A tetraspecific VHH-based neutralizing antibody modifies disease outcome in three animal models of Clostridium difficile infection

Clostridium difficile infection (CDI), a leading cause of nosocomial infection, is a serious disease in North America, Europe, and Asia. CDI varies greatly from asymptomatic carriage to life-threatening diarrhea, toxic megacolon, and toxemia. The incidence of community-acquired infection has increased due to the emergence of hypervirulent antibiotic-resistant strains. These new strains contribute to the frequent occurrence of disease relapse, complicating treatment, increasing hospital stays, and increasing morbidity and mortality among patients. Therefore, it is critical to develop new therapeutic approaches that bypass the development of antimicrobial resistance and avoid disruption of gut microflora. Here, we describe the construction of a single heteromultimeric VHH-based neutralizing agent (VNA) that targets the two primary virulence factors of Clostridium difficile, toxins A (TcdA) and B (TcdB). Designated VNA2-Tcd, this agent has subnanomolar toxin neutralization potencies for both C. difficile toxins in cell assays. When given systemically by parenteral administration, VNA2-Tcd protected against CDI in gnotobiotic piglets and mice and to a lesser extent in hamsters. Protection from CDI was also observed in gnotobiotic piglets treated by gene therapy with an adenovirus that promoted the expression of VNA2-Tcd

Microsporidiosis and Malnutrition in Children with Persistent Diarrhea, Uganda

We show that the microsporidian fungus Enterocytozoon bieneusi is associated with lower rates of weight gain in children in Uganda with persistent diarrhea. This relationship remained after controlling for HIV and concurrent cryptosporidiosis. Children with microsporidiosis were predicted to weigh 1.3 kg less than children without microsporidiosis at 5 years of age

Adenovirus vector expressing Stx1/Stx2-neutralizing agent protects piglets infected with Escherichia coli O157: H7 against fatal systemic intoxication

Hemolytic-uremic syndrome (HUS), caused by Shiga toxin (Stx)-producing Escherichia coli (STEC), remains untreatable. Production of human monoclonal antibodies against Stx, which are highly effective in preventing Stx sequelae in animal models, is languishing due to cost and logistics. We reported previously that the production and evaluation of a camelid heavy-chain-only V(H) domain (VHH)-based neutralizing agent (VNA) targeting Stx1 and Stx2 (VNA-Stx) protected mice from Stx1 and Stx2 intoxication. Here we report that a single intramuscular (i.m.) injection of a nonreplicating adenovirus (Ad) vector carrying a secretory transgene of VNA-Stx (Ad/VNA-Stx) protected mice challenged with Stx2 and protected gnotobiotic piglets infected with STEC from fatal systemic intoxication. One i.m. dose of Ad/VNA-Stx prevented fatal central nervous system (CNS) symptoms in 9 of 10 animals when it was given to piglets 24 h after bacterial challenge and in 5 of 9 animals when it was given 48 h after bacterial challenge, just prior to the onset of CNS symptoms. All 6 placebo animals died or were euthanized with severe CNS symptoms. Ad/VNA-Stx treatment had no impact on diarrhea. In conclusion, Ad/VNA-Stx treatment is effective in protecting piglets from fatal Stx2-mediated CNS complications following STEC challenge. With a low production cost and further development, this could presumably be an effective treatment for patients with HUS and/or individuals at high risk of developing HUS due to exposure to STEC

A Novel Strategy for Development of Recombinant Antitoxin Therapeutics Tested in a Mouse Botulism Model

Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant ‘targeting agent’ that binds a toxin at two unique sites and a ‘clearing Ab’ that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab VH (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit

Comparison of two label-free global quantitation methods, APEX and 2D gel electrophoresis, applied to the Shigella dysenteriae proteome

The in vitro stationary phase proteome of the human pathogen Shigella dysenteriae serotype 1 (SD1) was quantitatively analyzed in Coomassie Blue G250 (CBB)-stained 2D gels. More than four hundred and fifty proteins, of which 271 were associated with distinct gel spots, were identified. In parallel, we employed 2D-LC-MS/MS followed by the label-free computationally modified spectral counting method APEX for absolute protein expression measurements. Of the 4502 genome-predicted SD1 proteins, 1148 proteins were identified with a false positive discovery rate of 5% and quantitated using 2D-LC-MS/MS and APEX. The dynamic range of the APEX method was approximately one order of magnitude higher than that of CBB-stained spot intensity quantitation. A squared Pearson correlation analysis revealed a reasonably good correlation (R2 = 0.67) for protein quantities surveyed by both methods. The correlation was decreased for protein subsets with specific physicochemical properties, such as low Mr values and high hydropathy scores. Stoichiometric ratios of subunits of protein complexes characterized in E. coli were compared with APEX quantitative ratios of orthologous SD1 protein complexes. A high correlation was observed for subunits of soluble cellular protein complexes in several cases, demonstrating versatile applications of the APEX method in quantitative proteomics

Patterns of Genome Evolution among the Microsporidian Parasites Encephalitozoon cuniculi, Antonospora locustae and Enterocytozoon bieneusi

Microsporidia are intracellular parasites that are highly-derived relatives of fungi. They have compacted genomes and, despite a high rate of sequence evolution, distantly related species can share high levels of gene order conservation. To date, only two species have been analysed in detail, and data from one of these largely consists of short genomic fragments. It is therefore difficult to determine how conservation has been maintained through microsporidian evolution, and impossible to identify whether certain regions are more prone to genomic stasis.Here, we analyse three large fragments of the Enterocytozoon bieneusi genome (in total 429 kbp), a species of medical significance. A total of 296 ORFs were identified, annotated and their context compared with Encephalitozoon cuniculi and Antonospora locustae. Overall, a high degree of conservation was found between all three species, and interestingly the level of conservation was similar in all three pairwise comparisons, despite the fact that A. locustae is more distantly related to E. cuniculi and E. bieneusi than either are to each other.Any two genes that are found together in any pair of genomes are more likely to be conserved in the third genome as well, suggesting that a core of genes tends to be conserved across the entire group. The mechanisms of rearrangments identified among microsporidian genomes were consistent with a very slow evolution of their architecture, as opposed to the very rapid sequence evolution reported for these parasites

The piglet acute diarrhea model for evaluating efficacy of treatment and control of cryptosporidiosis

Cryptosporidium spp. are ranked as the second leading pathogens causing life-threatening diarrhea in children under 2 years of age. Although Cryptosporidium hominis causes three quarters of the cases of cryptosporidiosis, studies on C. hominis are limited since natural disease due to C. hominis is host-restricted to humans only. In this mini-review, we demonstrate the successfully adoption, propagation, and utility of the C. hominis strain TU502, isolated originally from an infant with diarrhea in Uganda, in gnotobiotic piglets. The TU502 C. hominis strain and the gnotobiotic piglet model currently are the only available preclinical tools to evaluate therapeutics that specifically target C. hominis. Infection in this gnotobiotic piglet model displays similar clinical symptoms of diarrhea observed in humans. Here we further describe how this unique model of acute diarrhea, can be used for drug discovery and testing of vaccine candidates against cryptosporidiosis. The shared anatomical, physiological and immunological characteristics between piglets and human infants makes the model ideal for evaluating the efficacy of therapeutics and vaccines against cryptosporidiosis as they become available

Recombinant Proteins of Cryptosporidium parvum Induce Proliferation of Mesenteric Lymph Node Cells in Infected Mice

Recombinant antigens of Cryptosporidium parvum, Cp900 and Cp40 but not Cp15, stimulated C. parvum-specific proliferative immune responses of mesenteric lymph node cells in C57BL/6J mice infected with different isolates (MD, GCH1, UCP, and IOWA) of C. parvum, indicating that both Cp900 and Cp40 are immunodominant targets of cellular immune responses during C. parvum infection