Higher levels of anti-IgE IgG antibodies are induced by immunization with IgE-Fel d 1 complexes compared to complexes containing deglycosylated IgE
We first investigated the IgE-specific IgG response induced by immunization with glycosylated (IgE-ICs) or deglycosylated (IgE(PNG)-IC) IgE in a complex with Fel d 1 (Figure 1A). Serum IgG anti-IgE antibodies were measured by ELISA (Fig. 1B-D). Both groups generated antibodies against IgE after immunization, which increased following the first and the second booster. However, mice immunized with IgE-ICs showed a higher anti-IgE IgG response than mice immunized with IgE(PNG)-IC. The IgG response against Fel d 1 and IgE(PNG) also increased after each boost, and the response was consistently higher in the group receiving IgE-ICs (Supp. Fig.1A-D).
IgE-specific plasma cells were quantified in lymphoid organs upon IgE-ICs or IgE(PNG)-ICs vaccination. For that, spleen and bone marrow (BM) were collected two weeks after the third injection (day 42) and analyzed for the presence of IgE-specific IgG secreting plasma cells. Bone marrow and spleen contained anti-IgE IgG secreting plasma cells (Fig. 1E, 1F). On average, 55 IgG secreting plasma cells were found per million bone marrow cells in the mice immunized with IgE-ICs compared to 22 in the deglycosylated group. In contrast, in the spleens of mice immunized with glycosylated and deglycosylated IgE similar number of IgG secreting plasma cells were found (Fig. 1G). The reduced levels of plasma cells, together with reduced avidities of the antibodies (see below), may explain the observed differences in antibody titers.
Furthermore, we investigated whether the glycosylation of IgE influences the formation of anti-IgE IgG subclasses. As in our previous study, IgG1 and IgG2b were the most frequent subclasses. Additionally, no significant difference between the glycosylated and deglycosylated groups was detectable (Supp. Fig 2A).
To investigate whether i.v. immunization with IgE-Fel d 1 immune complexes induce mucosal IgA; fecal pellets were collected before and after immunization and dissolved in PBS. ELISA results show a significantly higher anti-IgE IgA response in mice immunized with IgE-ICs compared to naïve mice or mice immunized with IgE(PNG)-IC (Supp. Fig. 2B).
In summary, triple vaccination with IgE-ICs without adjuvant induced a solid systemic humoral immune response against IgE, which was significantly stronger when glycosylated IgE was used as the immunogen. These results indicate that glycans play a role in modulating the anti-IgE IgG response.