title: Efficient single tobamoviral vector-based bioproduction of broadly neutralizing anti-HIV-1 monoclonal antibody VRC01 in Nicotiana benthamiana plants and utility of VRC01 in combination microbicides.
abstract: Broadly neutralizing monoclonal antibodies (bnMAbs) may offer powerful tools for HIV-1 preexposure prophylaxis, such as topical microbicides. However, this option is hampered due to expensive MAb biomanufacturing based on mammalian cell culture. To address this issue, we developed a new production system for bnMAb VRC01 in Nicotiana benthamiana plants using a tobamovirus replicon vector. Unlike conventional two-vector-based expression, this system was designed to overexpress full-length IgG1 from a single polypeptide by means of kex2p-like enzyme recognition sites introduced between the heavy and light chains. An enzyme-linked immunosorbent assay (ELISA) revealed that gp120-binding VRC01 IgG1 was maximally accumulated on 5 to 7 days following vector inoculation, yielding ~150 mg of the bnMAb per kg of fresh leaf material. The plant-made VRC01 (VRC01p) was efficiently purified by protein A affinity followed by hydrophobic-interaction chromatography. ELISA, surface plasmon resonance, and an HIV-1 neutralization assay demonstrated that VRC01p has gp120-binding affinity and HIV-1-neutralization capacity virtually identical to the human-cell-produced counterpart. To advance VRC01p's use in topical microbicides, we analyzed combinations of the bnMAb with other microbicide candidates holding distinct antiviral mechanisms in an HIV-1 neutralization assay. VRC01p exhibited clear synergy with the antiviral lectin griffithsin, the CCR5 antagonist maraviroc, and the reverse transcriptase inhibitor tenofovir in multiple CCR5-tropic HIV-1 strains from clades A, B, and C. In summary, VRC01p is amenable to robust, rapid, and large-scale production and may be developed as an active component in combination microbicides with other anti-HIV agents such as antiviral lectins, CCR5 antagonists, and reverse transcriptase inhibitors.
authors: Department of Pharmacology and Toxicology and James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, Kentucky, USA.
authors: Krystal Teasley
authors: KT
authors: Hamorsky
authors: Tiffany W
authors: TW
authors: Grooms-Williams
authors: Adam S
authors: AS
authors: Husk
authors: Lauren J
authors: LJ
authors: Bennett
authors: Kenneth E
authors: KE
authors: Palmer
authors: Nobuyuki
authors: N
authors: Matoba
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology / Q000494:pharmacology*; D057134:Antibodies, Neutralizing / Q000096:biosynthesis / Q000276:immunology / Q000494:pharmacology*; D000998:Antiviral Agents / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D002846:Chromatography, Affinity; D003510:Cyclohexanes / Q000494:pharmacology; D004338:Drug Combinations; D004357:Drug Synergism; D005822:Genetic Vectors; D015483:HIV Antibodies / Q000096:biosynthesis / Q000276:immunology / Q000494:pharmacology*; D015699:HIV Envelope Protein gp120 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects / Q000254:growth & development; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000276:immunology / Q000494:pharmacology*; D000077592:Maraviroc; D009500:Neutralization Tests; D063065:Organophosphonates / Q000494:pharmacology; D037121:Plant Lectins / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000494:pharmacology; D000068698:Tenofovir; D014026:Nicotiana / Q000235:genetics*; D017898:Tobamovirus / Q000235:genetics*; D014230:Triazoles / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000998:Antiviral Agents; D003510:Cyclohexanes; D004338:Drug Combinations; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D007074:Immunoglobulin G; D063065:Organophosphonates; D037121:Plant Lectins; D011994:Recombinant Proteins; D014230:Triazoles; D000068698:Tenofovir; D000225:Adenine; D000077592:Maraviroc; C499654:griffithsin protein, Griffithsia
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