Bacterial Toxin Fusion Proteins Elicit Mucosal Immunity against a Foot-and-Mouth Disease Virus Antigen When Administered Intranasally to Guinea Pigs

Peptides corresponding to the foot-and-mouth disease virus VP1 G-H loop are capable of inducing neutralizing antibodies in some species but are considered relatively poor immunogens, especially at mucosal surfaces. However, intranasal administration of antigens along with the appropriate delivery vehicle/adjuvant has been shown to induce mucosal immune responses, and bacterial enterotoxins have long been known to be effective in this regard. In the current study, two different carrier/adjuvant approaches were used to augment mucosal immunity to the FMDV O1 BFS G-H loop epitope, in which the G-H loop was genetically coupled to the E. coli LT-B subunit and coexpressed with the LTA2 fragment (LTA2B-GH), or the nontoxic pseudomonas exotoxin A (ntPE) was fused to LTA2B-GH at LT-A2 to enhance receptor targeting. Only guinea pigs that were inoculated intranasally with ntPE-LTA2B-GH and LTA2B-GH induced significant anti-G-H loop IgA antibodies in nasal washes at weeks 4 and 6 when compared to ovalbumin or G-H loop immunized animals. These were also the only groups that exhibited G-H loop-specific antigen-secreting cells in the nasal mucosa. These data demonstrate that fusion of nonreplicating antigens to LTA2B and ntPE-LTA2B has the potential to be used as carriers/adjuvants to induce mucosal immune responses against infectious diseases

Mucosal vaccination against Foot & Mouth Disease using bacterial toxins as mucosal adjuvants

Synthetic peptides derived from the G-H loop of the Foot-and Mouth Disease Virus (FMDV) capsid protein VP1 are relatively poor at recapitulating the native conformation present in the virus, and thus are often poor immunogens. We hypothesized that a candidate mucosal vaccine against FMDV could be developed using the non-toxic Pseudomonas aeruginosa exotoxin A (ntPE) and B subunit of E. coli heat labile enterotoxin (LTB) to deliver the G-H loop in its native conformation. PE, secreted by Pseudomonas aeruginosa is a mucosal adjuvant that binds to α2-macroglobulin receptor/Low density Lipoprotein receptor-related Protein (LRP1)/CD91 present on epithelial cells and APC plasma membranes to traverse the mucosal barrier. The LTB subunit, a 55KDa pentameric molecule is also a potent mucosal adjuvant and is responsible for binding to various eukaryotic cell receptors including GM1, glycosphingolipids, glycoprotein receptors, polyglycosilceramides and paraglobosides. ^ The chimeric protein ntPE-GH was generated by inserting the coding sequence of the G-H loop into an expression plasmid encoding ntPE, in place of the native Ib loop. Chimeric proteins LTA2B-GH and ntPE-LTA2B-GH were generated by inserting the coding sequence of the G-H loop at the C-terminus of LT where the toxic LTA1 domain is either removed or replaced with the protein of interest (ntPE) that allows the formation of holotoxin-like chimeras. These recombinant fusion proteins containing His6 tag were each expressed in E. coli , purified over a nickel resin and tested for their effectiveness in inducing anti-FMDV systemic and mucosal immune responses. In conclusion, this study demonstrates that the chimeric proteins ntPE-GH, LTA2B-GH and ntPE-LTA2B-GH could be used as a mucosal carriers/adjuvants to induce immune response against FMDV infection.

Selective Evolution of Ligands by Exponential Enrichment to Identify RNA Aptamers against Shiga Toxins

Infection with Shiga toxin- (Stx-) producing E. coli causes life threatening hemolytic uremic syndrome (HUS), a leading cause of acute renal failure in children. Of the two antigenically distinct toxins, Stx1 and Stx2, Stx2 is more firmly linked with the development of HUS. In the present study, selective evolution of ligands by exponential enrichment (SELEX) was used in an attempt to identify RNA aptamers against Stx1 and Stx2. After 5 rounds of selection, significant enrichment of aptamer pool was obtained against Stx2, but not against Stx1, using a RNA aptamer library containing 56 random nucleotides (N56). Characterization of individual aptamer sequences revealed that six unique RNA aptamers (mA/pC, mB/pA, mC, mD, pB, and pD) recognized Stx2 in a filter binding assay. None of these aptamers bound Stx1. Aptamers mA/pC, mB/pA, mC, and mD, but not pB and pD, partially blocked binding of Alexa 488-labeled Stx2 with HeLa cells in a flow cytometry assay. However, none of the aptamers neutralized Stx2-mediated cytotoxicity and death of HeLa cells

SB 9200, a novel agonist of innate immunity, shows potent antiviral activity against resistant HCV variants

SB 9200 is a novel, first-in-class oral modulator of innate immunity that is believed to act via the activation of the RIG-I and NOD2 pathways. SB 9200 has broad-spectrum antiviral activity against RNA viruses including hepatitis C virus (HCV), norovirus, respiratory syncytial virus, and influenza and has demonstrated activity against hepatitis B virus (HBV) in vitro and in vivo. In phase I clinical trials in chronically infected HCV patients, SB 9200 has been shown to reduce HCV RNA by up to 1.9 log10 . Here, we demonstrate the antiviral activity of SB 9200 against a HCV replicon system and patient derived virus. Using the HCV capture-fusion assay, we show that SB 9200 is active against diverse HCV genotypes and is also effective against HCV derived from patients who relapse following direct-acting antiviral treatment, including viruses containing known NS5A resistance-associated sequences. These data confirm the broad antiviral activity of SB 9200 and indicate that it may have clinical utility in HCV patients who have failed to respond to current antiviral regimens

A Microarray Biosensor for Multiplexed Detection of Microbes Using Grating-Coupled Surface Plasmon Resonance Imaging

Grating-coupled surface plasmon resonance imaging (GCSPRI) utilizes an optical diffraction grating embossed on a gold-coated sensor chip to couple collimated incident light into surface plasmons. The angle at which this coupling occurs is sensitive to the capture of analyte at the chip surface. This approach permits the use of disposable biosensor chips that can be mass-produced at low cost and spotted in microarray format to greatly increase multiplexing capabilities. The current GCSPRI instrument has the capacity to simultaneously measure binding at over 1000 unique, discrete regions of interest (ROIs) by utilizing a compact microarray of antibodies or other specific capture molecules immobilized on the sensor chip. In this report, we describe the use of GCSPRI to directly detect multiple analytes over a large dynamic range, including soluble protein toxins, bacterial cells, and viruses, in near real-time. GCSPRI was used to detect a variety of agents that would be useful for diagnostic and environmental sensing purposes, including macromolecular antigens, a nontoxic form of <i>Pseudomonas aeruginosa</i> exotoxin A (ntPE), <i>Bacillus globigii</i>, <i>Mycoplasma hyopneumoniae</i>, <i>Listeria monocytogenes</i>, <i>Escherichia coli</i>, and M13 bacteriophage. These studies indicate that GCSPRI can be used to simultaneously assess the presence of toxins and pathogens, as well as quantify specific antibodies to environmental agents, in a rapid, label-free, and highly multiplexed assay requiring nanoliter amounts of capture reagents