, its mean OD₄₅₀ value plus 3 times the standard deviation was calculated as the positive cut-off value, and above the red dotted line was the positive response. C The results of 398 cases were classified. Statistically significant differences were determined by the Kruskal‒Wallis test and Chi-square test: ns, no significance; ****, p < 0.0001

Fig. 2

Mapping linear B-cell epitopes of VP1 and VP2. A VP1 and B VP2 of the heatmap for screening linear B-cell epitopes identified in 20 double-positive subjects. The individual's specific IgG responses to 9 matrix pools and corresponding peptides were detected. For each individual, the highest IgG response to one pool or peptide in a pool was noted as "1" and considered as high response (HR), filled with a dark red mark, the other responses were evaluated by their relative strength against HR. The responses with strength between 81 and 99% of the HR was considered as a sub-high response and was marked in red. Total responses (TR) included all highest and sub-high responses. C Domain, sites and amino acid sequence information of all linear epitopes

Fig. 3

The IgG responses of epitopes were detected in more populations. A Specific IgG levels and B positive rates of each linear epitope were detected in 185 individuals. Irrelevant peptide OVA was used as a negative control, and its mean OD₄₅₀ plus 3 times the standard deviation was used as the positive cut-off value to calculate the positive rate. Statistically significant differences were determined by the Kruskal‒Wallis test: ****, p < 0.0001

Fig. 4

Sequence similarity of epitopes among common strains was performed. A VP1 and B VP2 epitope sequence conservation was analyzed in different genotypes of norovirus. Representative strains, mutant strains and recombinant strains of genotypes GII.4, GII.3, and GII.2 were selected for comparison. Each letter represents an amino acid residue, and the size of the letter reflects the conservation of this site

Fig. 5

VLP-HBGA binding antibody blockade assay. A Spatial structure of the VP1 protein and locations of linear epitopes. B Schematic of immunizing mice with epitope-KLH conjugation by Figdraw. C The titer of antiserum was detected by peptide ELISA. The control group was normal mouse serum immunized with NaCl and adjuvant. D Diagram of VLP-carbohydrate binding blockade assay by Figdraw. E The blocking efficiency of the antiserum, with normal mouse serum as the negative control and the Anti-Norovirus GII.4 (Absolute Antibody, NVB43.9) which have a block function as the positive control; mixed serum is a mixture of two epitope antisera