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.