Viruses of the familyCoronaviridaehave recently emerged through zoonotic transmission to become serious human pathogens. The pathogenic agent responsible for severe acute respiratory syndrome (SARS), the SARS coronavirus (SARS-CoV), is a member of this large family of positive-strand RNA viruses that cause a spectrum of disease in humans, other mammals, and birds. Since the publicized outbreaks of SARS in China and Canada in 2002-2003, significant efforts successfully identified the causative agent, host cell receptor(s), and many of the pathogenic mechanisms underlying SARS. With this greater understanding of SARS-CoV biology, many researchers have sought to identify agents for the treatment of SARS. Here we report the utility of the potent antiviral protein griffithsin (GRFT) in the prevention of SARS-CoV infection bothin vitroandin vivo. We also show that GRFT specifically binds to the SARS-CoV spike glycoprotein and inhibits viral entry. In addition, we report the activity of GRFT against a variety of additional coronaviruses that infect humans, other mammals, and birds. Finally, we show that GRFT treatment has a positive effect on morbidity and mortality in a lethal infection model using a mouse-adapted SARS-CoV and also specifically inhibits deleterious aspects of the host immunological response to SARS infection in mammals.

To prevent sexually transmitted HIV, the most desirable active ingredients of microbicides are antiretrovirals (ARVs) that directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals, which are costly to manufacture and deliver to resource-poor areas where effective microbicides are urgently needed. Here, we report a manufacturing breakthrough for griffithsin (GRFT), one of the most potent HIV entry inhibitors. This red algal protein was produced in multigram quantities after extraction from Nicotiana benthamiana plants transduced with a tobacco mosaic virus vector expressing GRFT. Plant-produced GRFT (GRFT-P) was shown as active against HIV at picomolar concentrations, directly virucidal via binding to HIV envelope glycoproteins, and capable of blocking cell-to-cell HIV transmission. GRFT-P has broad-spectrum activity against HIV clades A, B, and C, with utility as a microbicide component for HIV prevention in established epidemics in sub-Saharan Africa, South Asia, China, and the industrialized West. Cognizant of the imperative that microbicides not induce epithelial damage or inflammatory responses, we also show that GRFT-P is nonirritating and noninflammatory in human cervical explants and in vivo in the rabbit vaginal irritation model. Moreover, GRFT-P is potently active in preventing infection of cervical explants by HIV-1 and has no mitogenic activity on cultured human lymphocytes.

Summary: Plants have been proposed as an attractive alternative for pharmaceutical protein production to current mammalian or microbial cell‐based systems. Eukaryotic protein processing coupled with reduced production costs and low risk for mammalian pathogen contamination and other impurities have led many to predict that agricultural systems may offer the next wave for pharmaceutical product production. However, for this to become a reality, the quality of products produced at a relevant scale must equal or exceed the predetermined release criteria of identity, purity, potency and safety as required by pharmaceutical regulatory agencies. In this article, the ability of transient plant virus expression systems to produce a wide range of products at high purity and activity is reviewed. The production of different recombinant proteins is described along with comparisons with established standards, including high purity, specific activity and promising preclinical outcomes. Adaptation of transient plant virus systems to large‐scale manufacturing formats required development of virus particle and Agrobacterium inoculation methods. One transient plant system case study illustrates the properties of greenhouse and field‐produced recombinant aprotinin compared with an US Food and Drug Administration‐approved pharmaceutical product and found them to be highly comparable in all properties evaluated. A second transient plant system case study demonstrates a fully functional monoclonal antibody conforming to release specifications. In conclusion, the production capacity of large quantities of recombinant protein offered by transient plant expression systems, coupled with robust downstream purification approaches, offers a promising solution to recombinant protein production that compares favourably to cell‐based systems in scale, cost and quality.

Hepatitis C virus (HCV)-infected patients undergoing liver transplantation universally experience rapid reinfection of their new liver graft. Current treatment protocols do not prevent graft reinfection and, in addition, an accelerated disease progression is observed. In the present study, we have evaluated a novel strategy to prevent HCV infection using a lectin, griffithsin (GRFT) that specifically binds N-linked high-mannose oligosaccharides that are present on the viral envelope. The antiviral effect of GRFT was evaluated in vitro using the HCV pseudoparticle (HCVpp) and HCV cell culture (HCVcc) systems. We show here that preincubation of HCVpp and HCVcc with GRFT prevents infection of Huh-7 hepatoma cells. Furthermore, GRFT interferes with direct cell-to-cell transmission of HCV. GRFT acts at an early phase of the viral life cycle by interfering in a genotype-independent fashion with the interaction between the viral envelope proteins and the viral receptor CD81. The capacity of GRFT to prevent infection in vivo was evaluated using uPA ^+/+ -SCID mice (uPA stands for urokinase-type plasminogen activator) that harbor human primary hepatocytes in their liver (chimeric mice). In this proof-of-concept trial, we demonstrated that GRFT can mitigate HCV infection of chimeric mice. Treated animals that did become infected demonstrated a considerable delay in the kinetics of the viral infection. Our data demonstrate that GRFT can prevent HCV infection in vitro and mitigate HCV infection in vivo . GRFT treatment of chronically infected HCV patients undergoing liver transplantation may be a suitable strategy to prevent infection of the liver allograft.