Graham J. Hatch

Protective Effect of a Tuberculosis Subunit Vaccine Based on a Fusion of Antigen 85B and ESAT-6 in the Aerosol Guinea Pig Model

ABSTRACT A fusion protein of antigen 85B (Ag85B) and ESAT-6 administered in cationic lipid vesicles conferred a highly significant level of protection against Mycobacterium tuberculosis in the guinea pig aerosol model of infection. The protection was manifested as delayed clinical illness and prolonged survival. Neither Ag85B nor ESAT-6 (independently or as a cocktail) induced significant protection in this model.

Boosting with Poxviruses Enhances Mycobacterium bovis BCG Efficacy against Tuberculosis in Guinea Pigs

ABSTRACT Tuberculosis is rising in the developing world due to poor health care, human immunodeficiency virus type 1 infection, and the low protective efficacy of the Mycobacterium bovis BCG vaccine. A new vaccination strategy that could protect adults in the developing world from tuberculosis could have a huge impact on public health. We show that BCG boosted by poxviruses expressing antigen 85A induced unprecedented 100% protection of guinea pigs from high-dose aerosol challenge with Mycobacterium tuberculosis, suggesting a strategy for enhancing and prolonging the efficacy of BCG.

Mycobacterium tuberculosis arabinomannan–protein conjugates protect against tuberculosis

Lipoarabinomannan (LAM) is a major structural surface component of mycobacteria. Arabinomannan (AM) oligosaccharides derived from LAM of Mycobacterium tuberculosis H37Rv were isolated and covalently conjugated to tetanus toxoid (TT) or to short-term culture filtrate proteins (antigen 85B (Ag85B) or a 75kDa protein) from M. tuberculosis strain Harlingen. The different AM oligosaccharide (AMOs)-protein conjugate vaccine candidates proved to be highly immunogenic, inducing boosterable IgG responses against the AMOs portion of the conjugates in rabbits and guinea-pigs. Proliferation of T-cells from C57BL/6 mice immunized with the conjugates was seen upon in vitro stimulation with PPD. In C57BL/6 mice subcutaneous immunization with the AMOs-antigen 85B conjugate in alum provided significant protection compared to sham (alum only) immunized mice (P < 0.021) as estimated by long term survival against intravenous challenge with 10(5) M. tuberculosis H37Rv. Subcutaneous immunization followed by nasal boost with an AMOs-TT conjugate in Eurocine L3 adjuvant provided high (P < 0.025) protection as determined by long term survival after intranasal challenge with 10(5) virulent M. tuberculosis strain Harlingen. This level of protection was comparable to that obtained with the conventional live attenuated BCG vaccine. In guinea-pigs, immunization with AMOs-Ag85B in Eurocine L3 adjuvant followed by aerogenic challenge with M. tuberculosis H37Rv resulted in increased survival and reduced pathology in lungs and spleens relative to non-immunized animals.

Determination of lesion volume by MRI and stereology in a macaque model of tuberculosis

Sensitive and reproducible methods are needed to measure the impact on the host following experimental challenge with Mycobacterium tuberculosis, in order to determine the degree of protection conferred by new vaccines. Here we compare how well different clinical and post-mortem measures of disease burden predict the response by the host to increasing doses of M. tuberculosis in rhesus and cynomolgus macaques. The total lung and lesion volume was quantified from magnetic resonance imaging (MRI) digital stacks obtained from lungs of M. tuberculosis infected animals that were formalin fixed and scanned ex-vivo. The total lung lesion volume relative to the fixed whole lung volume was superior at indicating disease burden when compared to thoracic radiography, pathology scores, changes in body weight and temperature, as well as erythrocyte haemoglobin concentrations and sedimentation rate. The total lesion volume accurately reflected differences in challenge doses of M. tuberculosis that ranged from 30 to 500 CFU delivered by aerosol. The determination of total lesion volume from MR images demonstrated a species-dependent difference between rhesus and cynomolgus macaques in susceptibility to M. tuberculosis infection. MR stereology provides an accurate, quantifiable and relatively simple assessment, which can be easily standardized between laboratories and should form an essential component of the clinical assessment of disease progression, or vaccine efficacy.

Vaccination of Guinea Pigs with DNA Encoding the Mycobacterial Antigen MPB83 Influences Pulmonary Pathology but Not Hematogenous Spread following Aerogenic Infection with Mycobacterium bovis

ABSTRACT Protection of cattle against bovine tuberculosis by vaccination could be an important control strategy in countries where there is persistent Mycobacterium bovis infection in wildlife and in developing countries where it is not economical to implement a tuberculin test and slaughter control program. The main aim of such a vaccination strategy would be to reduce transmission of infection by reducing the lung pathology caused by infection and preventing seeding of the organism to organs from which M. bovis could be excreted. Recent reports of successful DNA vaccination against Mycobacterium tuberculosis in small-animal models have suggested that DNA vaccines act by reducing lung pathology without sensitizing animals to tuberculin testing. We therefore evaluated the ability of vaccines consisting of DNA encoding the mycobacterial antigens MPB83 and 85A to reduce lung pathology and prevent hematogenous spread in guinea pigs challenged with a low dose of aerosolized M. bovis. Vaccination with MPB83 DNA reduced the severity of pulmonary lesions, as assessed by histopathology, and resembled M. bovis BCG vaccination in this respect. However, unlike BCG vaccination, MPB83 DNA vaccination did not protect challenged guinea pigs from hematogenous spread of organisms to the spleen. In contrast, vaccination with antigen 85A DNA, a promising DNA vaccine for human tuberculosis, had no measurable protective effect against infection with M. bovis.

Survival of Mycobacterium tuberculosis during experimental aerosolization and implications for aerosol challenge models

Aims:  We undertook a series of experiments to investigate factors that contribute to variation in Mycobacterium tuberculosis viability and infectivity, during experimental aerosolization, with an aim to optimize a strategy to enable a more reproducible delivered dose within animal models of tuberculosis.

Efficacy of Liposome-Encapsulated Ciprofloxacin in a Murine Model of Q Fever

ABSTRACT Encapsulation of antibiotics may improve treatment of intracellular infections by prolonging antibiotic release and improving antibiotic uptake into cells. In this study, liposome-encapsulated ciprofloxacin for inhalation (CFI) was evaluated as a postexposure therapeutic for the treatment of Coxiella burnetii, the causative agent of Q fever. Intranasal treatment of male A/Jola (A/J) mice with CFI (50 mg/kg of body weight) once daily for 7 days protected mice against weight loss and clinical signs following an aerosol challenge with C. burnetii. In comparison, mice treated twice daily with oral ciprofloxacin or doxycycline (50 mg/kg) or phosphate-buffered saline (PBS) lost 15 to 20% body weight and exhibited ruffled fur, arched backs, and dehydration. Mice were culled at day 14 postchallenge. The weights and bacterial burdens of organs were determined. Mice treated with CFI exhibited reduced splenomegaly and reduced bacterial numbers in the lungs and spleen compared to mice treated with oral ciprofloxacin or doxycycline. When a single dose of CFI was administered, it provided better protection against body weight loss than 7 days of treatment with oral doxycycline, the current antibiotic of choice to treat Q fever. These data suggest that CFI has potential as a superior antibiotic to treat Q fever.

Efficacy Testing of Orally Administered Antibiotics against an Inhalational Bacillus anthracis Infection in BALB/C Mice

Anthrax is a potentially fatal disease in man, and events of the last decade have highlighted the possibility for Bacillus anthracis to be used as a biological weapon through deliberate release. Whilst outbreaks of anthrax occur in several countries, there are only a few areas in which outbreaks of inhalational anthrax occur, so it is not possible to test therapies using conventional clinical trials. To overcome these problems, the FDA published the ‘Animal Rule’ in 2002; a rule designed to permit licensure of drugs and biologics intended to reduce or prevent serious conditions caused by exposure to biological and other agents. Due to limited proven post-exposure prophylaxis after inhalational exposure for use in humans, it is essential to have a robust animal model of disease. In a series of three studies conducted in BALB/c mice, the efficacy of postexposure, orally administered antibiotics in preventing disease following inhalational exposure to B. anthracis was evaluated. Mice treated with either azithromycin or clarithromycin displayed a near identical disease progression to the control, untreated mice; in contrast, those treated with levofloxacin, amoxicillin and co-amoxiclav were generally protected with survival of 97%, 95% and 79%, respectively. The details of the assessments and future direction of these studies are presented.

Parenteral Administration of Capsule Depolymerase EnvD Prevents Lethal Inhalation Anthrax Infection

ABSTRACT Left untreated, inhalation anthrax is usually fatal. Vegetative forms of Bacillus anthracis survive in blood and tissues during infection due to elaboration of a protective poly-γ-d-glutamic acid (PDGA) capsule that permits uncontrolled bacterial growth in vivo, eventually leading to overwhelming bacillosis and death. As a measure to counter threats from multidrug-resistant strains, we are evaluating the prophylactic and therapeutic potential of the PDGA depolymerase EnvD, a stable and potent enzyme which rapidly and selectively removes the capsule from the surface of vegetative cells. Repeated intravenous administration of 10 mg/kg recombinant EnvD (rEnvD) to mice infected with lethal doses of B. anthracis Ames spores by inhalation prevented the emergence of symptoms of anthrax and death; all animals survived the 5-day treatment period, and 70% survived to the end of the 14-day observation period. In contrast to results in sham-treated animals, the lungs and spleen of rEnvD-dosed animals were free of gross pathological changes. We conclude that rEnvD has potential as an agent to prevent the emergence of inhalation anthrax in infected animals and is likely to be effective against drug-resistant forms of the pathogen.

Construction of a severely attenuated mutant of Mycobacterium tuberculosis for reducing risk to laboratory workers

The ability to construct defined deletions of Mycobacterium tuberculosis has allowed many genes involved in virulence to be identified. Deletion of nutritional genes leads to varying levels of attenuation, presumably reflecting the need for a particular molecule, and the availability (or lack) of that molecule in vivo. We have previously shown that M. tuberculosis mutants lacking either the trpD or ino1 gene are highly attenuated in mouse models of infection, but can grow when supplemented with tryptophan or inositol, respectively. In this paper we have constructed a double Delta trpDDelta ino1 mutant, and show that this is severely attenuated in SCID mouse and guinea pig models. As the strain will grow in the presence of supplements, we propose that this strain could be used for research and antigen preparative purposes, with reduced risks to laboratory workers.