West Nile Virus (WNV) causes infection in the central nervous system (CNS) in several vertebrate animal species. Humans infected with WNV can develop meningitis and encephalitis, and the elderly and immunocompromised are at greatest risk for severe neurological disease and death. New threats of WNV globally and the lack of available treatments warrant studies to develop effective therapeutics and production technologies that can rapidly transfer candidate therapies into the clinical care setting in a cost- conscious manner. We recently developed a plant-derived humanized MAb with promising therapeutic potential, with a desired human N-linked glycosylation pattern. This MAb (hE16) binds to a highly conserved epitope on the envelope protein of virtually all isolates worldwide and shows promising post-exposure therapeutic activity. Nonetheless, our studies show that peripheral delivery of hE16 has a limited window of efficacy in rodents: administration of a single dose of hE16 through an intravenous or intraperitoneal route at day 5 post infection or earlier improves survival rates. In comparison, delivery of hE16 directly into the brain at day 6 after infection improved protection against lethal WNV infection in hamsters. Our preliminary data show that a genetically engineered bifunctional MAb variant of hE16 (TfR-Bif) with the potential for enhanced crossing of the blood-brain barrier (BBB) can be expressed in CHO cells or plants. TfR-Bif retains its ability to bind and neutralize WNV, but importantly gains the ability to bind transferrin receptor (TfR) and endocytose into mouse brain endothelial cells. In the R21 phase of this grant, we will use the mouse TfR- specific TfR-Bif as a proof-of-principle in a mouse model of WNV infection. We will test the hypothesis that TfR-Bif can achieve higher levels in the CNS and extends the window of treatment against WNV encephalitis. To address critical safety issues in the brain, we will also identify TfR-Bif glycoforms that offer full bifunctionality with limited or specific Fc receptor (Fc?R) or C1q binding to eliminate potential antibody- dependent enhancement (ADE) of virus infection or pathogenic inflammation. In the R33 phase, we will develop an analogous bifunctional mAb with human therapeutic potential and test if plants provide distinctive advantages in safety, production cost, and scalability for large-scale production under cGMP. In addition to generating novel therapeutic reagents for WNV, this collaborative study will provides a platform for delivery of MAbs to the CNS, which should be applicable to the treatment of other infectious, inflammatory, or neoplastic diseases in the CNS.

Public Health Relevance

This study will generate novel therapeutic reagents for West Nile virus, provide a technology for delivery of therapeutics to the brain, and develop a cost-effective and scalable production technology using plants for protein therapeutics. Such technologies can be applied in the future to combat emerging infectious or non- infectious diseases or bioterrorist threats.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants Phase II (R33)
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Special Emphasis Panel (NSS)
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Repik, Patricia M
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Arizona State University-Tempe Campus
United States
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Lai, Huafang; Paul, Amber M; Sun, Haiyan et al. (2018) A plant-produced vaccine protects mice against lethal West Nile virus infection without enhancing Zika or dengue virus infectivity. Vaccine 36:1846-1852
Yang, Ming; Sun, Haiyan; Lai, Huafang et al. (2018) Plant-produced Zika virus envelope protein elicits neutralizing immune responses that correlate with protective immunity against Zika virus in mice. Plant Biotechnol J 16:572-580
Yang, Ming; Dent, Matthew; Lai, Huafang et al. (2017) Immunization of Zika virus envelope protein domain III induces specific and neutralizing immune responses against Zika virus. Vaccine 35:4287-4294
Yang, Ming; Lai, Huafang; Sun, Haiyan et al. (2017) Virus-like particles that display Zika virus envelope protein domain III induce potent neutralizing immune responses in mice. Sci Rep 7:7679
Sun, Haiyan; Chen, Qiang; Lai, Huafang (2017) Development of Antibody Therapeutics against Flaviviruses. Int J Mol Sci 19:
Dent, Matthew; Hurtado, Jonathan; Paul, Amber M et al. (2016) Plant-produced anti-dengue virus monoclonal antibodies exhibit reduced antibody-dependent enhancement of infection activity. J Gen Virol 97:3280-3290
Chen, Qiang (2016) Glycoengineering of plants yields glycoproteins with polysialylation and other defined N-glycoforms. Proc Natl Acad Sci U S A 113:9404-6
Fulton, Andrew; Lai, Huafang; Chen, Qiang et al. (2015) Purification of monoclonal antibody against Ebola GP1 protein expressed in Nicotiana benthamiana. J Chromatogr A 1389:128-32
Chen, Qiang (2015) Plant-made vaccines against West Nile virus are potent, safe, and economically feasible. Biotechnol J 10:671-80
Chen, Qiang; Lai, Huafang (2015) Gene delivery into plant cells for recombinant protein production. Biomed Res Int 2015:932161

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