Complement is a protein system in the plasma of humans and animals [
1]. After being activated, a series of important biological reactions generate several complement proteins that nonspecifically defend against invading pathogens [
2]. While complement protein C3 is a central component of the innate immune system, it also plays an important role in stimulating the humoral immune response [
1,
3]. At the point of convergence of three distinct pathways of complement activation, C3 is cleaved into C3a and C3b by the C3 convertase [
4]. Further proteolytic cleavage of C3b results in the formation of C3c and C3dg. The C3dg product can be further degraded by a variety of cellular proteases into C3d, a protein which attaches covalently to the surface of pathogens and upregulates B-cell responses [
4,
5]. Previous studies have demonstrated that C3d could enhance antigen recognition and specific immunoglobulin synthesis by antigen-specific B cells, as the antigen is taken up and processed via cell receptor 2 (CR2) by both antigen-specific and non-specific B cells [
6]. Subsequent investigations showed that three copies of murine C3d could dramatically enhance antibody responses to specific antigen, being 100-fold more effective than incomplete Freund's adjuvant [
7,
8]. Ross reported that C3d could enhance antibody responses directed toward a specific antigen encoded by a DNA vaccine [
9]. A DNA vaccine expressing a fusion of hemagglutinin (HA) from influenza virus or measles virus fused to three copies of the murine homologue of C3d (mC3d) achieved an early and efficient immune response in mice. Fusion to C3d has been shown to increase the immunogenicity of the capsular polysaccharide antigen of
Streptococcus pneumoniae[
10]. Using DNA vaccination, various forms of envelope (Env) proteins of the human immunodeficiency virus type 1 (HIV-1) fused at the carboxyl terminus with C3d of murine complement, generated high-titer, long-lasting, neutralizing antibodies in mice [
11]. In addition, the human homologue of C3d (hC3d) also enhanced anti-Env antibodies in rabbits when it was fused to sgp120 [
12]. Recently, Wang reported that the bovine homologue of C3d (boC3d) coupled to the E2 envelope protein of bovine viral diarrhea virus greatly enhanced immunogenicity in mice [
13]. Liu also reported that chicken C3d-P29 linked to the F gene of Newcastle disease virus (NDV) enhanced immunogenicity in chickens [
14]. Logan GJ found C3d (3)-fusion markedly increase antibody responses to the AAV-encoded model antigen (hen egg lysozyme) with greater than 50-fold enhancement in responses [
15]. Comparison of the human, mouse and bovine C3d sequences showed 84.1% amino acid homology between hC3d and mC3d and 80.5% homology between hC3d and boC3d, they either showed the function of immune adjuvant in mammalian model. Information on the function of avian C3d is scarce. Importantly, there are structural differences in the mammalian and avian immune systems, particularly the role of the bursa as one of the central immune organs in avian species.
Avian influenza (AI), caused by avian influenza virus (AIV), is a highly contagious disease of birds. Current AI vaccines induce antibodies against HA and neuraminidase (NA), two major surface glycoproteins expressed on the virus particles. However, due to rapid antigenic variation of HA and NA, AI vaccine can not protect avian against the new avian influenza virus strains. A vaccine that is less sensitive to the antigenic evolution of the virus would be a major improvement. As a result, vaccines have to be updated continuously to prevent disease emerging due to new viral strains. Hence, a vaccine that could induce broad cross protection against AIV would be desirable.
The Matrix protein 2 (M2) is an integral tetrameric membrane protein of AIV. Natural M2 protein is present in a few copies in the virus particle but in abundance on virus-infected cells. In contrast to hemagglutinin and neuraminidase, M2 is almost nonimmunogenic, and its sequence is highly conserved in all diverse subtypes of AIV. Several investigations have shown that the M2 protein has the potential to induce a broadly protective immunity against AIV. M2-specific antibodies have been shown to restrict virus growth in vitro and in vivo and thus have the potential of providing cross-reactive resistance to influenza type A virus infection [
16]. Frace reported that vaccination with the protein M2 was found to raise M2-specific serum antibodies and enhance viral clearance in mice challenged with homologous and heterologous influenza A viruses [
17]. Neirynck reported that the M2 domain was genetically fused to the hepatitis B virus core (HBc) protein to create fusion gene coding for M2HBc, and intraperitoneal or intranasal administration of purified M2HBc particles to mice provided 90-100% protection against a lethal virus challenge [
18]. Zhao designed a tetra-branched multiple antigenic peptide (MAP)-based vaccine, designated M2e-MAP, which contains the sequence overlapping the highly conserved extracellular domain of matrix protein 2 (M2e) of a HPAI H5N1 virus, animals test results showed that M2e-MAP vaccine induced strong M2e-specific IgG antibody responses following 3-dose immunization of mice with M2e-MAP in the presence of Freunds' or aluminium (alum) adjuvant [
19]. Rao SS have shown that vaccination with M2 in recombinant DNA and/or adenovirus vectors or with adjuvants confers protection against lethal challenge in the absence of HA, and also find that the protective efficacy of NP and M2 diminishes as the virulence and dose of the challenge virus are increased [
20]. However, M2 is only a minor protein component of AIV, and tends to induce a poor immune response [
21]. To overcome the shortcomings of M2-based vaccines, in this study we investigated the potential of M2-avian C3d fusion proteins to provide effective immunity.