Janet K. Dykes

Immunogenicity of Two Efficacious Outer Membrane Protein-Based Serogroup B Meningococcal Vaccines among Young Adults in Iceland

Serum bactericidal activity (SBA) and ELISA antibody levels elicited by two efficacious serogroup B meningococcal vaccines were measured in a controlled trial involving 408 15- to 20-year-olds. Subjects were given two doses at a 6-week interval of a serogroup B or control vaccine. Response was defined as > or = 4-fold rise in antibody level. After two doses of the Finlay Institute (Havana) vaccine at 12 months, the proportions of SBA and ELISA responders were not different from those of the control group (15% and 17% [vaccine] vs. 13% and 9% [control], P > .05). After two doses of the National Institute of Public Health (Oslo) vaccine, there were more SBA and ELISA responders than in the control group (47% and 34% [vaccine] vs. 10% and 1% [control]) or the Finlay Institute vaccine group (P < .05 for both). SBA and ELISA may be insensitive correlates for protective efficacy for some outer membrane protein-based serogroup B meningococcal vaccines.

Competition among Streptococcus pneumoniae for intranasal colonization in a mouse model

Widespread use of conjugate vaccines against Streptococcus pneumoniae, by reducing carriage of S. pneumoniae serotypes included in the vaccine, may result in an increase in nasopharyngeal carriage of - and disease from - nonvaccine serotypes of the same species. Mathematical models predict that the extent of such replacement will depend positively on the degree to which carriage of vaccine-type S. pneumoniae inhibits acquisition of nonvaccine-type pneumococci, and may depend negatively on the inhibition of vaccine-type pneumococci by nonvaccine-type pneumococci. We used a mouse model of intranasal carriage of pneumococci to test whether such inhibition occurs between different pneumococcal strains. Mice carrying a streptomycin-resistant derivative of S. pneumoniae BG9163 (serotype 6B) as a resident strain showed reduced levels of colonization when challenged intranasally by optochin-resistant derivatives of the same strain and of a serotype 23F pneumococcus, BG8826. Inhibition could be overcome by increasing the dose of the challenge strain. Carriage of optochin-resistant BG9163 did not inhibit acquisition of the streptomycin-resistant variant. Colonization by a challenge strain did not significantly affect the level of colonization with the resident strain. These results provide evidence that is consistent with several hitherto untested assumptions of mathematical models of serotype replacement and suggest that a biological mechanism exists that could account for serotype replacement that is observed in clinical trials. The findings provide a basis for further studies of in vivo interactions between strains of S. pneumoniae.

Correlation of Opsonophagocytosis and Passive Protection Assays Using Human Anticapsular Antibodies in an Infant Mouse Model of Bacteremia for Streptococcus pneumoniae

An infant mouse assay system for assessment of protective concentrations of human serum pneumococcal anticapsular antibodies is described. Passive immunization of anticapsular antibodies was evaluated for protection of infant mice challenged with Streptococcus pneumoniae serotypes 1, 4, 5, 6B, 18C, and 23A, with bacteremia as an end point. Protection was defined as no detectable bacteremia in 70% of mice 48 h after challenge. Type-specific anticapsular concentrations required for protection varied with serotype (</=0.05 to >0.4 microg/mL). Across serotypes, there was no significant correlation between human IgG concentration in mouse serum and protection from bacteremia or between IgG concentration and opsonophagocytic titer. Significant correlation (r=.84, P<.001) was observed between opsonophagocytic titer of human IgG antibody in mouse sera and protection from bacteremia. Thus, protective concentrations of anticapsular antibodies against bacteremia are serotype dependent. Opsonophagocytosis is a better predictor of in vivo protective capacity of pneumococcal anticapsular antibodies than are ELISA IgG antibody concentrations.

A Novel Botulinum Neurotoxin, Previously Reported as Serotype H, Has a Hybrid-Like Structure With Regions of Similarity to the Structures of Serotypes A and F and Is Neutralized With Serotype A Antitoxin

Botulism is a potentially fatal paralytic disease caused by the action of botulinum neurotoxin (BoNT) on nerve cells. There are 7 known serotypes (A-G) of BoNT and up to 40 genetic variants. Clostridium botulinum strain IBCA10-7060 was recently reported to produce BoNT serotype B (BoNT/B) and a novel BoNT, designated as BoNT/H. The BoNT gene (bont) sequence of BoNT/H was compared to known bont sequences. Genetic analysis suggested that BoNT/H has a hybrid-like structure containing regions of similarity to the structures of BoNT/A1 and BoNT/F5. This novel BoNT was serologically characterized by the mouse neutralization assay and a neuronal cell-based assay. The toxic effects of this hybrid-like BoNT were completely eliminated by existing serotype A antitoxins, including those contained in multivalent therapeutic antitoxin products that are the mainstay of human botulism treatment.

Inhibition of Pneumococcal Carriage in Mice by Subcutaneous Immunization with Peptides from the Common Surface Protein Pneumococcal Surface Adhesin A

Pneumococcal surface adhesin A (PsaA), a common protein expressed on all 90 pneumococcal serotypes, is a vaccine candidate. Three anti-PsaA monoclonal antibody phage display-expressed monopeptides (15 mers), in various formulations as lipidated or nonlipidated multiantigenic peptides or as bi- or tripeptide constructs, were studied in a mouse nasopharyngeal carriage model to determine the inhibitory effect of induced antibodies on carriage of pneumococcal serotypes 2, 4, and 6B. Antibodies to each of the various peptides tested reduced carriage of the 3 serotypes. Reduction in carriage by nonlipidated multiantigenic peptide antibodies was highly variable (39%-94%; mean, 59%; standard deviation [SD], 20.2%); however, more-consistent results were observed in mice immunized with lipidated (56%-98%; mean, 69%; SD, 13.6%) and combination or bipeptide (55%-91%; mean, 76%; SD, 13.1%) formulations. These peptides are immunogenic, and their induced antibodies reduce carriage in mice. PsaA peptides demonstrate potential for being important new vaccines against pneumococcal carriage, otitis media, and invasive pneumococcal disease.

Functional Characterization of Botulinum Neurotoxin Serotype H as a Hybrid of Known Serotypes F and A (BoNT F/A)

A unique strain of Clostridium botulinum (IBCA10-7060) was recently discovered which produces two toxins: botulinum neurotoxin (BoNT) serotype B and a novel BoNT reported as serotype H. Previous molecular assessment showed that the light chain (LC) of the novel BoNT most resembled the bont of the light chain of known subtype F5, while the C-terminus of the heavy chain (HC) most resembled the binding domain of serotype A. We evaluated the functionality of both toxins produced in culture by first incorporating an immunoaffinity step using monoclonal antibodies to purify BoNT from culture supernatants and tested each immune-captured neurotoxin with full-length substrates vesicle-associated membrane protein 2 (VAMP-2), synaptosomal-associated protein 25 (SNAP-25), syntaxin, and shortened peptides representing the substrates. The BoNT/B produced by this strain behaved as a typical BoNT/B, having immunoaffinity for anti-B monoclonal antibodies and cleaving both full length VAMP-2 and a peptide based on the sequence of VAMP-2 in the expected location. As expected, there was no activity toward SNAP-25 or syntaxin. The novel BoNT demonstrated immunoaffinity for anti-A monoclonal antibodies but did not cleave SNAP-25 as expected for BoNT/A. Instead, the novel BoNT cleaved VAMP-2 and VAMP-2-based peptides in the same location as BoNT/F5. This is the first discovery of a single botulinum neurotoxin with BoNT/A antigenicity and BoNT/F light chain function. This work suggests that the newly reported serotype H may actually be a hybrid of previously known BoNT serotype A and serotype F, specifically subtype F5.

First Report Worldwide of an Infant Botulism Case Due to Clostridium botulinum Type E

ABSTRACT Clostridium botulinum type E has been associated with botulism in adults but never in infants. Infant botulism type E cases have been associated with neurotoxigenic strains of C. butyricum. We report the first infant botulism case due to C. botulinum type E worldwide.

Utility of Botulinum Toxin ELISA A, B, E, F kits for clinical laboratory investigations of human botulism

The Botulinum Toxin ELISA effectively provided presumptive identification of toxin in 1381 investigation samples including clinical specimens, suspect foods, and cultures. Additionally, the ELISA detected all toxins produced by a panel of stock strains representing known subtypes and was negative for non-botulinum toxin producing Clostridium and enteric pathogens. ELISA results were reproducible both within the same kit (CV < 9%) and among different production lots (CV < 23%). Fifty-five of 57 laboratories correctly identified unknown samples in a multi-laboratory study. The ELISA provides a rapid, robust in vitro screening method which will reduce animal dependence during laboratory investigations of botulism.

Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain

Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes, serotypes A to G, based upon their selective neutralization by homologous antiserum, which is relevant for clinical and diagnostic purposes. Even though supportive care dramatically reduces the death rate of botulism, the only pharmaceutical intervention to reduce symptom severity and recovery time is early administration of antitoxin (antiserum raised against BoNTs). A recent report of a novel BoNT serotype, serotype H, raised concern of a “treatment-resistant” and highly potent toxin. However, the toxin’s chimeric structure and characteristics indicate a chimeric BoNT/FA. Here we describe the first characterization of this novel toxin in purified form. BoNT/FA was neutralized by available antitoxins, supporting classification as BoNT/FA. BoNT/FA required proteolytic activation to achieve full toxicity and had relatively low potency in mice compared to BoNT/A1 but surprisingly high activity in cultured neurons. ABSTRACT Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their toxicity in mice by homologous antibodies. Recently, a report of a potential eighth serotype of BoNT, designated “type H,” has been controversial. This novel BoNT was produced together with BoNT/B2 in a dual-toxin-producing Clostridium botulinum strain. The data used to designate this novel toxin as a new serotype were derived from culture supernatant containing both BoNT/B2 and novel toxin and from sequence information, although data from two independent laboratories indicated neutralization by antibodies raised against BoNT/A1, and classification as BoNT/FA was proposed. The sequence data indicate a chimeric structure consisting of a BoNT/A1 receptor binding domain, a BoNT/F5 light-chain domain, and a novel translocation domain most closely related to BoNT/F1. Here, we describe characterization of this toxin purified from the native strain in which expression of the second BoNT (BoNT/B) has been eliminated. Mass spectrometry analysis indicated that the toxin preparation contained only BoNT/FA and confirmed catalytic activity analogous to that of BoNT/F5. The in vivo mouse bioassay indicated a specific activity of this toxin of 3.8 × 107 mouse 50% lethal dose (mLD50) units/mg, whereas activity in cultured human neurons was very high (50% effective concentration [EC50] = 0.02 mLD50/well). Neutralization assays in cells and mice both indicated full neutralization by various antibodies raised against BoNT/A1, although at 16- to 20-fold-lower efficiency than for BoNT/A1. IMPORTANCE Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes, serotypes A to G, based upon their selective neutralization by homologous antiserum, which is relevant for clinical and diagnostic purposes. Even though supportive care dramatically reduces the death rate of botulism, the only pharmaceutical intervention to reduce symptom severity and recovery time is early administration of antitoxin (antiserum raised against BoNTs). A recent report of a novel BoNT serotype, serotype H, raised concern of a “treatment-resistant” and highly potent toxin. However, the toxin’s chimeric structure and characteristics indicate a chimeric BoNT/FA. Here we describe the first characterization of this novel toxin in purified form. BoNT/FA was neutralized by available antitoxins, supporting classification as BoNT/FA. BoNT/FA required proteolytic activation to achieve full toxicity and had relatively low potency in mice compared to BoNT/A1 but surprisingly high activity in cultured neurons.

Detection of Botulinum Toxins A, B, E, and F in Foods by Endopep-MS

Botulism is caused by exposure to botulinum neurotoxins (BoNTs). BoNTs are proteins secreted by some species of clostridia; these neurotoxins are known to interfere with nerve impulse transmission, thus causing paralysis. Botulism may be contracted through consumption of food either naturally or intentionally contaminated with BoNT. The human lethal dose of BoNT is not known but is estimated to be between 0.1 and 70 μg; thus, it is important to be able to detect small amounts of this toxin in foods to ensure food safety and to identify the source of an outbreak. Our laboratory previously reported on the development of Endopep-MS, a mass-spectrometric-based endopeptidase method for the detection and differentiation of BoNT. This method can detect BoNT at levels below the historic standard mouse bioassay in clinical samples such as serum, stool, and culture supernatants. We have now expanded this assay to detect BoNT in over 50 foods including representative products that were involved in actual botulism investigations. The foods tested by the Endopep-MS included those with various acidities, viscosities, and fat levels. Dairy and culturally diverse products were also included. This work demonstrates that the Endopep-MS method can be used to detect BoNT/A, /B, /E, and /F in foods at levels spiked below that of the limit of detection of the mouse bioassay. Furthermore, we successfully applied this method to investigate several foods associated with botulism outbreaks.