Hypersensitive Detection and Quantitation of BoNT/A by IgY Antibody against Substrate Linear-Peptide

Botulinum neurotoxin A (BoNT/A), the most acutely poisonous substance to humans known, cleave its SNAP-25 substrate with high specificity. Based on the endopeptidase activity, different methods have been developed to detect BoNT/A, but most lack ideal reproducibility or sensitivity, or suffer from long-term or unwanted interferences. In this study, we developed a simple method to detect and quantitate trace amounts of botulinum neurotoxin A using the IgY antibody against a linear-peptide substrate. The effects of reaction buffer, time, and temperature were analyzed and optimized. When the optimized assay was used to detect BoNT/A, the limit of detection of the assay was 0.01 mouse LD₅₀ (0.04 pg), and the limit of quantitation was 0.12 mouse LD₅₀/ml (0.48 pg). The findings also showed favorable specificity of detecting BoNT/A. When used to detect BoNT/A in milk or human serum, the proposed assay exhibited good quantitative accuracy (88% < recovery < 111%; inter- and intra-assay CVs < 18%). This method of detection took less than 3 h to complete, indicating that it can be a valuable method of detecting BoNT/A in food or clinical diagnosis.     

A Heterodimer of a VHH (Variable Domains of Camelid Heavy Chain-only) Antibody That Inhibits Anthrax Toxin Cell Binding Linked to a VHH Antibody That Blocks Oligomer Formation Is Highly Protective in an Anthrax Spore Challenge Model

Anthrax disease is caused by a toxin consisting of protective antigen (PA), lethal factor, and edema factor. Antibodies against PA have been shown to be protective against the disease. Variable domains of camelid heavy chain-only antibodies (VHHs) with affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified recognizing distinct, non-overlapping epitopes. One class recognizes domain 4 of PA at a well characterized neutralizing site through which PA binds to its cellular receptor. A second neutralizing VHH (JKH-C7) recognizes a novel epitope. This antibody inhibits conversion of the PA oligomer from “pre-pore” to its SDS and heat-resistant “pore” conformation while not preventing cleavage of full-length 83-kDa PA (PA83) by cell surface proteases to its oligomer-competent 63-kDa form (PA63). The antibody prevents endocytosis of the cell surface-generated PA63 subunit but not preformed PA63 oligomers formed in solution. JKH-C7 and the receptor-blocking VHH class (JIK-B8) were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA). This VNA displayed improved neutralizing potency in cell assays and protected mice from anthrax toxin challenge with much better efficacy than the separate component VHHs. The VNA protected virtually all mice when separately administered at a 1:1 ratio to toxin and protected mice against Bacillus anthracis spore infection. Thus, our studies show the potential of VNAs as anthrax therapeutics. Due to their simple and stable nature, VNAs should be amenable to genetic delivery or administration via respiratory routes.     

Mucosal vaccine targeting improves onset of mucosal and systemic immunity to botulinum neurotoxin A

Absence of suitable mucosal adjuvants for humans prompted us to consider alternative vaccine designs for mucosal immunization. Because adenovirus is adept in binding to the respiratory epithelium, we tested the adenovirus 2 fiber protein (Ad2F) as a potential vaccine-targeting molecule to mediate vaccine uptake. The vaccine component (the host cell-binding domain to botulinum toxin (BoNT) serotype A) was genetically fused to Ad2F to enable epithelial binding. The binding domain for BoNT was selected because it lies within the immunodominant H chain as a beta-trefoil (Hcbetatre) structure; we hypothesize that induced neutralizing Abs should be protective. Mice were nasally immunized with the Hcbetatre or Hcbetatre-Ad2F, with or without cholera toxin (CT). Without CT, mice immunized with Hcbetatre produced weak secretory IgA (sIgA) and plasma IgG Ab response. Hcbetatre-Ad2F-immunized mice produced a sIgA response equivalent to mice coimmunized with CT. With CT, Hcbetatre-Ad2F-immunized mice showed a more rapid onset of sIgA and plasma IgG Ab responses that were supported by a mixed Th1/Th2 cells, as opposed to mostly Th2 cells by Hcbetatre-dosed mice. Mice immunized with adjuvanted Hcbetatre-Ad2F or Hcbetatre were protected against lethal BoNT serotype A challenge. Using a mouse neutralization assay, fecal Abs from Hcbetatre-Ad2F or Hcbetatre plus CT-dosed mice could confer protection. Parenteral immunization showed that the inclusion of Ad2F enhances anti-Hcbetatre Ab titers even in the absence of adjuvant. This study shows that the Hcbetatre structure can confer protective immunity and that use of Hcbetatre-Ad2F gives more rapid and sustained mucosal and plasma Ab responses.     

Elimination of Botulinum Neurotoxin (BoNT) Type B from Drinking Water by Small-Scale (Personal-Use) Water Purification Devices and Detection of BoNT in Water Samples

Seven small-scale drinking water purification devices were evaluated for their capacity to eliminate botulinum neurotoxin (BoNT) type B from drinking water. Influent water inoculated with toxic Clostridium botulinum cultures and effluent purified water samples were tested for the presence of BoNT by using a standard mouse bioassay and two commercial rapid enzyme immunoassays (EIAs). The water purification devices based on filtration through ceramic or membrane filters with a pore size of 0.2 to 0.4 μm or irradiation from a low-pressure UV-lamp (254 nm) failed to remove BoNT from raw water (reduction of <0.1 log₁₀ units). A single device based on reverse osmosis was capable of removing the BoNT to a level below the detection limit of the mouse bioassay (reduction of >2.3 log₁₀ units). The rapid EIAs intended for the detection of BoNT from various types of samples failed to detect BoNT from aqueous samples containing an estimated concentration of BoNT of 396,000 ng/liter.     

Mucosal targeting of a BoNT/A subunit vaccine adjuvanted with a mast cell activator enhances induction of BoNT/A neutralizing antibodies in rabbits

Background

We previously reported that the immunogenicity of Hcβtre, a botulinum neurotoxin A (BoNT/A) immunogen, was enhanced by fusion to an epithelial cell binding domain, Ad2F, when nasally delivered to mice with cholera toxin (CT). This study was performed to determine if Ad2F would enhance the nasal immunogenicity of Hcβtre in rabbits, an animal model with a nasal cavity anatomy similar to humans. Since CT is not safe for human use, we also tested the adjuvant activity of compound 48/80 (C48/80), a mast cell activating compound previously determined to safely exhibit nasal adjuvant activity in mice.

Methods

New Zealand White or Dutch Belted rabbits were nasally immunized with Hcβtre or Hcβtre-Ad2F alone or combined with CT or C48/80, and serum samples were tested for the presence of Hcβtre-specific binding (ELISA) or BoNT/A neutralizing antibodies.

Results

Hcβtre-Ad2F nasally administered with CT induced serum anti-Hcβtre IgG ELISA and BoNT/A neutralizing antibody titers greater than those induced by Hcβtre + CT. C48/80 provided significant nasal adjuvant activity and induced BoNT/A-neutralizing antibodies similar to those induced by CT.

Conclusions

Ad2F enhanced the nasal immunogenicity of Hcβtre, and the mast cell activator C48/80 was an effective adjuvant for nasal immunization in rabbits, an animal model with a nasal cavity anatomy similar to that in humans.     

Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity

The paralytic disease botulism is caused by botulinum neurotoxins (BoNT), multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC) of BoNT serotype A (BoNT/A) was targeted for generation of monoclonal antibodies (mAbs) that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv) libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS). Affinities of the mAbs for BoNT/A LC ranged from a K_D value of 9.0×10⁻¹¹ M to 3.53×10⁻⁸ M (mean K_D 5.38×10⁻⁹ M and median K_D 1.53×10⁻⁹ M), as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC₅₀ values ranging from 8.3 ~73×10⁻⁹ M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors.     

Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in E. coli

Background

The 23-amino acid extracellular domain of matrix 2 protein (M2e) and the internal nucleoprotein (NP) of influenza are highly conserved among viruses and thus are promising candidate antigens for the development of a universal influenza vaccine. Various M2e- or NP-based DNA or viral vector vaccines have been shown to have high immunogenicity; however, high cost, complicated immunization procedures, and vector-specific antibody responses have restricted their applications. Immunization with an NP–M2e fusion protein expressed in Escherichia coli may represent an alternative strategy for the development of a universal influenza vaccine.

Methodology/Principal Findings

cDNA encoding M2e was fused to the 3′ end of NP cDNA from influenza virus A/Beijing/30/95 (H3N2). The fusion protein (NM2e) was expressed in E. coli and isolated with 90% purity. Mice were immunized with recombinant NM2e protein along with aluminum hydroxide gel and/or CpG as adjuvant. NM2e plus aluminum hydroxide gel almost completely protected the mice against a lethal (20 LD₅₀) challenge of heterologous influenza virus A/PR/8/34.

Conclusions/Significance

The NM2e fusion protein expressed in E. coli was highly immunogenic in mice. Immunization with NM2e formulated with aluminum hydroxide gel protected mice against a lethal dose of a heterologous influenza virus. Vaccination with recombinant NM2e fusion protein is a promising strategy for the development of a universal influenza vaccine.     

Small Molecular Compounds Inhibit HIV-1 Replication through Specifically Stabilizing APOBEC3G

APOBEC3G (hA3G) is a host inhibitor for human immunodeficiency virus, type 1 (HIV-1). However, HIV-1 Vif binds hA3G and induces its degradation. We have established a screening system to discover inhibitors that protect hA3G from Vif-mediated degradation. Through screening, compounds IMB-26 and IMB-35 were identified to be specific inhibitors for the degradation of hA3G by Vif. The inhibitors suppressed HIV-1 replication in hA3G-containing cells but not in those without hA3G. The anti-HIV effect correlated with the endogenous hA3G level. HIV-1 particles from hA3G(+) cells treated with IMB-26/35 contained a hA3G level higher than that from those without IMB-26/35 treatment and showed decreased infectivity. IMB-26/35 bound directly to the hA3G protein, suppressed Vif/hA3G interaction, and therefore protected hA3G from Vif-mediated degradation. The compounds were safe with an anti-HIV therapeutic index >200 in vitro. LD₅₀ of IMB-26 in mice was >1000 mg/kg (intraperitoneally). Therefore, IMB-26 and IMB-35 are novel anti-HIV leads working through specific stabilization of hA3G.     

High Antibody Titer against Apical Membrane Antigen-1 Is Required to Protect against Malaria in the Aotus Model

A Plasmodium falciparum 3D7 strain Apical Membrane Antigen-1 (AMA1) vaccine, formulated with AS02_A adjuvant, slowed parasite growth in a recent Phase 1/2a trial, however sterile protection was not observed. We tested this AS02_A, and a Montanide ISA720 (ISA) formulation of 3D7 AMA1 in Aotus monkeys. The 3D7 parasite does not invade Aotus erythrocytes, hence two heterologous strains, FCH/4 and FVO, were used for challenge, FCH/4 AMA1 being more homologous to 3D7 than FVO AMA1. Following three vaccinations, the monkeys were challenged with 50,000 FCH/4 or 10,000 FVO parasites. Three of the six animals in the AMA+ISA group were protected against FCH/4 challenge. One monkey did not become parasitemic, another showed only a short period of low level parasitemia that self-cured, and a third animal showed a delay before exhibiting its parasitemic phase. This is the first protection shown in primates with a recombinant P. falciparum AMA1 without formulation in Freund''s complete adjuvant. No animals in the AMA+AS02_A group were protected, but this group exhibited a trend towards reduced growth rate. A second group of monkeys vaccinated with AMA+ISA vaccine was not protected against FVO challenge, suggesting strain-specificity of AMA1-based protection. Protection against FCH/4 strain correlated with the quantity of induced antibodies, as the protected animals were the only ones to have in vitro parasite growth inhibitory activity of >70% at 1∶10 serum dilution; immuno-fluorescence titers >8,000; ELISA titers against full-length AMA1 >300,000 and ELISA titer against AMA1 domains1+2 >100,000. A negative correlation between log ELISA titer and day 11 cumulative parasitemia (Spearman rank r = −0.780, p value = 0.0001), further confirmed the relationship between antibody titer and protection. High titers of cross-strain inhibitory antibodies against AMA1 are therefore critical to confer solid protection, and the Aotus model can be used to down-select future AMA1 formulations, prior to advanced human trials.     

Mapping of the Synaptosome-Binding Regions on the Heavy Chain of Botulinum Neurotoxin A By Synthetic Overlapping Peptides Encompassing the Entire Chain

The purpose of this work was to map, on the heavy (H) chain of botulinum neurotoxin A (BoNT/A), the regions that bind to mouse brain synaptosomes (snps). We prepared 60 synthetic overlapping peptides that had uniform size and overlaps and encompassed the entire H chain (residues 499 to 1296) of BoNT/A. The ability of each peptide to inhibit the binding of ¹²⁵I-labeled BoNT/A to mouse brain snps was studied. The binding of ¹²⁵I-labeled BoNT/A to mouse brain snps was completely inhibited by free unlabeled BoNT/A, but not by unrelated proteins, indicating that the binding of BoNT/A to mouse brain snps was a specific event. Inhibition studies with the individual peptides showed that, on the H_N domain, inhibitory activities greater than 10% were exhibited, in decreasing order, by peptides 799–817, 659–677, 729–747, 533–551, 701–719, and 757–775. Lower inhibitory activities (between 5.6% and 8.7%) were exhibited by five other peptides, 463–481, 505–523, 519–537, 603–621 and 645–663. The remaining 18 H_N peptides had little or no inhibitory activity. In the H_C domain, peptides 1065–1083, 1163–1181 and 1275–1296 had the highest inhibitory activities (between 25% and 29%), followed (10–12% inhibitory activity) by peptides 1107–1125, 1191–1209 and 1233–1251. Two other peptides, 1079–1097 and 1177–1195, had very low (5.8% and 4.9 %) inhibitory activities. The remaining 23 H_C peptides had no inhibitory activity. Inhibition with mixtures of equimolar quantities of the most active 6 peptides of H_N, 5 of H_C or all 11 of H_N and H_C revealed that the peptides contain independent non-competing binding regions. Comparison of the locations of the snp-binding regions on the H-subunit with the regions that bind blocking mouse anti-BoNT/A Abs helped explain the protecting ability of these Abs. In the three-dimensional structure of BoNT/A, the snp-binding regions that completely coincide or significantly overlap with the antigenic regions occupy surface locations and most of them reside in the last half of the H_C domain. But some of the regions reside in the H_N domain and might play a role in the translocation event.     

The receptor binding domain of botulinum neurotoxin serotype C binds phosphoinositides

Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD₅₀ of ∼1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a “dual receptor” mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro domain. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides in both assays. Interactions with phosphoinositides may facilitate tighter binding between neuronal membranes and BoNT/C.     

Detection of Type A, B, E, and F Clostridium botulinum Neurotoxins in Foods by Using an Amplified Enzyme-Linked Immunosorbent Assay with Digoxigenin-Labeled Antibodies

An amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium botulinum complex neurotoxins was evaluated for its ability to detect these toxins in food. The assay was found to be suitable for detecting type A, B, E, and F botulinum neurotoxins in a variety of food matrices representing liquids, solid, and semisolid food. Specific foods included broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meat products, and dairy foods. The detection sensitivity of the test for these botulinum complex serotypes was found to be 60 pg/ml (1.9 50% lethal dose [LD₅₀]) for botulinum neurotoxin type A (BoNT/A), 176 pg/ml (1.58 LD₅₀) for BoNT/B, 163 pg/ml for BoNT/E (4.5 LD₅₀), and 117 pg/ml for BoNT/F (less than 1 LD₅₀) in casein buffer. The test could also readily detect 2 ng/ml of neurotoxins type A, B, E, and F in a variety of food samples. For specificity studies, the assay was also used to test a large panel of type A C. botulinum, a smaller panel of proteolytic and nonproteolytic type B, E, and F neurotoxin-producing Clostridia, and nontoxigenic organisms using an overnight incubation of toxin production medium. The assay appears to be an effective tool for large-scale screening of the food supply in the event of a botulinum neurotoxin contamination event.     

Hp1404, a New Antimicrobial Peptide from the Scorpion Heterometrus petersii

Antimicrobial peptides have attracted much interest as a novel class of antibiotics against a variety of microbes including antibiotics resistant strains. In this study, a new cationic antimicrobial peptide Hp1404 was identified from the scorpion Heterometrus petersii, which is an amphipathic α-helical peptide and has a specific inhibitory activity against gram-positive bacteria including methicillin-resistant Staphylococcus aureus. Hp1404 can penetrate the membrane of S. aureus at low concentration, and disrupts the cellular membrane directly at super high concentration. S. aureus does not develop drug resistance after multiple treatments with Hp1404 at sub MIC concentration, which is possibly associated with the antibacterial mechanism of the peptide. In addition, Hp1404 has low toxicity to both mammalian cells (HC₅₀  =  226.6 µg/mL and CC₅₀ > 100 µg/mL) and balb-c mice (Non-toxicity at 80 mg/Kg by intraperitoneal injection and LD₅₀  =  89.8 mg/Kg by intravenous injection). Interestingly, Hp1404 can improve the survival rate of the MRSA infected balb-c mice in the peritonitis model. Taken together, Hp1404 may have potential applications as an antibacterial agent.     

Mucosal Immunization with Live Attenuated Francisella novicida U112ΔiglB Protects against Pulmonary F. tularensis SCHU S4 in the Fischer 344 Rat Model

The need for an efficacious vaccine against Francisella tularensis is a consequence of its low infectious dose and high mortality rate if left untreated. This study sought to characterize a live attenuated subspecies novicida-based vaccine strain (U112ΔiglB) in an established second rodent model of pulmonary tularemia, namely the Fischer 344 rat using two distinct routes of vaccination (intratracheal [i.t.] and oral). Attenuation was verified by comparing replication of U112ΔiglB with wild type parental strain U112 in F344 primary alveolar macrophages. U112ΔiglB exhibited an LD₅₀>10⁷ CFU compared to the wild type (LD₅₀ = 5×10⁶ CFU i.t.). Immunization with 10⁷ CFU U112ΔiglB by i.t. and oral routes induced antigen-specific IFN-γ and potent humoral responses both systemically (IgG2a>IgG1 in serum) and at the site of mucosal vaccination (respiratory/intestinal compartment). Importantly, vaccination with U112ΔiglB by either i.t. or oral routes provided equivalent levels of protection (50% survival) in F344 rats against a subsequent pulmonary challenge with ∼25 LD₅₀ (1.25×10⁴ CFU) of the highly human virulent strain SCHU S4. Collectively, these results provide further evidence on the utility of a mucosal vaccination platform with a defined subsp. novicida U112ΔiglB vaccine strain in conferring protective immunity against pulmonary tularemia.     

Identification of a Nonstructural DNA-Binding Protein (DBP) as an Antigen with Diagnostic Potential for Human Adenovirus

Background

Human adenoviruses (HAdVs) have been implicated as important agents in a wide range of human illnesses. To date, 58 distinct HAdV serotypes have been identified and can be grouped into six species. For the immunological diagnosis of adenoviruses, the hexon protein, a structural protein, has been used. The potential of other HAdV proteins has not been fully addressed.

Methodology/Principal Findings

In this study, a nonstructural antigenic protein, the DNA binding protein (DBP) of human adenovirus 5 and 35 (Ad5, Ad35) - was identified using immunoproteomic technology. The expression of Ad5 and Ad35 DBP in insect cells could be detected by rhesus monkey serum antibodies and healthy adult human serum positive for Ad5 and Ad35. Recombinant DBPs elicited high titer antibodies in mice. Their conserved domain displayed immunological cross-reactions with heterologous DBP antibodies in Western blot assays. DBP-IgM ELISA showed higher sensitivity adenovirus IgM detection than the commercial Adenovirus IgM Human ELISA Kit. A Western blot method developed based on Ad5 DBP was highly consistent with (χ² =  44.9, P<0.01) the Western blot assay for the hexon protein in the detection of IgG, but proved even more sensitive.

Conclusions/Significance

The HAdV nonstructural protein DBP is an antigenic protein that could serve as an alternative common antigen for adenovirus diagnosis.     

Enhanced In Vivo Activity of Cefditoren in Pre-Immunized Mice against Penicillin-Resistant S. pneumoniae (Serotypes 6B, 19F and 23F) in a Sepsis Model

Background

Specific antibodies are likely to be present before S. pneumoniae infection. We explored cefditoren (CDN) total and free values of serum concentrations exceeding the MIC (t>MIC) related to efficacy in a mice sepsis model, and the effect of specific gammaglobulins on in-vitro phagocytosis and in-vivo efficacy.

Methodology/Principal Findings

We used three pneumococcal isolates (serotype, MIC of CDN): Strain 1 (6B, 1 µg/ml), Strain 2 (19F, 2 µg/ml) and Strain 3 (23F, 4 µg/ml). Hyperimmune serum (HS) was obtained from mice immunized with heat-inactivated strains. In-vitro, phagocytosis by HS diluted 1/10 in presence/absence of sub-inhibitory concentrations was measured by flow cytometry including fluorescent bacteria and a neutrophil cell line. In-vivo dose-ranging experiments with HS (dilutions 1/2–1/16) and CDN (6.25 mg/kg–100 mg/kg tid for 48 h) were performed to determine the minimal protective dilution/dose (highest survival) and the non-protective highest dilution/dose (highest mortality: HS-np dilution and CDN-np dose) over 7 days. Efficacy of CDN-np in animals pre-immunized with HS-np (combined strategy) was explored and blood bacterial clearance determined. The CDN measured protein binding was 86.9%. In-vitro, CDN significantly increased phagocytosis (vs. HS 1/10). In non pre-immunized animals, t>MIC values for CDN of ≈35% (total) and ≈19% (free) were associated with 100% survival. Significant differences in survival were found between HS-np alone (≤20%) or CDN-np alone (≤20%) vs. the combined strategy (90%, 60% and 60% for Stains 1, 2 and 3), with t>MIC (total/free) of 22.8%/14.3%, 26.8%/16.0%, and 22.4%/12.7% for Strains 1, 2 and 3, respectively. Prior to the second dose (8 h), median bacterial counts were significantly lower in animals surviving vs. dead at day 7.

Conclusions/Significance

In mice (CDN protein binding similar to humans) total t>MIC values of ≈35% (≈19% free) were efficacious, with a decrease in the required values in pre-immunized animals. This reinforces that immunoprotection to overcome resistance may provide lifesaving strategies.     

Nutritional characters in Ramularia species

Groups of mice were neonatally thymectomized and treated with antithymocyte serum (ATS) prior to challenge infection with viable yeast phase (YP)Histoplasma capsulatum G-17M. Moderate leucocytosis and moderate lymphopenia were seen in immunodeficient animals after infection. Surviving immunodeficient mice exhibited low levels of migration, inhibition activity, while peritoneal exudate cells and spleen cells harvested from surviving infected and untreated normal mice showed significant migration inhibition in the presence of histoplasmin antigen.The LD₅₀ values for YP cells ofH. capsulatum were 1.1×10⁶ for normal untreated mice, 6.0×10⁵ for thymectomized mice, and 6.3×10⁵ for ATS-treated mice. Thymectomized mice that also received ATS treatment exhibited an LD₅₀ of 1.7×10⁵ and were 6.5 times more susceptible to infection then normal mice. Mice which were either thymectomized or treated with ATS were 1.7 times as susceptible as normal mice to infection withH. capsulatum. The criterion of susceptibility is a decrease in the LD₅₀ value.     

Detection and Quantification of Botulinum Neurotoxin Type A by a Novel Rapid In Vitro Fluorimetric Assay

Botulinum neurotoxin type A (BoNT/A), the most poisonous substance known to humans, is a potential bioterrorism agent. The light-chain protein induces a flaccid paralysis through cleavage of the 25-kDa synaptosome-associated protein (SNAP-25), involved in acetylcholine release at the neuromuscular junction. BoNT/A is widely used as a therapeutic agent and to reduce wrinkles. The toxin is used at very low doses, which have to be accurately quantified. With this aim, internally quenched fluorescent substrates containing the fluorophore/repressor pair pyrenylalanine (Pya)/4-nitrophenylalanine (Nop) were developed. Nop and Pya were, respectively, introduced at positions 197 and 200 of the cleavable fragment (amino acids 187 to 203) of SNAP-25 (with norleucine at position 202 [Nle²⁰²]), which is acetylated at its N terminus and amidated at its C terminus. Cleavage of this peptide occurred between positions 197 and 198, as in SNAP-25, and was easily quantified by the strong fluorescence emission of the metabolite. To increase the assay sensitivity, the peptide sequence of the previous substrate was lengthened to account for exosite binding to BoNT/A. We synthesized the peptide PL50 (SNAP-25-NH₂ acetylated at positions 156 to 203 [Nop¹⁹⁷, Pya²⁰⁰, Nle²⁰²]) and its analogue PL51, in which all methionines were replaced by nonoxidizable Nle. Consistent with a large increase in affinity for BoNT/A, PL50 and PL51 exhibit catalytic efficiencies of 2.6 × 10⁶ M⁻¹ s⁻¹ and 8.85 × 10⁶ M⁻¹ s⁻¹, respectively, and behave as the best fluorigenic substrates of BoNT/A reported to date. Under optimized assay conditions, they allow simple quantification of as little as 100 and 60 pg of BoNT/A, respectively, within 2 h with a classical fluorimeter. Calibration of the method against the mouse 50% lethal dose assay unequivocally validates the enzymatic assay.The botulinum neurotoxin (BoNT) family consists of seven antigenically distinct serotypes, BoNT/A to BoNT/G, which act on the peripheral nervous system (19). Of these toxins, serotypes A, B, E, and F cause botulism in humans, a disease characterized by flaccid muscular paralysis. The neurotoxins are produced as single inactive polypeptides of 150 kDa, which are subsequently processed by proteolytic cleavage into biologically active di-chains (19). These forms consist of an approximately 50-kDa light chain (LC) linked by a disulfide bridge to a 100-kDa heavy chain (HC) that contains two domains, designated the binding and translocation domains. The neurotoxins reach their intracellular targets by translocating the LC into the cytosol after endocytosis via interaction of the HC with a high-affinity membrane-bound receptor complex (9, 20). The LC, which possesses a highly specific zinc-endopeptidase activity (29), then blocks the fusion of synaptic vesicles with the presynaptic membrane by selectively cleaving one of the three polypeptides involved in neuroexocytosis. BoNT/A, for instance, cleaves the 206-amino-acid, 25-kDa synaptosome-associated protein (SNAP-25) exclusively between the Q¹⁹⁷ and R¹⁹⁸ residues, thus inhibiting neurotransmitter release at the neuromuscular junction (37, 38).BoNT/A is recognized as the most toxic serotype; its oral 50% lethal dose (LD₅₀) for humans is estimated at 1 μg/kg of body weight (2). Because of this extreme toxicity and prolonged effect, BoNTs are classified by the Centers for Disease Control and Prevention (CDC) as one of the six highest-risk threat agents for bioterrorism in “category A” (27). In spite of this, BoNT/A and -B are widely used as therapeutic agents for the treatment of muscular and nerve disorders, as well as in the treatment of neurological diseases (14, 15, 28). There is also an increasing use of BoNT/A in esthetics for wrinkle reduction (4). Because of their high toxicity, BoNTs are used at very low concentrations, and procedures to be used for their detection and quantification in toxin preparations for medical applications or in the event of malevolent bioterrorist acts have to be highly sensitive, rapid, and easy to use; the use of all lengthy in vivo assays is excluded (2, 11). The advantage of the currently used pharmacotoxicological mouse LD₅₀ (MLD₅₀) assay, considered the gold standard assay, is that it provides the in vivo toxicity of a given botulinum toxin sample, whatever the nature of the infected medium. However, this assay is time-consuming, requires the use of a large number of animals, and has poor repeatability due to many fluctuant parameters involved in this method (22). Several in vitro assays have been reported for the detection of BoNT/A, relying either on mass spectrometry (3, 16), immunological detection (10, 25), or BoNT/A''s endopeptidase activity (12, 30). The advantage of the endopeptidase assay is that it measures and quantifies the “active” part of the toxin, which is directly responsible for neurotransmission inhibition. Various methods have been developed to quantify the BoNT/A proteolytic activity (12, 23, 32-33). Although some of these assays are very sensitive (11), they cannot be used for the field detection of BoNT/A, as they require a multistep procedure, and they are also not easily amenable to quantification of toxin preparations used for medical applications.In this paper, we have designed novel, specific, high-affinity, mimetic peptide substrates for BoNT/A using the internal-collision-induced fluorescence-quenching technique (13). This technique, the use of which has previously been successful in the design of peptide substrates for other Zn-metallopeptidases, e.g., ECE-1 (18) and BoNT/B (1, 26), involves the introduction of a fluorophore/repressor pair, here the highly fluorescent pyrenylalanine (Pya) along with a nitro-phenylalanine (Nop) repressor residue on each side of the cleavage site. Once the better positions of the fluorophore/repressor pair Pya/Nop were determined using a fragment of the SNAP-25 sequence from amino acids 187 to 203 [(187-203) SNAP-25] (30), the kinetic parameters of the peptide substrate were optimized and the stability of the final substrate, acetylated SNAP-25 from positions 156 to 203 [(Ac-156-203) SNAP-25] (Nop¹⁹⁷, Pya²⁰⁰, Nle²⁰²), also called PL50, was finally improved in PL51 by replacing the oxidizable methionine residues within the sequence with norleucines. Thus, the specificity constants (catalytic constant [k_cat]/Michaelis constant [K_m]) of PL50 and of its analogue PL51 were 2.6 × 10⁶ M⁻¹ s⁻¹ and 8.85 × 10⁶ M⁻¹ s⁻¹, respectively. The use of these novel high-affinity substrates provides a simple, one-step, specific, robust, and rapid enzymatic assay, thus fulfilling all the requirements for BoNT/A field detection and for BoNT/A''s quantification in preparations for medical applications.     

Inhibition of tonoplast ATPase by 2',3'-dialdehyde derivative of ATP

The 2′,3′-dialdehyde derivative of ATP (dial-ATP) has been shown to be an affinity label for the ATP binding site of the H⁺-ATPase from tonoplast of etiolated mung bean seedlings (Vigna radiata L.). The dial-ATP caused marked inactivation of enzymatic activities of both membrane-bound and soluble ATPase and its associated proton translocation. The inactivation was reversible, but could be stabilized by NaBH₄. The sodium dodecyl sulfatepolyacrylamide gel electrophoresis pattern revealed that the dial-ATP binding site was in the large (A) subunit of ATPase. The inhibition could be substantially protected by its physiological substrate ATP, pyrophosphate, and nucleotides in the decreasing order: ATP > pyrophosphate > ADP = AMP > GTP > CTP = UTP. A Lineweaver-Burk plot showed that the mode of inhibition was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first order kinetics with a K_i of 4.1 millimolar, a minimum inactivation half-time of 20 seconds, and a pseudo-first order rate constant of 0.035 s⁻¹. The double logarithmic plot of apparent rate constant versus dial-ATP concentration gave a slope of 0.927, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labeling studies with [³H]dial-ATP indicate that the incorporation of approximately 1 mole of dial-ATP per mole ATPase is sufficient to completely inhibit the ATPase. A working model of nonequivalent subunits for enzymatic mechanism of vacuolar ATPase is suggested.