Characterization of a Cynomolgus Macaque Model of Pneumonic Plague for Evaluation of Vaccine Efficacy.

The efficacy of a recombinant plague vaccine (rF1V) was evaluated in cynomolgus macaques (CMs) to establish the relationship among vaccine doses, antibody titers, and survival following an aerosol challenge with a lethal dose of Yersinia pestis strain Colorado 92. CMs were vaccinated with a range of rF1V doses on a three-dose schedule (days 0, 56, and 121) to provide a range of survival outcomes. The humoral immune response following vaccination was evaluated with anti-rF1, anti-rV, and anti-rF1V bridge enzyme-linked immunosorbent assays (ELISAs). Animals were challenged via aerosol exposure on day 149. Vaccine doses and antibody responses were each significantly associated with the probability of CM survival (P < 0.0001). Vaccination also decreased signs of pneumonic plague in a dose-dependent manner. There were statistically significant correlations between the vaccine dose and the time to onset of fever (P < 0.0001), the time from onset of fever to death (P < 0.0001), the time to onset of elevated respiratory rate (P = 0.0003), and the time to onset of decreased activity (P = 0.0251) postinfection in animals exhibiting these clinical signs. Delays in the onset of these clinical signs of disease were associated with larger doses of rF1V. Immunization with ≥ 12 µg of rF1V resulted in 100% CM survival. Since both the vaccine dose and anti-rF1V antibody titers correlate with survival, rF1V bridge ELISA titers can be used as a correlate of protection.

Establishment of a Swiss Webster mouse model of pneumonic plague to meet essential data elements under the animal rule.

A recombinant vaccine (rF1V) is being developed for protection against pneumonic plague. This study was performed to address essential data elements to establish a well-characterized Swiss Webster mouse model for licensing the rF1V vaccine using the FDA's Animal Rule. These elements include the documentation of challenge material characteristics, aerosol exposure parameters, details of the onset and severity of clinical signs, pathophysiological response to disease, and relevance to human disease. Prior to animal exposures, an evaluation of the aerosol system was performed to determine and understand the variability of the aerosol exposure system. Standardized procedures for the preparation of Yersinia pestis challenge material also were developed. The 50% lethal dose (LD(50)) was estimated to be 1,966 CFU using Probit analysis. Following the LD(50) determination, pathology was evaluated by exposing mice to a target LD(99) (42,890 CFU). Mice were euthanized at 12, 24, 36, 48, 60, and 72 h postexposure. At each time point, samples were collected for clinical pathology, detection of bacteria in blood and tissues, and pathology evaluations. A general increase in incidence and severity of microscopic findings was observed in the lung, lymph nodes, spleen, and liver from 36 to 72 h postchallenge. Similarly, the incidence and severity of pneumonia increased throughout the study; however, some mice died in the absence of pneumonia, suggesting that disease progression does not require the development of pneumonia. Disease pathology in the Swiss Webster mouse is similar to that observed in humans, demonstrating the utility of this pneumonic plague model that can be used by researchers investigating plague countermeasures.

Cynomolgus macaque model for pneumonic plague.

A recombinant vaccine (rF1V) is currently being developed for protection against pneumonic plague. An essential component in evaluating efficacy of the rF1V vaccine is the development of a well-understood animal model that shows similarity to human disease. The objective of this study was to determine the inhaled median lethal dose (LD50), evaluate the pathophysiology of disease and identify appropriate study endpoints in a cynomolgus macaque (CM) model of pneumonic plague. Eighteen CMs were challenged by head-only aerosol exposure with seven dosages of Yersinia pestis CO92. An LD50 of 24 colony forming units was estimated using Probit analysis. Disease pathology was evaluated by blood culture, clinical pathology, histopathology and telemetry. CMs that died became febrile following challenge and died 34-92 h after onset of fever. Bacteremia, increased respiration and heart rate, decreased blood pressure and loss of diurnal rhythm were also observed in conjunction with onset of fever. Histopathological examinations revealed significant findings in the lungs (intra-alveolar neutrophils and fibrinous pleuritis) consistent with pneumonic plague. These data indicate that the disease pathology observed in CMs following aerosol exposure to Y. pestis CO92 is similar to that of pneumonic plague in humans. Thus, the CM is an appropriate model to evaluate efficacy of a recombinant F1V vaccine candidate.

Protection in mice passively immunized with serum from cynomolgus macaques and humans vaccinated with recombinant plague vaccine (rF1V).

Passive transfer models were developed to evaluate the ability of antibodies generated in cynomolgus macaques and humans vaccinated with a recombinant plague vaccine (rF1V) to protect naïve Swiss Webster mice against pneumonic plague. Development of the passive transfer model is intended to support clinical and nonclinical development of the rF1V vaccine. To evaluate protection, unfractionated serum collected from rF1V vaccinated cynomolgus macaques and human volunteers with known antibody titers to rF1, rV and rF1V was transferred into naïve Swiss Webster mice via the intraperitoneal route. Results of these studies demonstrated that passive immunization protected mice from challenge or extended mean survival time and that the passive transfer assay can be used to evaluate the functional role of antibodies induced by rF1V vaccination in protection against aerosol exposure.

Construction of a rhamnose mutation in Bacillus anthracis affects adherence to macrophages but not virulence in guinea pigs.

Carbohydrate analyses of whole-spore extracts have confirmed the presence of rhamnose in the spore of the fully virulent Ames strain of Bacillus anthracis. A gene cluster containing loci with high homology to the rhamnose biosynthetic genes, rmlACBD, was identified within the B. anthracis chromosome. The first gene of this cluster, rmlA, was inactivated by forming a merodiploid cointegrate using an internal fragment of the gene within the Ames strain of B. anthracis to construct the mutant strain Ames-JAB1. Carbohydrate analysis of spores from this mutant demonstrated the loss of rhamnose. When assaying for spore infection of macrophages, we detected a significant decrease in the recovery with the Ames-JAB1 strain compared to the recovery with the Ames wild-type strain. When pre-treating macrophages with cytochalasin-D, spores of the mutant were further hindered in recovery, indicating that the spores were not able to bind as well to the macrophages. However, in guinea pigs challenge experiments, no difference in virulence was observed between the mutant and wild-type strains. These results suggest that the incorporation of rhamnose into the spore coat of B. anthracis is required for optimal interaction with macrophages but is not required for full virulence in this animal model.

Induction of opsonic antibodies to the gamma-D-glutamic acid capsule of Bacillus anthracis by immunization with a synthetic peptide-carrier protein conjugate.

The capsule of Bacillus anthracis, a polymer of gamma-D-glutamic acid, functions as a virulence determinant and is a poor immunogen. In this study we show that antibodies reactive with the B. anthracis capsule can be elicited in mice by immunization with a conjugate consisting of a synthetic gamma-D-glutamic acid nonamer peptide (gamma-D-glu9) covalently coupled to keyhole limpet hemocyanin. The serum response to gamma-D-glu9 was comprised primarily of IgG antibodies that recognized an epitope requiring a minimum of four gamma-linked D-glutamic acid residues. Antibodies to (gamma-D-glu9) bound to the surface of encapsulated B. anthracis cells and mediated opsonophagoctosis. These findings suggest that anti-capsular antibodies could mediate the clearance of vegetative B. anthracis cells in vivo. Thus, inclusion of an immunogenic capsular component as well as protective antigen in new anthrax vaccines would generate immune responses targeting both the bacteremic and toxigenic aspects of anthrax infection and thus may increase protective efficacy.

Vaccines and animal models for arboviral encephalitides.

Arthropod-borne viruses ("arboviruses") cause significant human illness ranging from mild, asymptomatic infection to fatal encephalitis or hemorrhagic fever. The most significant arboviruses causing human illness belong to genera in three viral families, Togaviridae, Flaviviridae, and Bunyaviridae. These viruses represent a significant public health threat to many parts of the world, and, as evidenced by the recent introduction of the West Nile virus (WNV) to the Western Hemisphere, they can no longer be considered specific to any one country or region of the world. Like most viral diseases, there are no specific therapies for the arboviral encephalitides; therefore, effective vaccines remain the front line of defense for these diseases. With this in mind, the development of new, more effective vaccines and the appropriate animal models in which to test them become paramount. In fact, for many important arboviruses (e.g. California serogroup and St. Louis encephalitis viruses), there are currently no approved vaccines available for human use. For others, such as the alphaviruses, human vaccines are available only as Investigational New Drugs, and thus are not in widespread use. On the other hand, safe and effective vaccines against tick-borne encephalitis virus (TBEV) and Japanese encephalitis virus (JEV) have been in use for decades. New challenges in vaccine development have been met with new technologies in vaccine research. Many of the newer vaccines are now being developed by recombinant DNA technology. For example, chimeric virus vaccines have been developed using infectious clone technology for many of the arboviruses including, WNV, JEV, and TBEV. Other successful approaches have involved the use of naked DNA encoding and subsequently expressing the desired protective epitopes. Naked DNA vaccines have been used for TBEV and JEV and are currently under development for use against WNV. The development of less expensive, more authentic animal models to evaluate new vaccines against arboviral diseases will become increasingly important as these new approaches in vaccine research are realized. This article reviews the current status of vaccines, both approved for use and those in developmental stages, against the major arboviral encephalitides causing human disease. In addition, research on animal models, both past and present, for these diseases are discussed.

Bacillus anthracis virulence in Guinea pigs vaccinated with anthrax vaccine adsorbed is linked to plasmid quantities and clonality.

Bacillus anthracis is a bacterial pathogen of great importance, both historically and in the present. This study presents data collected from several investigations and indicates that B. anthracis virulence is associated with the clonality and virulence of plasmids pXO1 and pXO2. Guinea pigs vaccinated with Anthrax Vaccine Adsorbed were challenged with 20 B. anthracis isolates representative of worldwide genetic diversity. These same isolates were characterized with respect to plasmid copy number by using a novel method of quantitative PCR developed for rapid and efficient detection of B. anthracis from environmental samples. We found that the copy numbers for both pXO1 and pXO2 differed from those in previously published reports. By combining the data on survival, plasmid copy numbers, and clonality, we developed a model predicting virulence. This model was validated by using a randomly chosen set of 12 additional B. anthracis isolates. Results from this study will be helpful in future efforts to elucidate the basis for variation in the virulence of this important pathogen.

The role of antibodies to Bacillus anthracis and anthrax toxin components in inhibiting the early stages of infection by anthrax spores.

Vaccines which are efficacious against anthrax, such as the human vaccine, Anthrax Vaccine Absorbed (AVA), contain the protective antigen (PA) component of the anthrax toxins as the major protective immunogen. Although AVA protects against inhalational anthrax, the immune responses to and role in protection of PA and possibly other antigens have yet to be fully elucidated. Sera from animals immunized with a toxin-producing, unencapsulated live vaccine strain of Bacillus anthracis have been reported to have anti-spore activities associated with the antitoxin humoral response. The authors performed studies to determine whether anti-PA antibody (Ab)-containing preparations stimulated spore uptake by phagocytes and suppressed the germination of spores in vitro. AVA- and PA-immune sera from several species enhanced the phagocytosis by murine peritoneal macrophages of spores of the virulent Ames and the Sterne vaccine strains. Antitoxin Abs appeared to contribute significantly, although not solely, to the enhanced uptake. Rabbit antisera to PA purified from either Sterne or a PA-producing pX01-cured recombinant, affinity-purified anti-PA IgG, and monkey antisera to AVA were used to assess the role of anti-PA ABS: Rabbit anti-PA Abs promoted the uptake of spores of the PA-producing strains Sterne, Ames and RP42, a mutant of Sterne producing only PA, but not of the pX01-Sterne-1 strain, Ames strain, or RP4, a mutant of Sterne with deletions in the loci encoding PA and the oedema factor (EF) toxin component and producing only the lethal factor toxin component. Rabbit anti-PA and monkey anti-AVA Abs also significantly inhibited spore germination in vitro compared to preimmune serum or medium. Spore-associated proteins recognized by anti-PA Abs were detected by electron microscopy and confirmed by immunoblotting of spore coat extracts. Thus, the anti-PA Ab-specific immunity induced by AVA has anti-spore activity and might have a role in impeding the early stages of infection with B. anthracis spores.