Antibody Responses in Mice Stimulated by Various Doses of the Potato-Derived Major Surface Antigen of Hepatitis B Virus^▿

The major surface antigen of hepatitis B virus (HBV) (HBsAg) is one of the best-developed examples of a plant-derived antigen (6, 9, 11, 13, 18); however, low expression levels prevent plant-derived antigens from being economically competitive (5, 15, 16). Average antigen expression levels are in the range of 0.01% to 0.4% of total soluble protein (TSP). Aspects related to eliciting protective antibody responses, e.g., oral administration doses (1, 10), efficient delivery of oral vaccines (3, 4), and functional effects of adjuvants (12), were identified to stimulate immune responses after vaccination with low levels of plant-derived antigen.

The objective here was to examine the effects of plant-based oral immunization on HBV-specific immune responses over a broad range of doses, from the lowest dose of 0.02 μg potato-derived HBsAg (P-HBsAg) to the maximum dose of 30 μg P-HBsAg. IgG humoral and IgA mucosal responses were observed at various P-HBsAg doses, and these results are discussed in relation to the optimization of plant-derived vaccines. Furthermore, the analysis of IgG subclass distribution following oral administration with varied doses of P-HBsAg was carried out to understand the mechanism of the immune response.

To evaluate the immunogenicity upon oral administration of various doses of P-HBsAg from a plant line showing the highest production of HBsAg (7, 18), mice were immunized orally with tuber extract on days 1, 7, and 14. The detailed procedure was described previously (18). Mice were immunized with 150 μg of yeast-derived HBsAg (Y-HBsAg; LG Life Sciences, Republic of Korea) mixed with 10 μg of Cholera toxin (CT; Sigma) as a positive control. Each concentration of P-HBsAg administered to mice was divided into one of the the following three groups: lower level, consisting of 0.02, 0.1, and 0.5 μg; middle level, consisting of 1.0, 2.5, and 5.0 μg; and higher level, consisting of 10 μg, 15 μg, and 30 μg. Only five concentrations (0.1, 1.0, 5.0, 10, and 30 μg) were shown among the nine concentrations depicted in the figures to help with understanding by using a concise arrangement. Antigen-specific IgG responses to representative doses in mouse sera against P-HBsAg were graphically monitored up to week 12 (Fig. 1a). The groups administered 10 μg and 30 μg of antigen stimulated slight primary responses of 41 mIU and 51 mIU, respectively, at 7 weeks before booster administration compared to responses of the mice immunized with potato extract from the nontransformed (NT) plant (NT group) (4 mIU). Mice administered higher levels of P-HBsAg (10, 15, and 30 μg) exhibited significantly increased immune responses after booster administration, with serum IgG levels of 446.23 ± 43.19 mIU, 513.33 ± 10.15 mIU, and 551.43 ± 14.09 mIU, respectively, at 12 weeks. The IgG titers of mice administered higher levels of antigen were similar at week 12. Mice receiving the middle dosage of P-HBsAg had augmented levels of 134.76 ± 16.94 mIU, 194.94 ± 8.52 mIU, and 282.81 ± 27.96 mIU for 1.0, 2.5, and 5.0 μg of antigen, respectively, in serum IgG titers by week 12. Mice administrated the smallest amount of P-HBsAg showed only a slight elevation of serum IgG titers compared with those of the NT group, with levels of 54.03 ± 2.75 mIU, 97.53 ± 0.92 mIU, and 113.77 ± 10.10 mIU for 0.02, 0.1, and 0.5 μg of antigen, respectively, after booster administration. No response was detected in the NT mice (16.36 ± 1.84 mIU), even after repeated or booster inoculations.

HBsAg-specific IgG subclasses of the serum samples were analyzed to characterize the IgG expression pattern by enzyme-linked immunosorbent assay (ELISA). In the group administered Y-HBsAg, IgG1 was observed with a level of 60% of the total IgG response, while the other subclasses (IgG2a, IgG2b, and IgG3) were not shown with significant levels (Fig. 2). A similar tendency was observed in the group administered higher doses of P-HBsAg (10, 15, and 30 μg). However, in the group administered lower doses of antigen (0.1, 1.0, and 5.0 μg), there were no obvious subclasses among the four subclasses. The IgG1 titer tended to increase with increasing the doses of potato extracts administered; otherwise, IgG2a, IgG2b, and IgG3 were nearly equally exhibited. None of the HBsAg-specific IgG subclasses could be detected after oral administration with untransformed potato extract.

Mice administered orally with smaller amounts of antigen did not show a significant increase in IgA values compared with those of the NT group. Mice administered 10 μg and 30 μg of P-HBsAg had significantly higher values of IgA, 0.348 ± 0.001 and 0.361 ± 0.001, respectively, at 12 weeks than the NT group (0.031 ± 0.001). In mice receiving the middle dosage (1.0, 2.5, and 5.0 μg) of P-HBsAg, IgA values steadily increased until at 11 weeks. At week 12, mice rapidly produced IgA antibody (levels of 0.247 ± 0.006, 0.257 ± 0.001, and 0.313 ± 0.003, respectively, for 1.0, 2.5, and 5.0 μg of antigen). Interestingly, immunized mice receiving middle and large amounts of antigen had slight primary immune responses before booster administration at 8 weeks (Fig. 3).

All analyses for statistically significant differences were performed with the Kruskal-Wallis test (R version 2.9), with P values of <0.05 considered significant.

To confirm the M cell uptake of P-HBsAg, transgenic and untransformed potato extract was administrated into the ligated small intestine loop including the Peyer's patches (PPs) of naive mice. Histological analysis showed Ulex europaeus agglutinin 1 (UEA-1)-positive M cells were found in the PP mucosa of both samples, but HBsAg S protein-positive M cells were found only in the transgenic potato extract-treated sample. Other histological changes were not found in either of the samples (Fig. 4).

In this study, we evaluated P-HBsAg-induced HBV-specific serum IgG and mucosal IgA responses stimulated by exposure to various doses of antigen. The strength of the immune response positively correlated with the antigen dose. Mice receiving more than 15 μg of antigen had titers of established antibody that were similar to or higher than those of mice receiving the positive Y-HBsAg; however, no responses to the booster were obtained in mice in the NT group. Although the IgG response in the 0.02-μg treatment group was low, 54 mIU, it was three times higher than that in the NT group. It indicates that the boosting response elicited in mice gavaged with P-HBsAg was indeed the result of the priming and establishment of immune memory to HBsAg presented in the gut (9). In the analysis of IgG subclasses, IgG1 was the predominant antibody isotype induced by the oral administration of P-HBsAg in BALB/c mice. Similar results have been found in other studies. In BALB/c mice, IgG1 was the predominant subclass response when the orally administered subunit vaccines were delivered with CT as an adjuvant (12). IgG1 has also been reported to be the predominant IgG subclass of anti-HBs (2, 14, 17).

The mucosal IgA antibody titers achieved in the feces of mice orally immunized with P-HBsAg were slightly higher than those of mice orally immunized with Y-HBsAg. Notably, there was no dramatic increase of the IgA immune response in the high-dose exposure groups (10, 15, and 30 μg) compared to the serum IgG immune response. A minor population of M cells within the follicle-associated epithelium (FAE) of intestinal PPs serves as a major portal for entry of exogenous antigens (8). By pinocytosis, they actively capture and transport antigens from the intestinal lumen to the connective tissue, where antigen-presenting cells (APCs) and B lymphocytes are usually present. The plasma cells derived from these lymphocytes secrete mostly IgA, which is transported through the epithelium toward the intestinal cavity. Therefore, uptake of the antigens by M cells is one of the requirements for a successful, targeted oral vaccine (3). In our study, we found M cell uptake of HBsAg S protein in the small intestine's PP mucosa; this may have an important role in the stimulation of systemic and mucosal immunity.

To our knowledge, this is the first work describing mice exposed to diverse doses of P-HBsAg. This study supports that the immunization protocol, oral administration with more than 15 μg of P-HBsAg, was sufficient to raise a substantial immune response. Also, these results suggest that one way to utilize plant-derived vaccines is to induce the immune response with low levels of antigen.