Virus-like particles (VLPs) derived from enteric pathogens like Norwalk virus (NV) are well suited to study oral immunization. We previously described stable transgenic plants that accumulate recombinant NV-like particles (rNVs) that were orally immunogenic in mice and humans. The transgenic approach suffers from long generation time and modest level of antigen accumulation. We now overcome these constraints with an efficient tobacco mosaic virus (TMV)-derived transient expression system using leaves of Nicotiana benthamiana. We produced properly assembled rNV at 0.8 mg/g leaf 12 days post-infection (dpi). Oral immunization of CD1 mice with 100 or 250 microg/dose of partially purified rNV elicited systemic and mucosal immune responses. We conclude that the plant viral transient expression system provides a robust research tool to generate abundant quantities of rNV as enriched, concentrated VLP preparations that are orally immunogenic.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are important pathogens that cause diverse diseases in humans and poultry. Some E. coli isolates from chicken feces contain ExPEC-associated virulence genes, so appear potentially pathogenic; they conceivably could be transmitted to humans through handling and/or consumption of contaminated meat. However, the actual extraintestinal virulence potential of chicken-source fecal E. coli is poorly understood. Here, we assessed whether fecal E. coli isolates from healthy production chickens could cause diseases in a chicken model of avian colibacillosis and three rodent models of ExPEC-associated human infections. From 304 E. coli isolates from chicken fecal samples, 175 E. coli isolates were screened by PCR for virulence genes associated with human-source ExPEC or avian pathogenic E. coli (APEC), an ExPEC subset that causes extraintestinal infections in poultry. Selected isolates genetically identified as ExPEC and non-ExPEC isolates were assessed in vitro for virulence-associated phenotypes, and in vivo for disease-causing ability in animal models of colibacillosis, sepsis, meningitis, and urinary tract infection. Among the study isolates, 13% (40/304) were identified as ExPEC; the majority of these were classified as APEC and uropathogenic E. coli, but none as neonatal meningitis E. coli. Multiple chicken-source fecal ExPEC isolates resembled avian and human clinical ExPEC isolates in causing one or more ExPEC-associated illnesses in experimental animal infection models. Additionally, some isolates that were classified as non-ExPEC were able to cause ExPEC-associated illnesses in animal models, and thus future studies are needed to elucidate their mechanisms of virulence. These findings show that E. coli isolates from chicken feces contain ExPEC-associated genes, exhibit ExPEC-associated in vitro phenotypes, and can cause ExPEC-associated infections in animal models, and thus may pose a health threat to poultry and consumers.

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The development of a vaccine to prevent norovirus infections has been focused on immunization at a mucosal surface, but has been limited by the low immunogenicity of self-assembling Norwalk virus-like particles (NV VLPs) delivered enterically or at nasal surfaces. Nasal immunization, which offers the advantage of ease of immunization, faces obstacles imposed by the normal process of mucociliary clearance, which limits residence time of applied antigens. Herein, we describe the use of a dry powder formulation (GelVac) of an inert in-situ gelling polysaccharide (GelSite) extracted from Aloe vera for nasal delivery of NV VLP antigen. Powder formulations, with or without NV VLP antigen, were similar in structure in dry form or when rehydrated in simulated nasal fluids. Immunogenicity of the dry powder VLP formulation was compared to equivalent antigen/adjuvant liquid formulations in animals. For the GelVac powder, we observed superior NV-specific serum and mucosal (aerodigestive and reproductive tracts) antibody responses relative to liquid formulations. Incorporation of TLR7 agonist gardiquimod in dry powder formulations did not enhance antibody responses, although its inclusion in liquid formulations did enhance VLP immunogenicity irrespective of the presence or absence of GelSite. We interpret these data as showing that GelSite-based dry powder formulations 1.) stabilize the immunogenic structural properties of VLPs and 2.) induce systemic and mucosal antibody titers which are equal or greater than those achieved by VLPs plus adjuvant in a liquid formulation. We conclude that in-situ gelation of the GelVac dry powder formulation at nasal mucosal surfaces delays mucociliary clearance and thereby prolongs VLP antigen exposure to immune effector sites.

The concept of using cholinesterase bioscavengers for prophylaxis against organophosphorous nerve agents and pesticides has progressed from the bench to clinical trial. However, the supply of the native human proteins is either limited (e.g., plasma-derived butyrylcholinesterase and erythrocytic acetylcholinesterase) or nonexisting (synaptic acetylcholinesterase). Here we identify a unique form of recombinant human butyrylcholinesterase that mimics the native enzyme assembly into tetramers; this form provides extended effective pharmacokinetics that is significantly enhanced by polyethylene glycol conjugation. We further demonstrate that this enzyme (but not a G117H/E197Q organophosphorus acid anhydride hydrolase catalytic variant) can prevent morbidity and mortality associated with organophosphorous nerve agent and pesticide exposure of animal subjects of two model species.countermeasures | nonconventional warfare agents | organophosphorous pesticides | protein engineering | transgenic plants B utyrylcholinesterase (BChE) is the major cholinesterase (ChE) in the serum of humans (1, 2). Although the closely related enzyme acetylcholinesterase (AChE) is well described as the primary synaptic regulator of cholinergic transmission, a definitive physiological role for BChE has not yet been demonstrated (3). BChE is catalytically promiscuous and hydrolyzes not only acetylcholine (ACh), but also longer-chain choline esters (e.g., butyrylcholine, its preferred substrate, and succinylcholine) and a variety of non-choline esters, such as acetylsalicylic acid (aspirin) and cocaine (4, 5). Moreover, BChE binds most environmentally occurring ChE inhibitors as well as man-made organophosphorous (OP) pesticides and nerve agents (NAs) (6, 7-10).The systemic biodistribution and affinity for ChE inhibitors allow endogenous BChE to provide broad-spectrum protection against various toxicants by their sequestration before they reach cholinergic synapses. However, under realistic high-dose exposure scenarios, BChE serum levels are too low to afford adequate protection, resulting in persistent cholinergic excitation due to irreversible inhibition of AChE and subsequent accumulation of ACh. Sublethal manifestations of this state include unregulated exocrine secretion and gastrointestinal hypermotility. Death usually results from unregulated stimulation at neuromuscular junction leading to hemodynamic instability and tetanic contraction of the respiratory muscles (11,12).Current OP poisoning therapy consists of atropine for muscarinic ACh receptor blockade and diazepam for symptomatic management of convulsions (12). Additionally, oxime therapy with 2-pralidoxime (2-PAM) can effectively reactivate some but not all OP-AChE adducts (13)(14)(15). This standard therapeutic approach can reduce mortality, but insufficiently prevents the incapacitation associated with OP toxicity (12, 16).Prophylaxis by administration of exogenous ChEs has proven successful in reducing OP-associated morbidity and mortality, but requires the availability of rel...

SUMMARY Pluripotent stem cell (PSC)-derived organoids have emerged as novel multicellular models of human tissue development but display immature phenotypes, aberrant tissue fates, and a limited subset of cells. Here, we demonstrate that integrated analysis and engineering of gene regulatory networks (GRNs) in PSC-derived multilineage human liver organoids direct maturation and vascular morphogenesis in vitro . Overexpression of PROX1 and ATF5 , combined with targeted CRISPR-based transcriptional activation of endogenous CYP3A4 , reprograms tissue GRNs and improves native liver functions, such as FXR signaling, CYP3A4 enzymatic activity, and stromal cell reactivity. The engineered tissues possess superior liver identity when compared with other PSC-derived liver organoids and show the presence of hepatocyte, biliary, endothelial, and stellate-like cell populations in single-cell RNA-seq analysis. Finally, they show hepatic functions when studied in vivo . Collectively, our approach provides an experimental framework to direct organogenesis in vitro by systematically probing molecular pathways and transcriptional networks that promote tissue development.

Ebola hemorrhagic fever is an acute and often deadly disease caused by Ebola virus (EBOV). The possible intentional use of this virus against human populations has led to design of vaccines that could be incorporated into a national stockpile for biological threat reduction. We have evaluated the immunogenicity and efficacy of an EBOV vaccine candidate in which the viral surface glycoprotein is biomanufactured as a fusion to a monoclonal antibody that recognizes an epitope in glycoprotein, resulting in the production of Ebola immune complexes (EICs). Although antigen–antibody immune complexes are known to be efficiently processed and presented to immune effector cells, we found that codelivery of the EIC with Toll-like receptor agonists elicited a more robust antibody response in mice than did EIC alone. Among the compounds tested, polyinosinic:polycytidylic acid (PIC, a Toll-like receptor 3 agonist) was highly effective as an adjuvant agent. After vaccinating mice with EIC plus PIC, 80% of the animals were protected against a lethal challenge with live EBOV (30,000 LD 50 of mouse adapted virus). Surviving animals showed a mixed Th1/Th2 response to the antigen, suggesting this may be important for protection. Survival after vaccination with EIC plus PIC was statistically equivalent to that achieved with an alternative viral vector vaccine candidate reported in the literature. Because nonreplicating subunit vaccines offer the possibility of formulation for cost-effective, long-term storage in biothreat reduction repositories, EIC is an attractive option for public health defense measures.

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