Structural Insights into Rational Design of Single-Domain Antibody-Based Antitoxins against Botulinum Neurotoxins.

Botulinum neurotoxin (BoNT) is one of the most acutely lethal toxins known to humans, and effective treatment for BoNT intoxication is urgently needed. Single-domain antibodies (VHH) have been examined as a countermeasure for BoNT because of their high stability and ease of production. Here, we investigate the structures and the neutralization mechanisms for six unique VHHs targeting BoNT/A1 or BoNT/B1. These studies reveal diverse neutralizing mechanisms by which VHHs prevent host receptor binding or block transmembrane delivery of the BoNT protease domain. Guided by this knowledge, we design heterodimeric VHHs by connecting two neutralizing VHHs via a flexible spacer so they can bind simultaneously to the toxin. These bifunctional VHHs display much greater potency in a mouse co-intoxication model than similar heterodimers unable to bind simultaneously. Taken together, our studies offer insight into antibody neutralization of BoNTs and advance our ability to design multivalent anti-pathogen VHHs with improved therapeutic properties

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

High-Resolution Epitope Positioning of a Large Collection of Neutralizing and Nonneutralizing Single-Domain Antibodies on the Enzymatic and Binding Subunits of Ricin Toxin

We previously produced a heavy-chain-only antibody (Ab) VH domain (VHH)-displayed phage library from two alpacas that had been immunized with ricin toxoid and nontoxic mixtures of the enzymatic ricin toxin A subunit (RTA) and binding ricin toxin B subunit (RTB) (D. J. Vance, J. M. Tremblay, N. J. Mantis, and C. B. Shoemaker, J Biol Chem 288:36538–36547, 2013, https://doi.org/10.1074/jbc.M113.519207). Initial and subsequent screens of that library by direct enzyme-linked immunosorbent assay (ELISA) yielded more than two dozen unique RTA- and RTB-specific VHHs, including 10 whose structures were subsequently solved in complex with RTA. To generate a more complete antigenic map of ricin toxin and to define the epitopes associated with toxin-neutralizing activity, we subjected the VHH-displayed phage library to additional “pannings” on both receptor-bound ricin and antibody-captured ricin. We now report the full-length DNA sequences, binding affinities, and neutralizing activities of 68 unique VHHs: 31 against RTA, 33 against RTB, and 4 against ricin holotoxin. Epitope positioning was achieved through cross-competition ELISAs performed with a panel of monoclonal antibodies (MAbs) and verified, in some instances, with hydrogen-deuterium exchange mass spectrometry. The 68 VHHs grouped into more than 20 different competition bins. The RTA-specific VHHs with strong toxin-neutralizing activities were confined to bins that overlapped two previously identified neutralizing hot spots, termed clusters I and II. The four RTB-specific VHHs with potent toxin-neutralizing activity grouped within three adjacent bins situated at the RTA-RTB interface near cluster II. These results provide important insights into epitope interrelationships on the surface of ricin and delineate regions of vulnerability that can be exploited for the purpose of vaccine and therapeutic development

Adenoviral expression of a bispecific VHH-based neutralizing agent that targets protective antigen provides prophylactic protection from anthrax in mice

Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 μg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 μg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 μg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors

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

STAT5 activation promotes progression and chemotherapy-resistance in early T-cell precursor acute lymphoblastic leukemia

IL-7 supports the growth and chemoresistance of T-cell acute lymphoblastic leukemia (T-ALL), particularly the early T-cell precursor subtype (ETP-ALL), which frequently has activating mutations of IL-7 signaling. STAT5 is an attractive therapeutic target because it is almost universally activated in ETP-ALL, even in the absence of mutations of upstream activators such as the IL-7R, JAK and FLT3. To examine the role of activated STAT5 in ETP-ALL, we have used a Lmo2-transgenic (Lmo2Tg) mouse model in which we can monitor chemoresistant pre-leukemia (pre-LSCs) and leukemia stem cells (LSCs) that drive T-ALL development and relapse following chemotherapy. Using IL-7R-deficient Lmo2Tg mice, we show that IL-7 signaling was not required for the formation of pre-LSCs but essential for their expansion and clonal evolution into LSCs to generate T-ALL. Activated STAT5B was sufficient for the development of T-ALL in IL-7R; Lmo2Tg mice, indicating that inhibition of STAT5 is required to block the supportive signals provided by IL-7. To further understand the role of activated STAT5 in LSCs of ETP-ALL, we developed a new transgenic mouse that enables T-cell specific and doxycycline-inducible expression of the constitutively activated STAT5B1∗6 mutant. Expression of STAT5B1∗6 in T-cells had no effect alone but promoted expansion and chemoresistance of LSCs in Lmo2Tg mice. Pharmacologic inhibition of STAT5 with Pimozide induced differentiation and loss of LSCs, whilst enhancing response to chemotherapy. Furthermore, Pimozide significantly reduced leukemia burden in vivo and overcame chemoresistance of patient-derived ETP-ALL xenografts. Overall, our results demonstrate that STAT5 is an attractive therapeutic target for eradicating LSCs in ETP-ALL

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

Placenta-Like Structure of the Aphid Endoparasitic Wasp Aphidius ervi: A Strategy of Optimal Resources Acquisition

Aphidius ervi (Hymenoptera: Braconidae) is an entomophagous parasitoid known to be an effective parasitoid of several aphid species of economic importance. A reduction of its production cost during mass rearing for inundative release is needed to improve its use in biological control of pests. In these contexts, a careful analysis of its entire development phases within its host is needed. This paper shows that this parasitoid has some characteristics in its embryological development rather complex and different from most other reported insects, which can be phylogenetically very close. First, its yolkless egg allows a high fecundity of the female but force them to hatch from the egg shell rapidly to the host hemocoel. An early cellularisation allowing a rapid differentiation of a serosa membrane seems to confirm this hypothesis. The serosa wraps the developing embryo until the first instar larva stage and invades the host tissues by microvilli projections and form a placenta like structure able to divert host resources and allowing nutrition and respiration of embryo. Such interspecific invasion, at the cellular level, recalls mammal's trophoblasts that anchors maternal uterine wall and underlines the high adaptation of A. ervi to develop in the host body

MagAO Imaging of Long-period Objects (MILO). II. A Puzzling White Dwarf around the Sun-like Star HD 11112

The version of record, Rodigas, T. J. et al, 'MagAO Imaging of long-period objects (MILO). II. A puzzling white dwarf around the sun-like star HD 11112', The Astrophysical Journal, 831:177, November 2016, is available online via doi: 10.3847/0004-637X/831/2/177 © 2016. The American Astronomical Society. All rights reserved.HD 11112 is an old, Sun-like star that has a long-term radial velocity (RV) trend indicative of a massive companion on a wide orbit. Here we present direct images of the source responsible for the trend using the Magellan Adaptive Optics system. We detect the object (HD 11112B) at a separation of 2\fasec 2 (100 AU) at multiple wavelengths spanning 0.6-4 \microns ~and show that it is most likely a gravitationally-bound cool white dwarf. Modeling its spectral energy distribution (SED) suggests that its mass is 0.9-1.1 \msun, which corresponds to very high-eccentricity, near edge-on orbits from Markov chain Monte Carlo analysis of the RV and imaging data together. The total age of the white dwarf is $>2\sigma$ discrepant with that of the primary star under most assumptions. The problem can be resolved if the white dwarf progenitor was initially a double white dwarf binary that then merged into the observed high-mass white dwarf. HD 11112B is a unique and intriguing benchmark object that can be used to calibrate atmospheric and evolutionary models of cool white dwarfs and should thus continue to be monitored by RV and direct imaging over the coming years.Peer reviewedFinal Published versio