It was alsoused to produce an IgG mAb, which efficiently assembled and retained its specific antigen binding house (Huang et al., 2010). (EIC) in produced assembled immunoglobulin, which was purified by ammonium sulfate precipitation and protein G affinity chromatography. Immune complex formation was confirmed by assays to show that this recombinant protein bound the match factor C1q. Size measurements of purified recombinant protein by dynamic light scattering and size exclusion chromatography also indicated complex formation. Subcutaneous immunization of BALB/C mice with purified EIC resulted in anti-Ebola computer virus antibody production at levels comparable to those obtained with a GP1 virus-like particle. These results show excellent potential Nilotinib (AMN-107) for a plant-expressed Nilotinib (AMN-107) EIC as a human vaccine. (Das et al., 2007), insect cells (Mellquist-Riemenschneider et al., 2003; Ye et al., 2006), and mammalian cells (Melito et al., 2008). Nilotinib (AMN-107) However, these systems are not optimal, and in order to reduce its toxicity around the host cell, GP1 expression in mammalian cells was regulated by an ecdysone inducible system (Melito et al., 2008). Recombinant immune complexes were originally expressed in tobacco plants via fusion of tetanus toxin fragment C (TTFC) to the heavy chain of a TTFC-binding IgG and co-expression with its light chain (Chargelegue et al., 2005). The TTFC immune complexes were shown to Nilotinib (AMN-107) bind to C1q, Fc receptor gamma RIIa (FcRIIa), and antigen presenting cells. Mice immunized with the recombinant TTFC immune complexes showed much higher antibody titers than those immunized with TTFC alone. This study exhibited the recombinant immune complex as a strong vaccine candidate and led us to pursue a similar strategy with Ebola GP1. In this study, we used the geminiviral replicon system derived from bean yellow dwarf computer virus (Huang et al., 2010) to produce Ebola immune complexes (EIC) in leaves using geminiviral replicons. The expression of viral Rep protein (C1/C2 gene) is required for amplification of the replicon (Laufs et al., 1995; Huang et al., 2010). The Rep cassette is usually contained in the complimentary sense orientation in the light chain vector pBYK3R (Fig. 1). The expression cassettes, driven by the dual-enhancer CaMV 35S promoter, are placed between the long intergenic region (LIR) and short intergenic region (SIR) in the viral-sense orientation, replacing the viral movement and coat protein genes. In the case of dual replicon vector pBYRH2GP1kdK3, the heavy chain-GP1 fusion and light chain cassettes are placed within different replicons oriented in tandem. In all cases we also co-expressed the gene silencing inhibitor p19 from tomato bushy stunt computer virus using the non-replicating expression vector pPSp19. We attempted Ebola GP1 protein expression without heavy chain fusion in herb leaves using pBYR6HGP1kd, which has a 6His usually tag at the N-terminus (Fig. 1). Ebola GP1 expressed from pBYR6HGP1kd produced strong necrosis in leaves, but fusing Rabbit Polyclonal to VPS72 GP1 to 6D8 mAb reduced the toxicity of GP1 (Fig. 2). Due to the considerable necrosis, were unable to obtain unfused GP1 in sufficient yield for immunization experiments. Open in a separate window Fig. 1 Schematic representation of the T-DNA region of the vectors used in this study. 35S/TEV5: CaMV 35S promoter with tobacco etch computer virus 5UTR; VSP3: soybean vspB gene 3 element, NPTII (yellow box): expression cassette encoding nptII gene for kanamycin resistance, LIR (reddish box) : long intergenic region of BeYDV genome, SIR (blue box): short intergenic region of BeYDV genome, C2/C1 : BeYDV ORFs C1 and C2 which encode for replication initiation protein (Rep) and RepA, LB and RB : the left and right borders of the T-DNA region. Open in a separate windows Fig. 2 Common phenotype of leaves on Nilotinib (AMN-107) day 5 expressing 6D8 mAb (1), Ebola GP1 (2), Ebola immune complex (EIC) (3), and GFP (4). leaves were co-infiltrated with pBYH2kdel+pBYK3R+p19 for 6D8 mAb expression (1), pBYR6HGP1kdel+p19 for Ebola GP1 expression (2), pBYRH2GP1kdelK3+p19 for EIC expression (3), and pBYGFP.R+p19 for GFP expression (4) at final OF600 = 0.25. The leaves were photographed on day 5 after infiltration. We compared the protein.
