In the present study, we describe a therapy for cat allergy based on immunization with the recombinant cat allergen Fel d1 displayed on VLPs derived from the bacteriophage Qβ (Qβ–Fel d1). The therapy is characterized by three key features: (a) Qβ–Fel d1 is highly immunogenic and a single vaccination is sufficient for therapy; (b) Qβ–Fel d1 is essentially nonreactogenic; and (c) the effector mechanism of the therapy is based upon the induction of allergen-specific IgGs.
Qβ-VLPs consist of 180 subunits of a 14-kD coat protein. These VLPs elicit strong B cell responses as a result of their highly organized and repetitive structures (Jegerlehner et al., 2002a
). This feature can be exploited to enhance the immunogenicity of self- and foreign antigens. Chemical coupling of antigens via a cysteine to surface lysine residues on the VLP renders these antigens equally repetitive and consequently immunogenic. Moreover, host cell RNA, a ligand for TLR3 and TLR7 (Kanzler et al., 2007
), is incorporated into the VLPs during self assembly of the coat proteins in E. coli
serving as a build-in adjuvant. Together, these features explain the strong IgG responses induced by a single injection of Qβ–Fel d1.
Interestingly, vaccination of sensitized mice with Qβ–Fel d1 resulted in a shutdown of memory IgE responses. Specifically, although a strong boosting of the IgE response was observed in sensitized control animals, no increase in sensitized and vaccinated mice was observed upon subsequent allergen challenge. There are different possibilities for how IgE responses might be down-regulated upon Qβ–Fel d1 vaccination. First, it is possible that binding of IgG–Fel d1 complexes to the FcγIIb receptor, which is also expressed on B cells, would mediate inhibition of B cell function (Muta et al., 1994
; Daëron et al., 1995a
). In this case, inhibition would specifically affect IgE and not IgG B cell responses because antigen-specific IgG is strongly boosted upon vaccination. However, using sensitized FcγRIIb−/−
mice for Qβ–Fel d1 vaccination, we found that despite the absence of the inhibitory FcγIIb receptor on B cells, vaccination with Qβ–Fel d1 still blocked IgE B cell memory responses (unpublished data). We therefore believe that an inhibitory FcγRIIb signaling on B cells is not the cause for the abrogated IgE B cell response upon antigen recall. A second possibility for how Qβ–Fel d1 vaccination could affect long-term IgE responses may be via a direct or indirect inhibitory effect of Qβ–Fel d1 on T helper cell responses, lowering IL-4 production and, consequently, IgE isotype switching. Indeed, ELISPOT assays performed with splenocytes from sensitized, vaccinated, and antigen-challenged mice showed that Qβ–Fel d1-vaccinated mice did not develop an increased IL-4 response upon allergen challenge, which is different from control mice. Thus, Qβ–Fel d1 seems to be able to block expansion of Th2 cells upon allergen challenge. We also performed IFN-γ ELISPOT assays but have not observed an increased Th1 response in Qβ–Fel d1-vaccinated mice (unpublished data). This suggests that the reduced Th2 response is not caused by a shift to Th1 cells. The mechanism of this inhibition will need to be further investigated. One further plausible explanation for the Qβ–Fel d1-mediated effect could be found in the composition of the Qβ-VLP itself. It is possible that only moderate, and not strong, cross-linking of surface Ig is able to drive IgE responses. Indeed, viruses rather than parasites exhibit highly organized surface structures in a manner similar to Fel d1 displayed on VLPs. IgE responses, however, protect against parasitic rather than viral infections. Furthermore, Qβ-VLPs contain RNA, a ligand for TLR 3/7, which also drives Th1 and IgG2a responses. Thus, the combination of high cross-linking activity with TLR7 ligands may be able to block IgE memory responses long term. Indeed, some of our preliminary results indicated that the shutdown of the allergen-specific IgE response was the result of the single-stranded RNA within the VLPs. Finally, available antigen may be limited in Qβ–Fel d1-vaccinated mice as a result of the presence of high anti–Fel d1 IgG titers abolishing the IgE response. The abrogated IgE response upon vaccination with Qβ–Fel d1 may have important clinical implications because exposure to allergen results in increased IgE levels in allergic individuals, which may worsen the allergy. Shutting down this allergen-induced IgE response may therefore facilitate long-term curing of the patients. Indeed, seasonal allergen exposure usually boosts IgE responses in allergic individuals, maintaining or even worsening the disease.
The observation that repetitive display of Fel d1 on the VLPs resulted in reduced ability to trigger mast cell degranulation may seem somewhat unexpected. Allergens, which are typically not multivalent, are not able to strongly cross-link IgE-Fcε
RI on mast cells. Hence, allergens displayed on VLPs are nonphysiological ligands for mast cells and may, therefore, not be able to properly activate the degranulation cascade. In fact, it is well known that supraoptimal levels of IgE cross-linking on mast cells lead to reduced rather than enhanced degranulation (Daëron and Lesourne, 2006
). Furthermore, because of the linkage of the allergen to the relatively large VLPs (30 nm), the in vivo distribution will be fundamentally different from free Fel d1. It is important to note that the failure of Qβ–Fel d1 to trigger mast cell degranulation is not the result of a failure of IgE antibodies to recognize the vaccine because both polyclonal and a panel of monoclonal Fel d1–specific IgE antibodies recognized Fel d1 on Qβ (unpublished data). Hence, the nonreactogenicity of Qβ–Fel d1 is not caused by an absence of recognition by IgE antibodies but by a failure to stimulate mast cells in a productive way.
In this context, T reg cells have been recently described as a regulator of mast cell activity by altering IgE receptor expression and signaling (Kashyap et al., 2008
). T reg cells may also activate mast cells to mediate local immune suppression (Lu et al., 2006
). In addition, the regulatory cytokine IL-10, which is produced by T reg cells, has been shown to silence mast cell function in vivo and in vitro (Grimbaldeston et al., 2007
; Kennedy Norton et al., 2008
). In contrast to these studies, we have found that our VLP-based vaccination primarily works through the induction of allergen-specific IgGs rather than T reg cells. This is based on our findings that transferred IgG antibodies could block mast cell degranulation in the mouse skin and that depletion of T reg cells with pc61 did not affect Qβ–Fel d1-mediated desensitization. It is known that administration of pc61 leads to incomplete T reg cell depletion because CD4+
cells cannot be depleted and may remain functionally present in vivo (Zelenay et al., 2005
; Couper et al., 2007
). We therefore performed experiments in mice in which all CD4+
T cells were depleted and, again, saw no effect on desensitization. Furthermore, IL-10 was also not required for the Qβ–Fel d1-induced desensitization. Although successful immunotherapy in humans may involve both T and B cells (Till et al., 2004
), as well as induction of T reg cells producing IL-10 (Francis et al., 2003
; Robinson et al., 2004
), our data demonstrate that these mediators are not important in the immediate type of allergic response upon Qβ–Fel d1 treatment in our mouse model.
Antigen-specific IgG1 was shown to induce anaphylaxis in several in vitro and in vivo studies (Ovary et al., 1970
; Oettgen et al., 1994
; Oshiba et al., 1996
; Dombrowicz et al., 1997
; Miyajima et al., 1997
; Ujike et al., 1999
). Yet we find in this paper that polyclonal antibodies specific for Fel d1 dominated by the IgG1 isotype not only fail to sensitize mice for anaphylaxis but even prevent IgE-mediated anaphylaxis upon allergen challenge. This difference may have several explanations. In the studies where IgG1-mediated anaphylaxis was observed, the reaction was induced by passive transfer of either antigen-specific IgE or IgG. These elegant studies clearly demonstrate that either activation of Fcε
RI–IgE or FcγRIII–IgG1 upon passive transfer and antigen stimulation was able to induce anaphylaxis in mice. Interestingly, IgG1-mediated active systemic anaphylaxis has so far only been demonstrated in Fcε
mice, which show increased anaphylactic responses caused by up-regulation of FcγRIII receptor expression (Dombrowicz et al., 1997
; Miyajima et al., 1997
). Under these nonphysiological conditions, IgG1 antibodies may preferentially bind to the FcγRIII, activating mast cell degranulation. In contrast to these studies, we used active sensitization with Fel d1 in WT mice. Under these conditions, mast cells/basophils express all relevant Fc receptors on their surface and IgG–Ag immune complexes will also bind to the inhibitory FcγRIIb, inhibiting IgE-mediated anaphylaxis. In addition, vaccination of sensitized mice results in specific IgG1 as well as IgG2a antibodies. The presence of Fel d1–specific IgG2a antibodies may have masked a potential ability of IgG1 antibodies to trigger anaphylactic reactions.
The results with FcγRIIB-deficient mice offer an explanation for how allergen-specific IgGs may inhibit allergic reactions. Interestingly, we found that IgG antibodies may inhibit the allergic reaction dependent on the allergen localization. The inhibitory FcγRIIb was essential for IgG-mediated inhibition of anaphylaxis upon systemic administration of the allergen. This suggests that because of the quick diffusion upon systemic exposure complete neutralization of the allergen may not be efficient by the circulating antibodies and some Fel d1 molecules will reach basophils. Nevertheless, Fel d1 will be complexed with antigen-specific IgG, engaging cell-bound FcγRIIb, capable of blunting Fcε
RI-mediated signals in WT mice. In contrast, local stimulation of mast cells in the skin was inhibited by polyclonal IgG antibodies even in the absence of negative FcγRIIb signaling. This suggests that Fel d1, which slowly diffuses within tissues, is recognized by specific IgG antibodies, covering up all available epitopes on the allergen, and may even result in local immune complexes, sequestering the allergen. Hence, Fel d1–specific IgG antibodies may simply neutralize the allergen in the periphery. Indeed, earlier studies with blood from allergic patients have suggested that IgG4 antibodies might exhibit blocking activities in tissues as they compete with IgE for binding to mast cells (García et al., 1993
). It has also been shown that serum obtained from subjects after antigen-specific immunotherapy could inhibit IgE-facilitated presentation of allergen to grass pollen–specific (van Neerven et al., 1999
; van Neerven et al., 2004
) or birch pollen–specific T cells (Wachholz et al., 2003
; Nouri-Aria et al., 2004
). Thus, anti–Fel d1-specific IgGs could also compete with IgE for antigen binding, inhibiting activation of mast cells in tissues in the absence of an inhibitory FcγIIb receptor. In summary, our data demonstrate that allergens displayed on VLPs have the potential to rapidly treat established allergies and delineate two different mechanisms how IgG antibodies are able to block type I allergic reactions.