Structure-Function Analysis of the A1 Subunit of Shiga Toxin 2 with Peptides That Target the P-Stalk Binding Site and Inhibit Activity.

Shiga toxin 2a (Stx2a) is the virulence factor of Escherichia coli (STEC), which is associated with hemolytic uremic syndrome, the leading cause of pediatric kidney failure. The A1 subunit of Stx2a (Stx2A1) binds to the conserved C-terminal domain (CTD) of the ribosomal P-stalk proteins to remove an adenine from the sarcin-ricin loop (SRL) in the 28S rRNA, inhibiting protein synthesis. There are no antidotes against Stx2a or any other ribosome-inactivating protein (RIP). The structural and functional details of the binding of Stx2A1 to the P-stalk CTD are not known. Here, we carry out a deletion analysis of the conserved P-stalk CTD and show that the last eight amino acids (P8) of the P-stalk proteins are the minimal sequence required for optimal affinity and maximal inhibitory activity against Stx2A1. We determined the first X-ray crystal structure of Stx2A1 alone and in complex with P8 and identified the exact binding site. The C-terminal aspartic acid of the P-stalk CTD serves as an anchor, forming key contacts with the conserved arginine residues at the P-stalk binding pocket of Stx2A1. Although the ricin A subunit (RTA) binds to the P-stalk CTD, the last aspartic acid is more critical for the interaction with Stx2A1, indicating that RIPs differ in their requirements for the P-stalk. These results demonstrate that the catalytic activity of Stx2A1 is inhibited by blocking its interactions with the P-stalk, providing evidence that P-stalk binding is an essential first step in the recruitment of Stx2A1 to the SRL for depurination.

Cloning and Functional Verification of Endogenous U6 Promoters for the Establishment of Efficient CRISPR/Cas9-Based Genome Editing in Castor (Ricinus communis).

Castor (Ricinus communis) seeds are rich in a type of hydroxy fatty acid called ricinoleic acid, which is in high demand for the production of plant-based plastics, lubricants, and hydraulic oils. However, the high content of ricin, a toxic protein, in these seeds has restricted further expansion in the area of castor cultivation. Therefore, the development of ricin-free castor is needed. Genome editing technology, although successfully applied in several plant species, is still in the developing stages in castor and awaits the identification of an endogenous U6 promoter with robust function. Here, we searched for U6 small nuclear RNA (snRNA) genes in the castor genome. This led to the identification of six U6 snRNA genes. The promoters of these U6 snRNA genes were cloned, and their function was examined in castor cells using the particle delivery method. The results showed that a U6 promoter length of approximately 300 bp from the transcription start site was sufficient to activate gene expression. This study provides insights into the endogenous castor U6 promoter sequences and outlines a method for verifying the function of U6 promoters in plants using the particle delivery system.

Crystal structure of ricin toxin A chain complexed with a highly potent pterin-based small-molecular inhibitor.

Ricin toxin A chain (RTA), from Ricinus communis, is a deadly protein that inactivates ribosomes by degrading an adenine residue at position 4324 in 28S rRNA. Recently, we have demonstrated that pterin-7-carboxamides with peptide pendants were potent RTA inhibitors. Among these, N-(pterin-7-carbonyl)glycyl-L-tyrosine (7PCGY) is the most potent RTA inhibitor as a small organic molecule. However, despite this fascinating inhibitory activity, the mode of interaction of 7PCGY with RTA remains elusive. This study aimed to elucidate the factors responsible for the high RTA inhibitory activity of 7PCGY based on X-ray crystallographic analysis. Herein, we report the successfully resolved X-ray crystal structure of 7PCGY/RTA complexes, revealing that the interaction between the phenolic hydroxy group in 7PCGY and Asn78 of RTA through a hydrogen bonding and the conformational change of Tyr80 and Asn122 are responsible for the high RTA inhibitory activity of 7PCGY.

Ribosome inactivating proteins in insects: HGT, gene expression, and functional implications.

Ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that depurinate an adenine residue in the conserved alpha-sarcin/ricin loop (SRL) of rRNA, inhibiting protein synthesis. Previously, we reported the existence of these toxins in insects, whose presence is restricted to mosquitoes from the Culicinae subfamily (e.g., Aedes aegypti) and whiteflies from the Aleyrodidae family (e.g., Bemisia tabaci). Both groups of genes are derived from two independent horizontal gene transfer (HGT) events and are evolving under purifying selection. Here, we report and characterize the occurrence of a third HGT event in the Sciaroidea superfamily, which supports the recurrent acquisition of RIP genes by insects. Transcriptomic experiments, available in databases, allowed us to describe the temporal and spatial expression profiles for these foreign genes in these organisms. Furthermore, we found that RIP expression is induced after infection with pathogens and provided, for the first time, transcriptomic evidence of parasite SRL depurination. This evidence suggests a possible role of these foreign genes as immune effectors in insects.

Sites of vulnerability on ricin B chain revealed through epitope mapping of toxin-neutralizing monoclonal antibodies.

Ricin toxin's B subunit (RTB) is a multifunctional galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that promotes uptake and intracellular trafficking of ricin's ribosome-inactivating subunit (RTA) into mammalian cells. Structurally, RTB consists of two globular domains (RTB-D1, RTB-D2), each divided into three homologous sub-domains (α, ß, γ). The two carbohydrate recognition domains (CRDs) are situated on opposite sides of RTB (sub-domains 1α and 2γ) and function non-cooperatively. Previous studies have revealed two distinct classes of toxin-neutralizing, anti-RTB monoclonal antibodies (mAbs). Type I mAbs, exemplified by SylH3, inhibit (~90%) toxin attachment to cell surfaces, while type II mAbs, epitomized by 24B11, interfere with intracellular toxin transport between the plasma membrane and the trans-Golgi network (TGN). Localizing the epitopes recognized by these two classes of mAbs has proven difficult, in part because of RTB's duplicative structure. To circumvent this problem, RTB-D1 and RTB-D2 were expressed as pIII fusion proteins on the surface of filamentous phage M13 and subsequently used as "bait" in mAb capture assays. We found that SylH3 captured RTB-D1 (but not RTB-D2) in a dose-dependent manner, while 24B11 captured RTB-D2 (but not RTB-D1) in a dose-dependent manner. We confirmed these domain assignments by competition studies with an additional 8 RTB-specific mAbs along with a dozen a single chain antibodies (VHHs). Collectively, these results demonstrate that type I and type II mAbs segregate on the basis of domain specificity and suggest that RTB's two domains may contribute to distinct steps in the intoxication pathway.

Induction of Male Sterility in Tobacco by Anther-Specific Expression of the Gene for Ricin Enzymatic Subunit A Chain RTA.

Targeted gene expression in plants allows us to further study biological traits of interest, such as reproductive and developmental processes. Here, the tobacco TA29 anther-specific promoter was used to direct the expression of the ricin enzymatic subunit A (RTA) in transgenic tobacco plants, phenotypic analysis of the resulting positive transgenic tobacco (Nicotiana tabacum L.) plants demonstrated that RTA expression led to a reduction in pistil length and shriveling of anthers, as well as the grayish-brown color of anthers, the reduced pollen viability and male sterility. For the first time, a plant-derived ricin gene enzymatic subunit A (RTA) expression system under the tissue-specific promoter was demonstrated to be sensitive and efficient in controlling plant sterility and creating male-sterile materials. Consequently, it could be used to control other agronomic traits and produce hybrid seeds in plants in the future.

Leucine 232 and hydrophobic residues at the ribosomal P stalk binding site are critical for biological activity of ricin.

Ricin interacts with the ribosomal P stalk to cleave a conserved adenine from the α-sarcin/ricin loop (SRL) of the rRNA. Ricin toxin A chain (RTA) uses Arg235 as the most critical arginine for binding to the P stalk through electrostatic interactions to facilitate depurination. Structural analysis showed that a P2 peptide binds to a hydrophobic pocket on RTA and the last two residues form hydrogen bonds with Arg235. The importance of hydrophobic residues relative to Arg235 in the interaction with the P stalk in vivo and on the toxicity of RTA is not known. Here, we mutated residues in the hydrophobic pocket to analyze their contribution to toxicity and depurination activity in yeast and in mammalian cells. We found that Leu232, Tyr183 and Phe240 contribute cumulatively to toxicity, with Leu232 being the most significant. A quadruple mutant, Y183A/L232A/R235A/F240A, which combined mutations in critical hydrophobic residues with R235A completely abolished the activity of RTA, indicating that Arg235 and hydrophobic residues are required for full biological activity. Y183A and F240A mutants had reduced activity on RNA, but higher activity on ribosomes compared with R235A in vitro, suggesting that they could partially regain activity upon interaction with ribosomes. These results expand the region of interaction between RTA and the P stalk critical for cellular activity to include the hydrophobic pocket and provide the first evidence that interaction of P stalk with the hydrophobic pocket promotes a conformational rearrangement of RTA to correctly position the active site residues for catalytic attack on the SRL.

Selection and validation of castor bean (Ricinus communis) reference genes for quantitative PCR (RT-qPCR) in developing and germinating seeds and expression pattern of four ricin-family genes.

This study aims to expand the set of internal control genes used for RT-qPCR experiments with Castor bean (Ricinus communis) seeds by evaluating candidate genes across several seed tissues and developmental stages. Nine reference genes were selected, including actin-11 (ACT11), tubulin alpha-2 (Tα2), elongation factor 1-alpha (EF1-α), protein phosphatase 2A-2 (PP2A2), polyubiquitin-3 (PUB3) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Biological samples consisted of R. communis seeds in 15 stages of maturation and germination. We demonstrate that PP2A2, PUB3 and EF1-α are the most stably expressed genes across the tested conditions and therefore appropriate for RT-qPCR. Subsequently, those reference genes were used for the analysis of the expression of four R. communis ricin-family genes. In developing seeds, the highest ricin expression levels was seen in the nucellus and in the endosperm, whereas in germinating seeds a peak expression occurs 4-6 days after germination. The four tested ricin isoforms exhibited differential expression patterns across tissues and seed developmental stages, which may indicate distinct biological roles for each ricin gene.

Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation.

Glycosylation is a fundamental modification of proteins and membrane lipids. Toxins that utilize glycans as their receptors have served as powerful tools to identify key players in glycosylation processes. Here, we carried out Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated genome-wide loss-of-function screens using two related bacterial toxins, Shiga-like toxins (Stxs) 1 and 2, which use a specific glycolipid, globotriaosylceramide (Gb3), as receptors, and the plant toxin ricin, which recognizes a broad range of glycans. The Stxs screens identified major glycosyltransferases (GTs) and transporters involved in Gb3 biosynthesis, while the ricin screen identified GTs and transporters involved in N-linked protein glycosylation and fucosylation. The screens also identified lysosomal-associated protein transmembrane 4 alpha (LAPTM4A), a poorly characterized four-pass membrane protein, as a factor specifically required for Stxs. Mass spectrometry analysis of glycolipids and their precursors demonstrates that LAPTM4A knockout (KO) cells lack Gb3 biosynthesis. This requirement of LAPTM4A for Gb3 synthesis is not shared by its homolog lysosomal-associated protein transmembrane 4 beta (LAPTM4B), and switching the domains between them determined that the second luminal domain of LAPTM4A is required, potentially acting as a specific "activator" for the GT that synthesizes Gb3. These screens also revealed two Golgi proteins, Transmembrane protein 165 (TMEM165) and Transmembrane 9 superfamily member 2 (TM9SF2), as shared factors required for both Stxs and ricin. TMEM165 KO and TM9SF2 KO cells both showed a reduction in not only Gb3 but also other glycosphingolipids, suggesting that they are required for maintaining proper levels of glycosylation in general in the Golgi. In addition, TM9SF2 KO cells also showed defective endosomal trafficking. These studies reveal key Golgi proteins critical for regulating glycosylation and glycolipid synthesis and provide novel therapeutic targets for blocking Stxs and ricin toxicity.

Construction and characterization of the recombinant immunotoxin RTA-4D5-KDEL targeting HER2/neu-positive cancer cells and locating the endoplasmic reticulum.

The specific targeting of immunotoxins enables their wide application in cancer therapy. The A-chain of the ricin protein (RTA) is an N-glycosidase that catalyzes the removal of adenine from the 28S rRNA, preventing protein translation and leading to cell death. Ricin is highly toxic but can only exert its toxic effects from within the cytoplasm. In this study, we linked the anti-HER2 single-chain variable fragment 4D5 scFv and the endoplasmic reticulum-targeting peptide KDEL to the C-terminal of the RTA to construct immunotoxin RTA-4D5-KDEL. In vitro experiments showed that the anticancer effect of RTA-4D5-KDEL towards ovarian cancer cells SKOV-3 increased 440-fold and 28-fold relative to RTA and RTA-4D5, respectively. RTA-4D5-KDEL had a strong inhibitory effect on HER2-overexpressing SKOV-3 cells and caused little damage to normal HEK-293 cells and H460 lung cancer cells. Immunofluorescence experiments showed that the immunotoxin RTA-4D5 could specifically bind to SKOV-3 cells, but not to normal cells HEK-293. The immunotoxin RTA-4D5-KDEL could rapidly localize the recombinant protein to the endoplasmic reticulum. These results suggest that the recombinant immunotoxin RTA-4D5-KDEL has a strong inhibitory effect on ovarian cancer cells that overexpress HER2 but little harm to the normal cells.

Expression of novel fusion antiviral proteins ricin a chain-pokeweed antiviral proteins (RTA-PAPs) in Escherichia coli and their inhibition of protein synthesis and of hepatitis B virus in vitro.

BACKGROUND: Ricin A chain (RTA) and Pokeweed antiviral proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential production amenability and sub-toxic antiviral value of novel fusion proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral protein isoform 1 from seeds (RTA-PAPS1) was produced in an E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and protein synthesis inhibitory activity assayed by comparison to the production of a control protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. The fusion protein was further optimized using in silico tools, produced in an E. coli in vivo expression system, purified by a three-step process from soluble lysate and confirmed in a protein synthesis inhibition activity assay. RESULTS: Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded protein was bioactive with a 50% protein synthesis inhibitory concentration (IC50) of 0.06 nM (3.63 ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03 nM (1.82 ng/ml) and a therapeutic index of > 21,818 with noticeable steric hindrance. Results also showed that the optimized protein ricin A chain mutant-Pokeweed antiviral protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function on protein synthesis inhibition activity, with an IC50 of 0.03 nM (1.82 ng/ml), and with minimal, if any, steric hindrance. CONCLUSIONS: Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant gain of function on protein synthesis inhibition and anti-HBV activities in vitro with a high therapeutic index and, thus, merit further development as potential potent antiviral agents against chronic HBV infection to be used as a standalone or in combination with existent therapies.

Bio-detoxification of ricin in castor bean (Ricinus communis L.) seeds.

Ricin is a highly toxic ribosome-inactivating lectin occurring in the seeds of castor bean (Ricinus communis L.). Castor bean grows throughout tropical and sub-tropical regions and is a very important crop due to its high seed content of ricinoleic acid, an unusual fatty acid, which has several industrial applications. However, due to the presence of the toxin, castor bean can cause death after the exposure of animals to low doses of ricin through skin contact, injection, inhalation or oral routes. Aiming to generate a detoxified genotype, we explored the RNAi concept in order to silence the ricin coding genes in the endosperm of castor bean seeds. Results indicated that ricin genes were effectively silenced in genetically modified (GM) plants, and ricin proteins were not detected by ELISA. Hemagglutination activity was not observed with proteins isolated from GM seeds. In addition, we demonstrated that seed proteins from GM plants were not toxic to rat intestine epithelial cells or to Swiss Webster mice. After oil extraction, bio-detoxified castor bean cake, which is very rich in valuable proteins, can be used for animal feeding. Gene silencing would make castor bean cultivation safer for farmers, industrial workers and society.

Selective cytotoxicity of a novel immunotoxin based on pulchellin A chain for cells expressing HIV envelope.

Immunotoxins (ITs), which consist of antibodies conjugated to toxins, have been proposed as a treatment for cancer and chronic infections. To develop and improve the ITs, different toxins such as ricin, have been used, aiming for higher efficacy against target cells. The toxin pulchellin, isolated from the Abrus pulchellus plant, has similar structure and function as ricin. Here we have compared two plant toxins, recombinant A chains from ricin (RAC) and pulchellin (PAC) toxins, for their ability to kill HIV Env-expressing cells. In this study, RAC and PAC were produced in E. coli, and chromatographically purified, then chemically conjugated to two different anti-HIV monoclonal antibodies (MAbs), anti-gp120 MAb 924 or anti-gp41 MAb 7B2. These conjugates were characterized biochemically and immunologically. Cell internalization was studied by flow cytometry and confocal microscopy. Results showed that PAC can function within an effective IT. The ITs demonstrated specific binding against native antigens on persistently HIV-infected cells and recombinant antigens on Env-transfected cells. PAC cytotoxicity appears somewhat less than RAC, the standard for comparison. This is the first report that PAC may have utility for the design and construction of therapeutic ITs, highlighting the potential role for specific cell targeting.

Evaluation of lumazine synthase from Bacillus anthracis as a presentation platform for polyvalent antigen display.

Polyvalent antigen display is an effective strategy to enhance the immunogenicity of subunit vaccines by clustering them in an array-like manner on a scaffold system. This strategy results in a higher local density of antigens, increased high avidity interactions with B cells and other antigen presenting cells, and therefore a more effective presentation of vaccine antigens. In this study, we used lumazine synthase (LS), an icosahedral symmetry capsid derived from Bacillus anthracis, as a scaffold to present 60 copies of a linear B cell epitope (PB10) from the ricin toxin fused to the C terminus of LS via four different linkers. We then investigated the effects of linker length, linker rigidity and formaldehyde crosslinking on the protein assembly, conformational integrity, thermal stability, in vitro antibody binding, and immunogenicity in mice. Fusion of the PB10 peptide onto LS, with varying linker lengths, did not affect protein assembly, thermal stability or exposure of the epitope, but had a minor impact on protein conformation. Formaldehyde crosslinking considerably improved protein thermal stability with only minor impact on protein conformation. All LS_PB10 constructs, when administered to mice by injection without adjuvant, elicited measurable anti-ricin serum IgG titers, although the titers were not sufficient to confer protection against a 10× lethal dose ricin challenge. This work sheds light on the biophysical properties, immunogenicity and potential feasibility of LS from B. anthracis as a scaffold system for polyvalent antigen display.

Ricin uses arginine 235 as an anchor residue to bind to P-proteins of the ribosomal stalk.

Ricin toxin A chain (RTA) binds to stalk P-proteins to reach the α-sarcin/ricin loop (SRL) where it cleaves a conserved adenine. Arginine residues at the RTA/RTB interface are involved in this interaction. To investigate the individual contribution of each arginine, we generated single, double and triple arginine mutations in RTA. The R235A mutation reduced toxicity and depurination activity more than any other single arginine mutation in yeast. Further reduction in toxicity, depurination activity and ribosome binding was observed when R235A was combined with a mutation in a nearby arginine. RTA interacts with the ribosome via a two-step process, which involves slow and fast interactions. Single arginine mutations eliminated the fast interactions with the ribosome, indicating that they increase the binding rate of RTA. Arginine residues form a positively charged patch to bind to negatively charged residues at the C-termini of P-proteins. When electrostatic interactions conferred by the arginines are lost, hydrophobic interactions are also abolished, suggesting that the hydrophobic interactions alone are insufficient to allow binding. We propose that Arg235 serves as an anchor residue and cooperates with nearby arginines and the hydrophobic interactions to provide the binding specificity and strength in ribosome targeting of RTA.

Genetic diversity and population structure of castor (Ricinus communis L.) germplasm within the US collection assessed with EST-SSR markers.

Castor is an important oilseed crop and although its oil is inedible, it has multiple industrial and pharmaceutical applications. The entire US castor germplasm collection was previously screened for oil content and fatty acid composition, but its genetic diversity and population structure has not been determined. Based on the screening results of oil content, fatty acid composition, and country origins, 574 accessions were selected and genotyped with 22 polymorphic EST-SSR markers. The results from cluster analysis, population structure, and principal component analysis were consistent, and partitioned accessions into four subpopulations. Although there were certain levels of admixtures among groups, these clusters and subpopulations aligned with geographic origins. Both divergent and redundant accessions were identified in this study. The US castor germplasm collection encompasses a moderately high level of genetic diversity (pairwise dissimilarity coefficient = 0.53). The results obtained here will be useful for choosing accessions as parents to make crosses in breeding programs and prioritizing accessions for regeneration to improve germplasm management. A subset of 230 accessions was selected and will be planted in the field for establishing a core collection of the US castor germplasm. Further evaluation of the US castor germplasm collection is also discussed.

Crystal Structure of Ribosome-Inactivating Protein Ricin A Chain in Complex with the C-Terminal Peptide of the Ribosomal Stalk Protein P2.

Ricin is a type 2 ribosome-inactivating protein (RIP), containing a catalytic A chain and a lectin-like B chain. It inhibits protein synthesis by depurinating the N-glycosidic bond at α-sarcin/ricin loop (SRL) of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation center of the ribosome. Here, we present the 1.6 Å crystal structure of Ricin A chain (RTA) complexed to the C-terminal peptide of the ribosomal stalk protein P2, which plays a crucial role in specific recognition of elongation factors and recruitment of eukaryote-specific RIPs to the ribosomes. Our structure reveals that the C-terminal GFGLFD motif of P2 peptide is inserted into a hydrophobic pocket of RTA, while the interaction assays demonstrate the structurally untraced SDDDM motif of P2 peptide contributes to the interaction with RTA. This interaction mode of RTA and P protein is in contrast to that with trichosanthin (TCS), Shiga-toxin (Stx) and the active form of maize RIP (MOD), implying the flexibility of the P2 peptide-RIP interaction, for the latter to gain access to ribosome.

Ricin-B-lectin enhances microsporidia Nosema bombycis infection in BmN cells from silkworm Bombyx mori.

Nosema bombycis is an obligate intracellular parasitic fungus that utilizes a distinctive mechanism to infect Bombyx mori Spore germination can be used for host cell invasion; however, the detailed mechanism remains to be elucidated. The ricin-B-lectin (RBL) gene is significantly differentially regulated after N. bombycis spore germination, and NbRBL might play roles in spore germination and infection. In this study, the biological function of NbRBL was examined. Protein sequence analysis showed that NbRBL is a secreted protein that attaches to carbohydrates. The relative expression level of the NbRBL gene was low during the first 30 h post-infection (hpi) in BmN cells, and high expression was detected from 42 hpi. Gene cloning, prokaryotic expression, and antibody preparation for NbRBL were performed. NbRBL was detected in total and secreted proteins using western blot analysis. Subcellular localization analysis showed that NbRBL is an intracellular protein. Spore adherence and infection assays showed that NbRBL could enhance spore adhesion to BmN cells; the proliferative activities of BmN cells incubated with anti-NbRBL were higher than those in negative control groups after N. bombycis infection; and the treatment groups showed less damage from spore invasion. We therefore, propose that NbRBL is released during spore germination, enhances spore adhesion to BmN cells, and contributes to spore invasion.

Intranasal Immunization with Influenza Virus-Like Particles Containing Membrane-Anchored Cholera Toxin B or Ricin Toxin B Enhances Adaptive Immune Responses and Protection against an Antigenically Distinct Virus.

Vaccination is the most effective means to prevent influenza virus infection, although current approaches are associated with suboptimal efficacy. Here, we generated virus-like particles (VLPs) composed of the hemagglutinin (HA), neuraminidase (NA) and matrix protein (M1) of A/Changchun/01/2009 (H1N1) with or without either membrane-anchored cholera toxin B (CTB) or ricin toxin B (RTB) as molecular adjuvants. The intranasal immunization of mice with VLPs containing membrane-anchored CTB or RTB elicited stronger humoral and cellular immune responses when compared to mice immunized with VLPs alone. Administration of VLPs containing CTB or RTB significantly enhanced virus-specific systemic and mucosal antibody responses, hemagglutination inhibiting antibody titers, virus neutralizing antibody titers, and the frequency of virus-specific IFN-γ and IL-4 secreting splenocytes. VLPs with and without CTB or RTB conferred complete protection against lethal challenge with a mouse-adapted homologous virus. When challenged with an antigenically distinct H1N1 virus, all mice immunized with VLPs containing CTB or RTB survived whereas mice immunized with VLPs alone showed only partial protection (80% survival). Our results suggest that membrane-anchored CTB and RTB possess strong adjuvant properties when incorporated into an intranasally-delivered influenza VLP vaccine. Chimeric influenza VLPs containing CTB or RTB may represent promising vaccine candidates for improved immunological protection against homologous and antigenically distinct influenza viruses.

An in vitro evaluation of epigallocatechin gallate (eGCG) as a biocompatible inhibitor of ricin toxin.

The catechin, epigallocatechin gallate (eGCG), found in green tea, has inhibitory activity against a number of protein toxins and was investigated in relation to its impact upon ricin toxin (RT) in vitro. The IC(50) for RT was 0.08±0.004 ng/mL whereas the IC(50) for RT+100 µM eGCG was 3.02±0.572 ng/mL, indicating that eGCG mediated a significant (p<0.0001) reduction in ricin toxicity. This experiment was repeated in the human macrophage cell line THP-1 and IC(50) values were obtained for RT (0.54±0.024 ng/mL) and RT+100 µM eGCG (0.68±0.235 ng/mL) again using 100 µM eGCG and was significant (p=0.0013). The documented reduction in ricin toxicity mediated by eGCG was found to be eGCG concentration dependent, with 80 and 100 µg/mL (i.e. 178 and 223 µM respectively) of eGCG mediating a significant (p=0.0472 and 0.0232) reduction in ricin toxicity at 20 and 4 ng/ml of RT in Vero and THP-1 cells (respectively). When viability was measured in THP-1 cells by propidium iodide exclusion (as opposed to the MTT assays used previously) 10 ng/mL and 5 ng/mL of RT was used. The addition of 1000 µM and 100 µM eGCG mediated a significant (p=0.0015 and <0.0001 respectively) reduction in ricin toxicity relative to an identical concentration of ricin with 1 µg eGCG. Further, eGCG (100 µM) was found to reduce the binding of RT B chain to lactose-conjugated Sepharose as well as significantly (p=0.0039) reduce the uptake of RT B chain in Vero cells. This data suggests that eGCG may provide a starting point to refine biocompatible substances that can reduce the lethality of ricin.