8 TLR7/8 signalling broadens antibody repertoire, affinity, and avidity
Despite the above-described ability of low affinity allergen-specific IgG antibodies to block type I hypersensitivity responses, it remains common thinking that high affinity antibodies would be more effective to also neutralize the allergen. In addition, induction of high affinity antibodies continues to be a general and common goal in vaccinology. For that, induction of germinal centers (GC), also known as secondary follicles, is a key goal, as GCs are the anatomical location of hypermutation and antibody affinity maturation. Induction of GCs is driven by antigen deposition on follicular dendritic cells (FDCs) and effective stimulation of B cells. Repetitive display of antigens in general and allergens specifically has been found to induce strong and rapid B cell responses due to effective B cell receptor cross-linking as well as engagement of the innate immune system30. Indeed, natural IgM recognizes repetitive epitopes on viruses and virus-like particles (VLPs), causing activation of the classical pathway of complement (via C1q), leading to the deposition of these particles on FDCs and to GC-reactions 16. In addition, VLPs may package RNA from E. coli during VLP production, when assembling inside bacteria. This RNA has not only been observed to effectively drive class switching to IgG and IgA but also to increase the affinity of the induced antibodies as well as to facilitate maintenance of a broad immunoglobulin repertoire against both VLPs as well as displayed allergens32. In a recent preclinical model of peanut allergy, the presence of RNA in VLPs (Cucumber mosaic virus-like particle; CuMVTT) displaying the peanut allergen Ara h 2 (CuMVTT-Ara h 2; VLP Peanut) proved essential for induction of protective IgG responses against peanut allergy; here the role of TLR-signalling and its influence on the vaccine’s efficacy profile was explored in TLR7 knock out (KO) mice as well as VLPs loaded with low amounts of RNA33. Unexpectedly the total amount of Ara h 2 specific antibodies was not affected by TLR 7 signalling; in contrast, when mice were immunized with the product containing approximately half the total RNA content, a significant reduction of Ara h 2 specific IgG titers was observed in Wild Type and TLR 7 KO mice. Not only the total amount of IgG antibodies was affected but also IgG subclasses and the number of high avidity IgG antibodies (Figure 3). In essence, Ara h 2 specific total IgG responses were highly dependent on the VLP Peanut carried RNA. The observed difference in low RNA versus lack of TLR 7/8 is explained by the fact that the RNA loaded in VLPs also engages TLR 3, contributing to the overall immunogenicity profile. Indeed, the bacterial RNA contained in VLP Peanut exists in both single- and double-stranded forms, stimulating TLR 7 and TLR 3. A missing TLR 7 signal can therefore partially be compensated by a TLR 3-derived signal as both TLRs are expressed in B cells34,35.
Additionally, there seems to be a different dependency on the VLP-carried RNA between antigen- and carrier-specific immune responses. The observation that VLP-specific antibodies were less dependent on TLR signalling than Ara h 2 specific antibodies may be explained by the fact that the CuMVTT subunits are more densely packed at a lower distance than the Ara h 2 molecules, overcoming TLR dependence. This indicates that the overall IgG response is dictated by a 2-dimensional integral of TLR stimulation and antigen density. We have previously seen that B cell activation may be driven by integrated overall signals, as TLR-signalling could overcome IL-21-dependence of B cell responses36; an observation reminiscent of the observed independent of TLR-signalling.
Hence, repetitive display of allergens on VLPs packaged with RNA appears an attractive way to increase induction and maintenance of high affinity antibodies (Figure 4). In studies on mast cell interaction with VLPs displaying Fel d 1 or peanut allergens, a substantially reduced interaction with IgE bound to mast cells was observed and an even more pronounced failure to significantly activate the FcεRI mediated signalling cascade. Specifically, under conditions where similar or higher amounts of allergen were bound to IgE on mast cells, free allergens induced strong cellular activation while allergens on VLPs failed to do so13,14. Together, it was therefore an attractive choice to bring Ara h 2 displayed on VLPs (VLP Peanut) into clinical development24.