Although IgG2a is the most potent Ab isotype in the host response to viral and bacterial infections, the regulation of class switch recombination to IgG2a in vivo is not yet well understood. Recognition of pathogen-associated molecular patterns by dendritic cells expressing TLRs, like TLR7, recognizing ssRNA, or TLR9, recognizing DNA rich in nonmethylated CG motifs (CpG), favors induction of Th1 responses. It is generally assumed that these Th1 responses are responsible for the TLR-mediated induction of IgG2a. Using virus-like particles loaded with CpGs, we show here that TLR9 ligands can directly stimulate B cells to undergo isotype switching to IgG2a. Unexpectedly, TLR9 expression in non-B cells did not affect isotype switching in the Ab response against virus-like particles. Thus, TLR9 can regulate isotype switching to IgG2a directly by interacting with B cells rather than indirectly by inducing Th1 responses.

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IL-21 produced by follicular Th (Tfh) cells is an important regulator of Tfh cell development and B cell responses, including germinal center (GC) formation. However, whether defective GC formation and Ab responses are a consequence of impaired Tfh cells development or a B cell-intrinsic defect in IL-21–deficient mice requires clarification. To address this question, we generated chimeric mice lacking IL-21R exclusively on B cells. In this study, we demonstrate that GC reaction and B cell responses induced by immunization with virus-like particles were strongly reduced in both global and B cell-specific IL-21R–deficient mice. Interestingly, the presence of TLR7 ligand within virus-like particles largely restored defective GC reaction and Ab responses in global as well as in B cell-specific IL-21R–deficient mice. Hence, IL-21 acts directly on B cells and cooperates with TLR signaling for optimal B cell responses.

This mouse model is able to recognize immunogenic modifications of human IgG1. While the degree of stress-induced aggregation varies for different mAbs, our findings using a particular mAb (mAb1) demonstrate that non-covalently modified aggregates do not break tolerance, contrary to widely held opinion. The immunogenic potential of soluble aggregates of human IgG strongly depends on the presence of neo-epitopes resulting from harsh stress conditions, i.e. extensive exposure to artificial light.

Intranasal (i.n.) immunization aims to induce local as well as systemic immune responses. In the present study, we assessed a vaccine platform based on virus-like particles (VLP) derived from the RNA phage Qb for i.n. immunization. We found that both i.n. and subcutaneous (s.c.) administration of Qb-VLP elicited strong and comparable specific IgG responses in serum and lung. Surprisingly, both routes also induced high levels of specific IgA in serum. In contrast, only i.n. administration of Qb-VLP resulted in local IgA production in the lung. Efficient induction of B cell responses by i.n. administration of VLP was further supported by the presence of large numbers of germinal centers (GC) as well as memory B cells in the spleen and plasma cells in the bone marrow. Results obtained for the VLP itself could be extended to an antigen covalently attached to it. Specifically, i.n. immunization of mice with VLP displaying the influenza virus derived ectodomain of the M2 protein resulted in strong M2-specific antibody responses as well as anti-viral protection. In contrast, i.n. immunization with VLP displaying p33 peptide, the major CTL epitope of lymphocytic choriomeningitis virus, induced relatively inefficient cytotoxic T cell responses, resulting in low numbers of specific T cells and poor effector cell differentiation. Taken together, these results suggest that effective antibody-based vaccines are achievable by i.n. administration of Qb-VLP displaying specific antigens.