Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana

Abstract

Summary Filoviruses (Ebola and Marburg viruses) cause severe and often fatal hemorrhagic fever in humans and non-human primates. The US Centers for Disease Control identify Ebola and Marburg viruses as “category A” pathogens (defined as posing a risk to national security as bioterrorism agents), which has lead to a search for vaccines that could prevent the disease. Because the use of such vaccines would be in the service of public health, the cost of production is an important component of their development. The use of plant biotechnology is one possible way to cost-effectively produce subunit vaccines. In this work, a geminiviral replicon system was used to produce an Ebola immune complex (EIC) in Nicotiana benthamiana. Ebola glycoprotein (GP1) was fused at the C-terminus of the heavy chain of humanized 6D8 IgG monoclonal antibody, which specifically binds to a linear epitope on GP1. Co-expression of the GP1-heavy chain fusion and the 6D8 light chain using a geminiviral vector in leaves of Nicotiana benthamiana 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 the recombinant protein bound the complement 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 virus antibody production at levels comparable to those obtained with a GP1 virus-like particle. These results show excellent potential for a plant-expressed EIC as a human vaccine.

A nonreplicating subunit vaccine protects mice against lethal Ebola virus challenge

“…These molecules self-interacted to form an EIC in which the variable region of the antibody domain recognized its cognate epitope in the GP1 of another molecule. C1q, a protein component of the complement cascade of mammalian innate immune systems, bound to these complexes, indicating their association as immune complexes (14). We compared murine antibody fused to EIC GP1 (m-EIC) vs. humanized antibody fused to EIC GP1 (h-EIC) in anticipation that the human "scaffold protein" (the antibody domain of h-EIC) might influence the immunogenicity of the attached GP1.…”

“…When coexpressed in tobacco, they assembled h-6D8 (7). With the availability of expression vectors for both m-6D8 and h-6D8 in hand, we designed a plant-optimized DNA sequence encoding GP1, by using codons that are preferred in tobacco and removing spurious mRNA-processing signals (14). We created genetic fusions of the GP1 coding sequence to the C terminus of the HC (both murine and humanized forms), and coexpressed each with the gene for the corresponding LC in N. benthamiana (14).…”

“…With the availability of expression vectors for both m-6D8 and h-6D8 in hand, we designed a plant-optimized DNA sequence encoding GP1, by using codons that are preferred in tobacco and removing spurious mRNA-processing signals (14). We created genetic fusions of the GP1 coding sequence to the C terminus of the HC (both murine and humanized forms), and coexpressed each with the gene for the corresponding LC in N. benthamiana (14). The resulting fusion protein complex isolated from these plants contained both light and heavy antibody chains, but with the latter extended by the protein encoded by the GP1 sequence.…”

“…In one example, doses of serum albumin administered bound to syngeneic antibodies were fivefold more immunogenic than free antigen (measured as antigen-specific Tcell proliferation) (20). In our previous study of EIC expression in plants (14), we did find that vaccination with the isolated complex resulted in production of EBOV-specific antibodies.…”

“…Although the potential value of a GP1-based subunit vaccine has been long appreciated, the commercial-scale manufacture of this antigen has been a technical obstacle. We have overcome this hurdle by fusing GP1 to a carrier protein that stabilizes the GP1 in the host production cell, but also provides a scaffold that allows easy purification of the antibody backbone with standard pharmaceutical protocols (protein A column as a primary step) (14).…”