Characterization of Therapeutic human monoclonal antibodies against SARS The discovery that a corona virus is the causative agent of SARS and its rapid genomic sequencing has created opportunities to develop effective modalities of prevention and treatment against this virus. Of particular interest is the spike glycoprotein (S protein), which is expressed as a surface projection. The S protein is involved in viral attachment, cell-cell fusion, the induction of protective immune response in virus neutralization. This proposal will focus on developing human monoclonal antibodies that can be used for prophylaxis, and/or post-exposure treatment of SARS-CoV infection. We have already generated a panel of hybridomas capable of producing neutralizing human monoclonal antibodies (HmAbs) in close collaboration with Abgenix Inc using their XenoMouse. These hybridomas have been cloned and 19 HmAbs have been produced, purified and re-tested in a neutralization assay. In addition, we have already produced several recombinant fragments of the S protein and obtained overlapping peptides that span the S protein, and used them to determine binding specificities of HmAbs.
In aim -1 we will further characterize the neutralizing ability of human monoclonal antibodies against SARS-CoV in vitro and determine their relative binding affinities and specificities using a number of different current approaches.
In aim -2, we will identify human monoclonal antibodies that can potentially neutralize a broad range of clinical isolates by testing them in a viral entry and a cell fusion assay using different pseudotyped viruses expressing mutant S proteins that contain amino acid substitutions identified in over 90 clinical isolates from around the world. These studies are expected to result in the characterization of HmAbs with potential as passive immunotherapeutics against SARS-CoV infection in humans.
The current proposal will explore the potential utility of passive immunization against SARS-CoV infection, which is acute and can cause severe disease often leading to mortality. This will be accomplished by characterizing a large panel of human monoclonal antibodies that we have already produced against SARSCoV to provide instantaneous protection. The proposed approach is novel, since it uses human Abs rather than Igs of animal origin or serum from convalescent patients or vaccinated human subjects. In this regard, we have taken advantage of the XenoMouse technology, and generated over 50 different hybridomas capable of producing neutralizing Abs against SARS-CoV. We will use multiple approaches to characterize the binding specificities and relative affinities of these human monoclonal antibodies (HmAbs), and select a subset of neutralizing antibodies targeted to the most stable areas of the S protein. These antibodies can be subsequently tested for their ability confer protection against SARS-CoV challenge in experimental animals.
| Coughlin, Melissa M; Prabhakar, Bellur S (2012) Neutralizing human monoclonal antibodies to severe acute respiratory syndrome coronavirus: target, mechanism of action, and therapeutic potential. Rev Med Virol 22:2-17 |
