A Single Functional Group Substitution in C5a Breaks B Cell and T Cell Tolerance and Protects Against Experimental Arthritis

Abstract

Objective Deficiency in complement factor 5 (C5) protects against arthritis development. However, there is currently no approach translating these findings successfully into arthritis therapy, as by targeting the key component, C5a. Methods We generated an anti-C5a vaccine by incorporating the unnatural amino acid p-nitrophenylalanine (4NPA) at selected sites into murine C5a. C5a-4NPA variants were screened for their immunogenicity on different arthritis susceptiple MHCII backgrounds. A vaccine candidate was tested for its impact on disease in the collagen induced arthritis (CIA) mouse model. Immunity towards endogenous C5a as well as type II collagen was monitored and characterized. Results The replacement of a single tyrosine residue in position 35 (Y35) by 4NPA generated an anti-C5a vaccine, which partly protected mice from developing CIA while strongly ameliorating severity of clinical disease. Although differing in just three atoms from wtC5a, C5aY354NPA induced loss of T and B cell tolerance towards the endogenous protein in mice expressing MHCII H2q molecules. Despite differential B cell epitope recognition, antibodies induced by either wtC5a or C5aY354NPA both neutralized C5a. Thus, anti-wtC5a IgG titers during arthritis priming were potentially critical for disease protection as high titers of C5a-neutralizing antibodies post disease onset were unable to reverse the arthritis course. Conclusion Our results suggest the most effective anti-C5a treatment in arthritis to be accomplished by a preventive vaccination strategy, while conventional biological or small molecule treatments targeting the C5a:C5aR axis risk missing the optimal therapeutic window for intervention during the subclinical priming phase of the disease.

Allosterism in human complement component 5a (hC5a): a damper of C5a receptor (C5aR) signaling

“…It is also noteworthy that while Arg 37 display the highest fluctuation in h C5a, the Tyr 36 display the highest fluctuation in m C5a. Interestingly, mutation of Tyr 36 in m C5a (Figure 2(c)) to NPA (p-nitrophenyl alanine) has recently been shown to produce an anti-m C5a vaccine (Kessel et al, 2014), effective against the collagen-induced arthritis (Kessel et al, 2014). Similarly, previous mutational studies have also identified Arg 37 as a potential pharmacophore that affect the potency of h C5a toward C5aR (Kola, Baensch, Bautsch, Klos, & Kohl, 1999;Toth et al, 1994).…”

Immunological effects and therapeutic role of C5a in cancer

“…The vaccine itself lead to a loss of B and T-cell tolerance to C5a in mice whose cells expressed the particular receptor “class II MHC H-2(q).” Both the indigenous C5a and modified C5a induced antibodies capable of effectively neutralizing C5a. The high titers of IgG prevented disease development, but did not reverse the course of ongoing disease [75]. …”

De novo generation of specific human IgGs by in vitro immunization using autologous proteins containing immunogenic p-nitrophenylalanine

“…15 In addition, pNO 2 Phe-containing self-antigens induced a protective cross-reactive immune response without the need for strong adjuvants and resulted in high titers for at least 4 months in mice. 16 Thus, we decided to assess whether endogenous protein variants containing pNO 2 Phe are able to trigger a humoral immune response in vitro.…”