Morly Fisher

Protective Antigen as a Correlative Marker for Anthrax in Animal Models

ABSTRACT The most aggressive form of anthrax results from inhalation of airborne spores of Bacillus anthracis and usually progresses unnoticed in the early stages because of unspecific symptoms. The only reliable marker of anthrax is development of bacteremia, which increases with disease progress. Rapid diagnosis of anthrax is imperative for efficient treatment and cure. Herein we demonstrate that the presence and level of a bacterial antigen, the protective antigen (PA), a component of B. anthracis toxins, in host sera can serve as a reliable marker of infection. This was tested in two animal models of inhalation anthrax, rabbits and guinea pigs infected by intranasal instillation of Vollum spores. In both models, we demonstrated qualitative and quantitative correlations between levels of bacteremia and PA concentrations in the sera of sick animals. The average time to death in infected animals was about 16 h after the appearance of bacteremia, leaving a small therapeutic window. As the time required for immunodetection of PA can be very short, the use of this marker will be beneficial for faster diagnosis and treatment of inhalation anthrax.

Shr Is a Broad-Spectrum Surface Receptor That Contributes to Adherence and Virulence in Group A Streptococcus

ABSTRACT Group A streptococcus (GAS) is a common hemolytic pathogen that produces a range of suppurative infections and autoimmune sequelae in humans. Shr is an exported protein in GAS, which binds in vitro to hemoglobin, myoglobin, and the hemoglobin-haptoglobin complex. We previously reported that Shr is found in association with whole GAS cells and in culture supernatant. Here, we demonstrate that cell-associated Shr could not be released from the bacteria by the muralytic enzyme mutanolysin and was instead localized to the membrane. Shr was available, however, on the exterior of GAS, exposed to the extracellular environment. In vitro binding and competition assays demonstrated that in addition to hemoprotein binding, purified Shr specifically interacts with immobilized fibronectin and laminin. The absence of typical fibronectin-binding motifs indicates that a new protein pattern is involved in the binding of Shr to the extracellular matrix. Recombinant Lactococcus lactis cells expressing Shr on the bacterial surface gained the ability to bind to immobilized fibronectin, suggesting that Shr can function as an adhesin. The inactivation of shr resulted in a 40% reduction in the attachment to human epithelial cells in comparison to the parent strain. GAS infection elicited a high titer of Shr antibodies in sera from convalescent mice, demonstrating that Shr is expressed in vivo. The shr mutant was attenuated for virulence in an intramuscular zebrafish model system. In summary, this study identifies Shr as being a new microbial surface component recognizing adhesive matrix molecules in GAS that mediates attachment to epithelial cells and contributes to the infection process.

Defense From the Group A Streptococcus by Active and Passive Vaccination With the Streptococcal Hemoprotein Receptor

BACKGROUND The worldwide burden of the Group A Streptococcus (GAS) primary infection and sequelae is considerable, although immunization programs with broad coverage of the hyper variable GAS are still missing. We evaluate the streptococcal hemoprotein receptor (Shr), a conserved streptococcal protein, as a vaccine candidate against GAS infection. METHODS Mice were immunized intraperitoneally with purified Shr or intranasally with Shr-expressing Lactococcus lactis. The resulting humoral response in serum and secretions was determined. We evaluated protection from GAS infection in mice after active or passive vaccination with Shr, and Shr antiserum was tested for bactericidal activity. RESULTS A robust Shr-specific immunoglobulin (Ig) G response was observed in mouse serum after intraperitoneal vaccination with Shr. Intranasal immunization elicited both a strong IgG reaction in the serum and a specific IgA reaction in secretions. Shr immunization in both models allowed enhanced protection from systemic GAS challenge. Rabbit Shr antiserum was opsonizing, and mice that were administrated with Shr antiserum prior to the infection demonstrated a significantly higher survival rate than did mice treated with normal rabbit serum. CONCLUSIONS Shr is a promising vaccine candidate that is capable of eliciting bactericidal antibody response and conferring immunity against systemic GAS infection in both passive and active vaccination models.

Involvement of TLR2 in innate response to Bacillus anthracis infection.

The involvement of TLR2 receptor in the innate response to infection with Bacillus anthracis was investigated. We studied the response to virulent or attenuated Vollum strains in either in vitro assays using macrophage cultures, or in an in vivo model comparing the sensitivity of Syrian hamster cells (expressing normal TLR2) to Chinese hamster cells (lacking functional TLR2) to infection by the various B. anthracis strains. Phagocytosis experiments with murine cell cultures or primary macrophages from both hamster strains, using virulent or attenuated Tox+Cap -, Tox-Cap+ or Tox-Cap- spores indicated that the secretion of TNF-α was induced by all the bacterial spores and purified spore antigens. In contrast, capsular antigens induce secretion of TNF-α only by Syrian hamster macrophages indicating the involvement of a functional TLR2 in macrophage activation. Challenge experiments with both hamster strains by intranasal spore inoculation, indicated that, while both strains are equally sensitive to infection with the virulent strain, the Chinese hamster demonstrated a higher sensitivity to infection with the toxinogenic or encapsulated strains. In conclusion, our findings imply that TLR2 has an important role in the attempt of the innate immunity to control B. anthracis infection, although TNF-α secretion was found to be mediated by both TLR2-dependent and TLR2-independent pathways.

The search for early markers of plague: evidence for accumulation of soluble Yersinia pestis LcrV in bubonic and pneumonic mouse models of disease.

Markers of the early stages of plague, a rapidly progressing deadly disease, are crucial for enabling the onset of an effective treatment. Here, we show that V-antigen protein (LcrV) is accumulated in the serum of Yersinia pestis-infected mice before bacterial colonization of the spleen and dissemination to blood, in a model of bubonic plague. LcrV accumulation is detected earlier than that of F1 capsular antigen, an established marker of disease. In a mouse model of pneumonic plague, LcrV can be determined in the bronchoalveolar lavage fluid somewhat later than F1, but before dissemination of Y. pestis to the blood. Thus, determination of soluble LcrV is suggested as a potential useful tool for monitoring disease progression in both bubonic and pneumonic plague. Moreover, it may be of particular advantage in cases of infections with F1 nonproducing strains.

Highly Stable Lyophilized Homogeneous Bead-Based Immunoassays for On-Site Detection of Bio Warfare Agents from Complex Matrices

This study shows the development of dry, highly stable immunoassays for the detection of bio warfare agents in complex matrices. Thermal stability was achieved by the lyophilization of the complete, homogeneous, bead-based immunoassay in a special stabilizing buffer, resulting in a ready-to-use, simple assay, which exhibited long shelf and high-temperature endurance (up to 1 week at 100 °C). The developed methodology was successfully implemented for the preservation of time-resolved fluorescence, Alexa-fluorophores, and horse radish peroxidase-based bead assays, enabling multiplexed detection. The multiplexed assay was successfully implemented for the detection of Bacillus anthracis, botulinum B, and tularemia in complex matrices.

Simultaneous Immunodetection of Anthrax, Plague, and Tularemia from Blood Cultures by Use of Multiplexed Suspension Arrays

ABSTRACT Multiplexed detection technologies are becoming increasingly important given the possibility of bioterrorism attacks, for which the range of suspected pathogens can vary considerably. In this work, we describe the use of Luminex MagPlex magnetic microspheres for the construction of two multiplexed diagnostic suspension arrays, enabling antibody-based detection of bacterial pathogens and their related disease biomarkers directly from blood cultures. The first 4-plex diagnostic array enabled the detection of both anthrax and plague infections using soluble disease biomarkers, including protective antigen (PA) and anthrax capsular antigen for anthrax detection and the capsular F1 and LcrV antigens for plague detection. The limits of detection (LODs) ranged between 0.5 and 5 ng/ml for the different antigens. The second 2-plex diagnostic array facilitated the detection of Yersinia pestis (LOD of 1 × 106 CFU/ml) and Francisella tularensis (LOD of 1 × 104 CFU/ml) from blood cultures. Inoculated, propagated blood cultures were processed (15 to 20 min) via 2 possible methodologies (Vacutainer or a simple centrifugation step), allowing the direct detection of bacteria in each sample, and the entire assay could be performed in 90 min. While detection of bacteria and soluble markers from blood cultures using PCR Luminex suspension arrays has been widely described, to our knowledge, this study is the first to demonstrate the utility of the Luminex system for the immunodetection of both bacteria and soluble markers directly from blood cultures. Targeting both the bacterial pathogens as well as two different disease biomarkers for each infection, we demonstrated the benefit of the multiplexed developed assays for enhanced, reliable detection. The presented arrays could easily be expanded to include antibodies for the detection of other pathogens of interest in hospitals or labs, demonstrating the applicability of this technology for the accurate detection and confirmation of a wide range of potential select agents.

A Rapid Method for Bacillus anthracis Genotyping

Classification and differentiation of Bacillus anthracis isolates by genetic markers play an important role in the study of anthrax epidemiology. We have applied a PCR based method – Random Amplification of Polymorphic DNA (RAPD) to identify twenty-five B. anthracis genetic markers. These markers allowed for classification of the studied strains into five different groups. Three selected RAPD markers were cloned and sequenced. Typical integration of the three markers allowed for specific definition of the five RAPD derived genotypes. To test the universal power of these markers to discriminate between diverse B. anthracis strains, the nucleotide sequence of each marker was searched against all available B. anthracis genome sequences (both finished and unfinished). The three markers system could differentiate between strains belonging to the genetic groups Aβ, A1a, A1b, A4 and B1 (as defined by Keim et al., 2000; Maho et al., 2006) and gave rise to a unique combination for group A3, but couldn’t distinguish between the sub groups A3a and A3b. In addition, this system could not distinguish between groups B2 and C (identical three marker combination). In an attempt to improve the resolution of this system we introduced a fourth marker. In silico analysis revealed that the resulting four markers system could now differentiate between group B2 and C, adding a second genotype to groups A1a, A3a and A4. This four marker system could potentially provide an accurate, simple, and inexpensive agarose-based system for classification B. anthracis strains in laboratories involved in research of this bacterium.