Defining a serological correlate of protection in rabbits for a recombinant anthrax vaccine

In these studies, a serological correlate of protection against anthrax was identified in New Zealand white (NZW) rabbits that had been given one or two injections of various amounts of recombinant protective antigen (rPA) combined with aluminum hydroxide adjuvant (Alhydrogel). Rabbits were subsequently challenged by the aerosol route with spores of the Ames isolate of Bacillus anthracis. Results suggested that the antibody response, as determined by the quantitative anti-rPA IgG ELISA and toxin neutralizing antibody (TNA) assay, were significant predictors (P<0.0015) of protection against a B. anthracis aerosol spore challenge in rabbits.     

An intranasal vaccine targeting both the Bacillus anthracis toxin and bacterium provides protection against aerosol spore challenge in rabbits

An intranasal vaccine targeting the Bacillus anthracis toxin and vegetative bacterium was tested for the ability to protect immunized rabbits against aerosol B. anthracis spore exposure. Rabbits were vaccinated intranasally with PA-based vaccines formulated as dry powders with or without chitosan (ChiSys, Archimedes Development Limited), a compound that exhibits muco-adhesive properties, or as a liquid. Formulations also contained MPL adjuvant and PA. Some vaccines contained PA conjugated to a 10-mer peptide of the poly-d-glutamic acid capsule of B. anthracis. Rabbits were immunized on days 0 and 28 and aerosol challenged with an average 250LD50 Ames spores on day 85. Serum antibody was measured before and after challenge. Significant anti-PA serum IgG levels were obtained, particularly with use of ChiSys based formulations. PA-Conj induced significant anti-capsule responses, although a formulation containing free capsule peptide did not. All immunized rabbits survived the challenge, but differences in morbidity, as evidenced by anorexia, between vaccine groups were observed. Only rabbits immunized with PA+PA-Conj appeared normal throughout the post-challenge observation period (14 days), while all that received PA with the free capsule peptide appeared ill at times as evidenced by a failure to eat normally. One negative control rabbit received a lower inhaled spore dose (183LD50) and survived the challenge, although it was anorexic post-challenge. It also had a high level of anti-LF antibodies in its convalescent serum (5400 U/ml), indicating an extensive infection. In contrast, 75% of the immunized rabbits had no LF-specific antibody in their post-challenge sera, and the rest had low levels (< or = 138 U/ml), indicating that infections resulting in toxin production were avoided or greatly reduced. Thus, intranasal immunization with a chitosan-based powder vaccine combining PA and capsule epitopes provided superior protection against B. anthracis infection compared to a single antigen (PA) vaccine, as evidenced by a reduction in morbidity and prevention of death.     

Recombinant Bacillus anthracis spore proteins enhance protection of mice primed with suboptimal amounts of protective antigen

Inactivated Bacillus anthracis spores given with protective antigen (PA) contribute to immunity against anthrax in several animal models. Antiserum raised against whole irradiated B. anthracis spores has been shown to have anti-germination and opsonic activities in vitro. Based on these observations, we hypothesized that surface-exposed spore proteins might serve as supplemental components of a PA-based anthrax vaccine. The protective anti-spore serum was tested for reactivity with recombinant forms of 30 proteins known, or believed to be, present within the B. anthracis exosporium. Eleven of those proteins were reactive with this antiserum, and, subsequently a subset of this group was used to generate rabbit polyclonal antibodies. These sera were evaluated for recognition of the immunogens on intact spores generated from Sterne strain, as well as from an isogenic mutant lacking the spore surface protein Bacillus collagen-like antigen (BclA). The data were consistent with the notion that the antigens in question were located beneath BclA on the basal surface of the exosporium. A/J mice immunized with either the here-to-for hypothetical protein p5303 or the structural protein BxpB, each in combination with subprotective levels of PA, showed enhanced protection against subcutaneous spore challenge. While neither anti-BxpB or anti-p5303 antibodies reduced the rate of spore germination in vitro, both caused increased uptake and lead to a higher rate of destruction by phagocytic cells. We conclude that by facilitating more efficient phagocytic clearance of spores, antibodies against individual exosporium components can contribute to protection against B. anthracis infection.     

Simple and rapid method for detection of bacterial spores in powder useful for first responders

The need for a rapid method by which first responders can screen for the presence of spores in powder samples has been increased since the anthrax attack of 2001. The majority of powders that were sampled in the context of that attack were hoaxes and did not contain Bacillus anthracis. The large number of samples overwhelmed the analysis capacity of public health laboratories. A rapid screening method for determining the presence of viable spores would eliminate much laboratory work and expedite procedures for identification if spores were detected. In the study reported here, Bacillus thuringiensis was used as a surrogate for B. anthracis to investigate if heat shock followed by luminescence analysis would allow rapid quantification of viable spores. The aim of the study was to investigate the best and fastest heat shock conditions that would trigger the breakdown of endospore dormancy of Bacillus species and give a higher luminescence signal. Heat shock and luminescence do not identify the type of spores in a powder sample, but they can detect the presence of viable spores in near real time. Different types of germinant were tested at different conditions (germination duration, temperature, concentration). The rapid method was compared with the traditional plate count method. The results showed that the rapid method can be used as a rapid (< 15 min), sensitive (< 100 spores), and inexpensive detection technique. The rapid method can be applied on site when suspicious powder is found and enables decision making for further identification.     

Protecting building environments from airborne chemical,biologic, or radiologic attacks

In November 2001, following the discovery that letters containing Bacillus anthracis had been mailed to targeted locations in the United States, the Secretary of the U.S. Department of Health and Human Services requested site assessments of an array of public- and private-sector buildings by a team of engineers and scientists from CDC's National Institute for Occupational Safety and Health (NIOSH). In November 2001, this team assessed six buildings, including a large hospital and medical research facility, a museum, a transportation building, two large office buildings, and an office/laboratory building. In January 2002, additional building assessments were conducted at CDC campuses in Atlanta and, in April 2002, at a large, urban transportation facility. A total of 59 buildings were evaluated during this 5-month period.     

Efficacy of a human anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin

The efficacy of a licensed human anthrax vaccine (Anthrax Vaccine Adsorbed (AVA)) was tested in guinea pigs, rabbits, and rhesus macaques against spore challenge by Bacillus anthracis isolates of diverse geographical origin. Initially, groups of Hartley guinea pigs were vaccinated at 0 and 4 weeks with AVA, then challenged intramuscularly at 10 weeks with spores from 33 isolates of B. anthracis. Survival among the vaccinated groups varied from 6 to 100%, although there were no differences in mean time to death among the groups. There was no correlation between isolate virulence and variable number tandem repeat category or protective antigen genotype identified. New Zealand white rabbits were then vaccinated with AVA at 0 and 4 weeks, and challenged at 10 weeks by aerosol with spores from six of the isolates that were highly virulent in vaccinated guinea pigs. AVA completely protected the rabbits from four of the isolates, and protected 90% of the animals from the other two isolates. Subsequently, two of these six isolates were then used to challenge rhesus macaques, previously vaccinated with AVA at 0 and 4 weeks, and challenged at 10 weeks by aerosol. AVA protected 80 and 100% of the animals from these two isolates. These studies demonstrated that, although AVA confers variable protection against different B. anthracis isolates in guinea pigs, it is highly protective against these same isolates in both rabbits and rhesus macaques.     

A single immunization with a dry powder anthrax vaccine protects rabbits against lethal aerosol challenge

Here we confirm that intranasal (IN) dry powder anthrax vaccine formulations are able to protect rabbits against aerosol challenge 9 weeks after a single immunization. The optimum dose of rPA in our dry powder anthrax vaccine formulation in rabbits was experimentally determined to be 150microg and therefore was chosen as the target dose for all subsequent experiments. Rabbits received a single dose of either 150microg rPA, 150microg rPA+150microg of a conjugated 10-mer peptide representing the Bacillus anthracis capsule (conj), or 150microg of conj alone. All dry powder formulations contained MPL and chitosan (ChiSys). Significant anti-rPA titers and anthrax lethal toxin neutralizing antibody (TNA) levels were seen with both rPA containing vaccines, although rPA-specific IgG and TNA levels were reduced in rabbits immunized with rPA plus conj. Nine weeks after immunization, rabbits were exposed to a mean aerosol challenge dose of 278 LD50 of Ames spores. Groups immunized with rPA or with rPA+conj had significant increases in survivor proportions compared to the negative control group by Logrank test (p=0.0001 and 0.003, respectively), and survival was not statistically different for the rPA and rPA+conj immunized groups (p=0.63). These data demonstrate that a single immunization with our dry powder anthrax vaccine can protect against a lethal aerosol spore challenge 9 weeks later.     

Efficacy and durability of Bacillus anthracis bacteriophages used against spores

Antibiotics and vaccines help fight anthrax disease, but there are no anthrax spore control methods suitable for use in environments where humans are present. The work reported in this article indicates that bacteriophages may help reduce risk from anthrax spores. Dose-response studies demonstrated that higher concentrations of mixed Bacillus anthracis bacteriophages (3.5 x 10(8) plaque-forming units per milliliter) inhibited subsequent growth of bacteria when sprayed on B. anthracis spores. Phages also were tested for durability under conditions designed to simulate environments possibly encountered during mass phage production, storage, and use against anthrax spores. They remained infectious at temperatures from -20 degrees C to 37 degrees C, under filtration, aerosolization, and treatments with perspiration and blood. Phages were sensitive to temperatures over 55 degrees C and to desiccation. Ultraviolet light reduced spore viability more than phage infectivity under similar conditions. The potential for personal or environmental decontamination of anthrax spores with phages is discussed.     

Inadvertent laboratory exposure to Bacillus anthracis--California, 2004

On June 9, 2004, the California Department of Health Services (CDHS) was notified of possible inadvertent exposure to Bacillus anthracis spores at Children's Hospital Oakland Research Institute (CHORI), where workers were evaluating the immune response of mice to B. anthracis. This report summarizes the subsequent investigation by CDHS and CDC, including assessment of exposures, administration of postexposure chemoprophylaxis, and serologic testing of potentially exposed workers. The findings underscore the importance of using appropriate biosafety practices and performing adequate sterility testing when working with material believed to contain inactivated B. anthracis organisms.     

Minimizing pathogenic bacteria, including spores, in indoor air

Five experiments were conducted to assess whether aerosolized bacteria, including spores, respond like particulate contaminants to the primary (electrical) forces that control the distribution of small particulate contaminants in indoor air. Such response would suggest an approach to minimizing infection in offices, hospitals, nursing homes, and other facilities. It also would have implications for protection against intentionally introduced pathogenic bacteria, including spores. The experiments used two different genera and five different strains of bacteria, including spores. Micrococcus luteus was used as a surrogate for Gram-positive cocci, because M. luteus is similar in size, shape, and cell wall composition to staphylococci, streptococci, and enterococci. Similarly, spore-forming and vegetative Bacillus subtilis were used as surrogates for Gram-positive bacilli such as Bacillus anthracis. The experiments were conducted in a dedicated aerosol physics test facility with culture-based measurements made at timed intervals. The results indicate that the organisms do respond like particulate contaminants to typical electrical forces in a room.     

Assessment of electrical charge on airborne microorganisms by a new bioaerosol sampling method

Bioaerosol sampling is necessary to monitor and control human exposure to harmful airborne microorganisms. An important parameter affecting the collection of airborne microorganisms is the electrical charge on the microorganisms. Using a new design of an electrostatic precipitator (ESP) for bioaerosol sampling, the polarity and relative strength of the electrical charges on airborne microorganisms were determined in several laboratory and field environments by measuring the overall physical collection efficiency and the biological collection efficiency at specific precipitation voltages and polarities. First, bacteria, fungal spores, and dust dispersed from soiled carpets were sampled in a walk-in test chamber. Second, a simulant of anthrax-causing Bacillus anthracis spores was dispersed and sampled in the same chamber. Third, bacteria were sampled in a small office while four adults were engaged in lively discussions. Fourth, bacteria and fungal spores released from hay and horse manure were sampled in a horse barn during cleanup operations. Fifth, bacteria in metalworking fluid droplets were sampled in a metalworking simulator. It was found that the new ESP differentiates between positively and negatively charged microorganisms, and that in most of the tested environments the airborne microorganisms had a net negative charge. This adds a signature to the sampled microorganisms that may assist in their identification or differentiation, for example, in an anti-bioterrorism network.     

Anthrax--continuous threat to humans and animals

Gram-positive, spore-forming, aerobic bacterium Bacillus anthracis is an etiological agent of anthrax a disease very dangerous to humans and all warm-blooded animals. The spore forms are markedly resistant to unfavourable environmental extremes of heat, cold, desiccation, chemicals, irradiation etc. The vegetative forms characterised virulence factors: the antiphagocytic poly-gamma-D-polipeptide capsule and three proteins, edema factor (EF), lethal factor (LF) and protective antigen (PA). Anthrax is mainly transmitted from animals to man through food of animal origin, animal products and contamination of the environment with B. anthracis and its spores. There are three types of this disease: cutaneous, intestinal and inhalation anthrax. Research on anthrax as a biological weapon began more then 80 years ago. Depending on the target chosen and the scale of the attack the anthrax spores may by used to contaminate of foodstuffs or liquids and water. The aerosolised release of anthrax spore can cause illness with a high fatality rate.     

Immunization against anthrax using Bacillus subtilis spores expressing the anthrax protective antigen

Protective immunity to anthrax can be achieved by antibodies raised against the secreted protective antigen (PA) and this forms the basis of the current acellular vaccines for human use. Bacillus subtilis spores have previously been used for delivery of heterologous antigens by the oral and nasal routes and their intrinsic heat-stability make them attractive vaccine vehicles. In this study we have expressed PA, or segments of PA, in B. subtilis using two strategies. First, display on the spore coat, and second, in the germinated spore (or vegetative cell). Using parenteral delivery we show that recombinant spores can be used to confer protective immunity in a murine model using an in vitro toxin neutralization assay and a challenge experiment with the latter showing protection to 100 median lethal dose of B. anthracis spores. PA must be secreted from the live bacterium or alternatively displayed on the spore surface to confer protective immunity. Intracellular expression of PA failed to confer protective immunity. The highest levels of protective immunity were achieved when PA was displayed on the spore surface as well as in the germinating spore.     

二氧化氯消毒剂杀菌效果的实验观察

目的：通过实验室试验研究二氧化氯消毒剂杀灭枯草杆菌黑色变种芽孢、金黄色葡萄球菌和大肠杆菌的最低有效剂量。方法：依据《消毒技术规范》（2002年版）采用悬液定量试验法进行了实验室观察。结果：在3％和0．3％牛血清白蛋白条件下，二氧化氯含量分别为15.2mg／L，作用1min和10min对大肠杆菌平均杀灭对数值均〉5．00；在3％和0．3％牛血清白蛋白条件下，二氧化氯含量分别为20、6mg／L，作用1min对金黄色葡萄球菌平均杀灭对数值均〉5．00；在3％和o．3％牛血清白蛋白条件下，二氧化氯含量分别为100、60mg／L，作用5min对枯草杆菌黑色变种芽孢平均杀灭对数值均〉5．00。结论：实验室的结果略低于生产企业申报检测时的二氧化氯消毒剂作用浓度，在不同实验室有效剂量也有差异，可能由于二氧化氯生产原料和工艺不同所致，最终应以实验数据为准。     

Cytoplasmic delivery of antigens, by Bacillus subtilis enhances Th1 responses

Endospores of the Gram-positive bacterium, Bacillus subtilis, have been used successfully for delivery of antigens where the immunogen is expressed on the spore surface. In this work the spore has been engineered to deliver antigens to the cytoplasm of macrophages by expressing listeriolysin O (LLO) or a derivative, LLO(L461T), that is stable at neutral pH, from the B. subtilis vegetative cell. Following phagocytosis spores were shown to germinate in the phagosome enabling secretion of LLO/LLO(L461T) and entry of the bacterium into the cytosol. We have shown that in the cytosol B. subtilis proliferates before eventually being destroyed. Immunisation of mice with spores that co-expressed LLO with Protective Antigen (PA) of Bacillus anthracis generated an increase in IgG2a against PA, toxin-neutralising activity coupled with specific IFN-gamma and IL-12 (and reduced IL-4) responses of splenocytes, both indicative of an enhanced Th1 response. Enhanced Th1 responses via LLO co-expression of antigen by B. subtilis spores may be a useful strategy to improve vaccine performance.     

Anthrax vaccine efficacy in golden Syrian hamsters

The efficacy of a licensed human anthrax vaccine (anthrax vaccine adsorbed, AVA) was tested in golden Syrian hamsters against a virulent Bacillus anthracis spore challenge. Groups of golden Syrian hamsters were vaccinated at either 0 and 4 weeks or 0, 4 and 8 weeks, then challenged subcutaneously (s.c.) at 10 weeks with spores of various B. anthracis isolates. Although ELISA and toxin neutralization assays demonstrated high titers, none of the AVA-vaccinated hamsters were protected from challenge or demonstrated a significantly extended time to death compared to that of control animals. The results of the study demonstrate that the golden Syrian hamster is not an appropriate model for investigating human anthrax vaccine efficacy.     

Inactivation of Bacillus anthracis spores

After the intentional release of Bacillus anthracis through the U.S. Postal Service in the fall of 2001, many environments were contaminated with B. anthracis spores, and frequent inquiries were made regarding the science of destroying these spores. We conducted a survey of the literature that had potential application to the inactivation of B. anthracis spores. This article provides a tabular summary of the results.     

Genetic immunization against anthrax

The objective of this study was to determine whether a DNA prime-protein boost immunization against the Bacillus anthracis protective antigen (PA) and lethal factor (LF) antigens could induce a protective immune response against significant aerosol challenge in the rabbit model. Rabbits were vaccinated with different regimens of DNA vaccines (Table 1) and aerosol challenged with B. anthracis spores, Ames strain, with an average dose of 50 LD(50s) with a range from 18 to 169 LD(50s.) Of the five vaccinated rabbits that survived, two were immunized intramuscularly (i.m.) with DNA followed with a protein boost and three were immunized subcutaneous (s.q.) with recombinant protein. A major factor predicting survival was the ability of the animal to mount a lasting antibody response to PA. Rabbit sera were collected prior to and following aerosol challenge and titrated for PA antibodies by indirect ELISA. The results of this study indicate that DNA-based immunization against PA and LF induces significant protective immunity against aerosol challenge in the rabbit model and compares favorably with protein-based immunization.     

巢式PCR快速检测炭疽芽孢杆菌

目的：快速简便地检测炭疽芽孢杆菌。方法：细菌培养物经反复冻融，SDS，蛋白酶K和煮沸处理后，作为PCR模板，根据炭疽芽孢杆菌质粒pX01中水肿因子（EF）基因设计两对引物，采用巢式PCR（nestedPCR)扩增目的基因，结果：从炭疽芽孢杆菌模板中成功扩增出1247bp的特异片段，而未在炭疽芽孢杆菌无毒株，蜡样芽孢杆菌和枯草杆菌模板中扩增出相应条带；第一次扩增能检出的最低细菌量是10^3拷贝；经再次巢式PCR，扩增出208bp的特异片段，最低检出的细菌数为10个拷贝，敏感性提高了100倍。结论：巢式PCR是一种快速，特异，敏感的检测炭疽芽孢杆菌的方法。     

Opening a bacillus anthracis-containing envelope,Capitol Hill,Washington, D.C.: the public health response

On October 15, 2001, a U.S. Senate staff member opened an envelope containing Bacillus anthracis spores. Chemoprophylaxis was promptly initiated and nasal swabs obtained for all persons in the immediate area. An epidemiologic investigation was conducted to define exposure areas and identify persons who should receive prolonged chemoprophylaxis, based on their exposure risk. Persons immediately exposed to B. anthracis spores were interviewed; records were reviewed to identify additional persons in this area. Persons with positive nasal swabs had repeat swabs and serial serologic evaluation to measure antibodies to B. anthracis protective antigen (anti-PA). A total of 625 persons were identified as requiring prolonged chemoprophylaxis; 28 had positive nasal swabs. Repeat nasal swabs were negative at 7 days; none had developed anti-PA antibodies by 42 days after exposure. Early nasal swab testing is a useful epidemiologic tool to assess risk of exposure to aerosolized B. anthracis. Early, wide chemoprophylaxis may have averted an outbreak of anthrax in this population.