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.