It has been previously shown that mice subjected to an aerosol exposure to Yersinia pestis and treated with ?-lactam antibiotics after a delay of 42 h died at an accelerated rate compared to controls. It was hypothesized that endotoxin release in antibiotic-treated mice accounted for the accelerated death rate in the mice exposed to aerosol Y. pestis. Imipenem, a ?-lactam antibiotic, binds to penicillin binding protein 2 with the highest affinity and produces rounded cells. The binding of imipenem causes cells to lyse quickly and thereby to release less free endotoxin. Two imipenem regimens producing fractions of time that the concentration of free, unbound drug was above the MIC (fT>MIC) of approximately 25% (6/24 h) and 40% (9.5/24 h) were evaluated. In the postexposure prophylaxis study, the 40% and 25% regimens produced 90% and 40% survivorship, respectively. In the 42-h treatment study, both regimens demonstrated a 40 to 50% survivorship at therapy cessation and some deaths thereafter, resulting in a 30% survivorship. As this was an improvement over the results with other ?-lactams, a comparison of both endotoxin and cytokine levels in mice treated with imipenem and ceftazidime (a ?-lactam previously demonstrated to accelerate death in mice during treatment) was performed and supported the original hypotheses; however, the levels observed in animals treated with ciprofloxacin (included as an unrelated antibiotic that is also bactericidal but should cause little lysis due to a different mode of action) were elevated and significantly (7-fold) higher than those with ceftazidime.
The current candidate vaccine against Yersinia pestis infection consists of two subunit proteins: the capsule protein or F1 protein and the low calcium response V protein or V-antigen. Little is known of the recognition of the vaccine by the host's innate immune system and how it affects the acquired immune response to the vaccine. Thus, we vaccinated Toll-like receptor (Tlr) 2, 4, and 2/4-double deficient, as well as signal adaptor protein Myd88-deficient mice. We found that Tlr4 and Myd88 appeared to be required for an optimal immune response to the F1-V vaccine but not Tlr2 when compared to wild-type mice. However, there was a difference between the requirement for Tlr4 and MyD88 in vaccinated animals. When F1-V vaccinated Tlr4 mutant (lipopolysaccharide tolerant) and Myd88-deficient mice were challenged by aerosol with Y. pestis CO92, all but one Tlr4 mutant mice survived the challenge, but no vaccinated Myd88-deficient mice survived the challenge. Spleens from these latter nonsurviving mice showed that Y. pestis was not cleared from the infected mice. Our results suggest that MyD88 appears to be important for both an optimal immune response to F1-V and in protection against a lethal challenge of Y. pestis CO92 in F1-V vaccinated mice.
In the future, we may be faced with the need to provide treatment for an emergent biological threat against which existing vaccines and drugs have limited efficacy or availability. To prepare for this eventuality, our objective was to use a metabolic network-based approach to rapidly identify potential drug targets and prospectively screen and validate novel small-molecule antimicrobials. Our target organism was the fully virulent Francisella tularensis subspecies tularensis Schu S4 strain, a highly infectious intracellular pathogen that is the causative agent of tularemia and is classified as a category A biological agent by the Centers for Disease Control and Prevention. We proceeded with a staggered computational and experimental workflow that used a strain-specific metabolic network model, homology modeling and X-ray crystallography of protein targets, and ligand- and structure-based drug design. Selected compounds were subsequently filtered based on physiological-based pharmacokinetic modeling, and we selected a final set of 40 compounds for experimental validation of antimicrobial activity. We began screening these compounds in whole bacterial cell-based assays in biosafety level 3 facilities in the 20th week of the study and completed the screens within 12 weeks. Six compounds showed significant growth inhibition of F. tularensis, and we determined their respective minimum inhibitory concentrations and mammalian cell cytotoxicities. The most promising compound had a low molecular weight, was non-toxic, and abolished bacterial growth at 13 µM, with putative activity against pantetheine-phosphate adenylyltransferase, an enzyme involved in the biosynthesis of coenzyme A, encoded by gene coaD. The novel antimicrobial compounds identified in this study serve as starting points for lead optimization, animal testing, and drug development against tularemia. Our integrated in silico/in vitro approach had an overall 15% success rate in terms of active versus tested compounds over an elapsed time period of 32 weeks, from pathogen strain identification to selection and validation of novel antimicrobial compounds.
Immunization with the Yersinia pestis F1 and LcrV proteins improves survival in mouse and non-human primate models of pneumonic plague. F1- and LcrV-specific antibodies contribute to protection, however, the mechanisms of antibody-mediated defense are incompletely understood and serum antibody titers do not suffice as quantitative correlates of protection. Previously we demonstrated roles for tumor necrosis factor-alpha (TNF?) and gamma-interferon (IFN?) during defense against conditionally attenuated pigmentation (pgm) locus-negative Y. pestis. Here, using intranasal challenge with fully virulent pgm-positive Y. pestis strain CO92, we demonstrate that neutralizing TNF? and IFN? interferes with the capacity of therapeutically administered F1- or LcrV-specific antibody to reduce bacterial burden and increase survival. Moreover, using Y. pestis strain CO92 in an aerosol challenge model, we demonstrate that neutralizing TNF? and IFN? interferes with protection conferred by immunization with recombinant F1-LcrV fusion protein vaccine (p<0.0005). These findings establish that TNF? and IFN? contribute to protection mediated by pneumonic plague countermeasures targeting F1 and LcrV, and suggest that an individuals capacity to produce these cytokines in response to Y. pestis challenge will be an important co-determinant of antibody-mediated defense against pneumonic plague.
Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr) V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs) against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252) and two anti-V-specific human mAb (m253, m254) by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.
The potential use of Yersinia pestis as a bioterror agent is a great concern. Development of a stable powder vaccine against Y. pestis and administration of the vaccine by minimally invasive methods could provide an alternative to the traditional liquid formulation and intramuscular injection. We evaluated a spray-freeze-dried powder vaccine containing a recombinant F1-V fusion protein of Y. pestis for vaccination against plaque in a mouse model. Mice were immunized with reconstituted spray-freeze-dried F1-V powder via intramuscular injection, microneedle-based intradermal delivery, or noninvasive intranasal administration. By intramuscular injection, the reconstituted powder induced serum antibody responses and provided protection against lethal subcutaneous challenge with 1,000 50% lethal doses of Y. pestis at levels equivalent to those elicited by unprocessed liquid formulations (70 to 90% protection). The feasibility of intradermal and intranasal delivery of reconstituted powder F1-V vaccine was also demonstrated. Overall, microneedle-based intradermal delivery was shown to be similar in efficacy to intramuscular injection, while intranasal administration required an extra dose of vaccine to achieve similar protection. In addition, the results suggest that seroconversion against F1 may be a better predictor of protection against Y. pestis challenge than seroconversion against either F1-V or V. In summary, we demonstrate the preclinical feasibility of using a reconstituted powder F1-V formulation and microneedle-based intradermal delivery to provide protective immunity against plague in a mouse model. Intranasal delivery, while feasible, was less effective than injection in this study. The potential use of these alternative delivery methods and a powder vaccine formulation may result in substantial health and economic benefits.
The current U.S. Department of Defense candidate plague vaccine is a fusion between two Yersinia pestis proteins: the F1 capsular protein, and the low calcium response (Lcr) V-protein. We hypothesized that an immunomodulator, such as CpG oligodeoxynucleotide (ODN)s, could augment the immune response to the plague F1-V vaccine in a mouse model for plague. CpG ODNs significantly augmented the antibody response and efficacy of a single dose of the plague vaccine in murine bubonic and pneumonic models of plague. In the latter study, we also found an overall significant augmentation the immune response to the individual subunits of the plague vaccine by CpG ODN 2006. In a long-term, prime-boost study, CpG ODN induced a significant early augmentation of the IgG response to the vaccine. The presence of CpG ODN induced a significant increase in the IgG2a subclass response to the vaccine up to 5 months after the boost. Our studies showed that CpG ODNs significantly augmented the IgG antibody response to the plague vaccine, which increased the probability of survival in murine models of plague (P<0.0001).
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