The rLVS ΔcapB/iglABC vaccine provides potent protection in Fischer rats against inhalational tularemia caused by various virulent Francisella tularensis strains.

Francisella tularensis is one of the several biothreat agents for which a licensed vaccine is needed. To ensure vaccine protection is achieved across a range of virulent F. tularensis strains, we assembled and characterized a panel of F. tularensis isolates to be utilized as challenge strains. A promising tularemia vaccine candidate is rLVS ΔcapB/iglABC (rLVS), in which the vector is the LVS strain with a deletion in the capB gene and which additionally expresses a fusion protein comprising immunodominant epitopes of proteins IglA, IglB, and IglC. Fischer rats were immunized subcutaneously 1-3 times at 3-week intervals with rLVS at various doses. The rats were exposed to a high dose of aerosolized Type A strain Schu S4 (FRAN244), a Type B strain (FRAN255), or a tick derived Type A strain (FRAN254) and monitored for survival. All rLVS vaccination regimens including a single dose of 107 CFU rLVS provided 100% protection against both Type A strains. Against the Type B strain, two doses of 107 CFU rLVS provided 100% protection, and a single dose of 107 CFU provided 87.5% protection. In contrast, all unvaccinated rats succumbed to aerosol challenge with all of the F. tularensis strains. A robust Th1-biased antibody response was induced in all vaccinated rats against all F. tularensis strains. These results demonstrate that rLVS ΔcapB/iglABC provides potent protection against inhalational challenge with either Type A or Type B F. tularensis strains and should be considered for further analysis as a future tularemia vaccine.

Functional assays to screen and select monoclonal antibodies that target Yersinia pestis.

Yersinia pestis is a gram-negative bacterium that causes plague in animals and humans. Depending on the route of disease transmission, the bacterium can cause an acute, often fatal disease that has a narrow window for treatment with antibiotics. Additionally, antibiotic resistant strains have been identified, emphasizing the need for novel treatments. Antibody therapy is an appealing option that can direct the immune system to clear bacterial infections. Advances in biotechnology have made both engineering and producing antibodies easier and more affordable. In this study, two screening assays were optimized to evaluate the ability of antibodies to promote phagocytosis of Y. pestis by macrophages and to induce a cytokine signature in vitro that may be predictive of protection in vivo. We evaluated a panel of 21 mouse monoclonal antibodies targeting either the anti-phagocytic capsule F1 protein or the LcrV antigen, which is part of the type 3 secretion system that facilitates translocation of virulence factors into the host cell, using two functional assays. Anti-F1 and anti-LcrV monoclonal antibodies both increased bacterial uptake by macrophages, with greater uptake observed in the presence of antibodies that were protective in the mouse pneumonic plague model. In addition, the protective anti-F1 and anti-LcrV antibodies produced unique cytokine signatures that were also associated with in vivo protection. These antibody-dependent characteristics from in vitro functional assays will be useful in down-selecting efficacious novel antibodies that can be used for treatment of plague.

Polyclonal Antibodies Derived from Transchromosomic Bovines Vaccinated with the Recombinant F1-V Vaccine Increase Bacterial Opsonization In Vitro and Protect Mice from Pneumonic Plague.

Plague is an ancient disease that continues to be of concern to both the public health and biodefense research communities. Pneumonic plague is caused by hematogenous spread of Yersinia pestis bacteria from a ruptured bubo to the lungs or by directly inhaling aerosolized bacteria. The fatality rate associated with pneumonic plague is significant unless effective antibiotic therapy is initiated soon after an early and accurate diagnosis is made. As with all bacterial pathogens, drug resistance is a primary concern when developing strategies to combat these Yersinia pestis infections in the future. While there has been significant progress in vaccine development, no FDA-approved vaccine strategy exists; thus, other medical countermeasures are needed. Antibody treatment has been shown to be effective in animal models of plague. We produced fully human polyclonal antibodies in transchromosomic bovines vaccinated with the recombinant F1-V plague vaccine. The resulting human antibodies opsonized Y. pestis bacteria in the presence of RAW264.7 cells and afforded significant protection to BALB/c mice after exposure to aerosolized Y. pestis. These data demonstrate the utility of this technology to produce large quantities of non-immunogenic anti-plague human antibodies to prevent or possibly treat pneumonic plague in human.

The BALB/c Mouse Model for the Evaluation of Therapies to Treat Infections with Aerosolized Burkholderia pseudomallei.

Burkholderia pseudomallei, the causative agent of the disease melioidosis, has been isolated from the environment in 45 countries. The treatment of melioidosis is complex, requiring lengthy antibiotic regimens, which can result in the relapse of the disease following treatment cessation. It is important that novel therapies to treat infections with B. pseudomallei be assessed in appropriate animal models, and discussions regarding the different protocols used between laboratories are critical. A 'deep dive' was held in October 2020 focusing on the use of the BALB/c mouse model and the inhalational route of infection to evaluate new antibiotic therapies.

Efficacy of delafloxacin against the biothreat pathogen Bacillus anthracis.

OBJECTIVES: To evaluate the in vitro activity and in vivo efficacy of delafloxacin against Bacillus anthracis, the causative agent of anthrax. METHODS: MICs were obtained according to CLSI guidelines for 30 virulent isolates and 14 attenuated antibiotic-resistant strains. For the in vivo efficacy study, mice were administered delafloxacin (30-62.5 mg/kg) subcutaneously, or ciprofloxacin (30 mg/kg) intraperitoneally beginning at either 24 or 48 ±â€Š1 h post-challenge (post-exposure prophylaxis) and continued every 12 h for 14 days with study termination on day 30. The mean inhaled dose in the study was approximately 103 × LD50 equivalents, and the range was 87-120 × LD50. RESULTS: Delafloxacin (MIC90 = 0.004 mg/L) was 16-fold more potent than ciprofloxacin (MIC90 = 0.06 mg/L) against a 30-strain set of virulent B. anthracis. Against a panel of attenuated antibiotic-resistant strains, delafloxacin demonstrated potency ≥128-fold over that observed with ciprofloxacin. When evaluated in vivo, mice treated with all delafloxacin doses tested at 24 h post-challenge demonstrated equivalent survival compared with mice treated with the positive control ciprofloxacin. Because of the high challenge dose of spores, mice treated at 48 h showed rapid and high mortality in all groups including the positive control. Surviving animals in all delafloxacin- and ciprofloxacin-treated groups (24 and 48 h) showed complete splenic clearance of infection and <2.2 × 103 cfu/g lung tissue. CONCLUSIONS: Given the high bar set by the 100 × LD50 challenge dose in this study, the results from delafloxacin treatment are promising for the treatment of inhaled anthrax.

Efficacy of Treatment with the Antibiotic Novobiocin against Infection with Bacillus anthracis or Burkholderia pseudomallei.

The microbial pathogens Burkholderia pseudomallei and Bacillus anthracis are unrelated bacteria, yet both are the etiologic agents of naturally occurring diseases in animals and humans and are classified as Tier 1 potential biothreat agents. B. pseudomallei is the gram-negative bacterial agent of melioidosis, a major cause of sepsis and mortality globally in endemic tropical and subtropical regions. B. anthracis is the gram-positive spore-forming bacterium that causes anthrax. Infections acquired by inhalation of these pathogens are challenging to detect early while the prognosis is best; and they possess innate multiple antibiotic resistance or are amenable to engineered resistance. Previous studies showed that the early generation, rarely used aminocoumarin novobiocin was very effective in vitro against a range of highly disparate biothreat agents. The objective of the current research was to begin to characterize the therapeutic efficacy of novobiocin in mouse models of anthrax and melioidosis. The antibiotic was highly efficacious against infections by both pathogens, especially B. pseudomallei. Our results supported the concept that specific older generation antimicrobials can be effective countermeasures against infection by bacterial biothreat agents. Finally, novobiocin was shown to be a potential candidate for inclusion in a combined pre-exposure vaccination and post-exposure treatment strategy designed to target bacterial pathogens refractory to a single medical countermeasure.

Evaluation of two different vaccine platforms for immunization against melioidosis and glanders.

Burkholderia pseudomallei and the closely related species, Burkholderia mallei, produce similar multifaceted diseases which range from rapidly fatal to protracted and chronic, and are a major cause of mortality in endemic regions. Besides causing natural infections, both microbes are Tier 1 potential biothreat agents. Antibiotic treatment is prolonged with variable results, hence effective vaccines are urgently needed. The purpose of our studies was to compare candidate vaccines that target both melioidosis and glanders to identify the most efficacious one(s) and define residual requirements for their transition to the non-human primate aerosol model. Studies were conducted in the C57BL/6 mouse model to evaluate the humoral and cell-mediated immune response and protective efficacy of three Burkholderia vaccine candidates against lethal aerosol challenges with B. pseudomallei K96243, B. pseudomallei MSHR5855, and B. mallei FMH. The recombinant vaccines generated significant immune responses to the vaccine antigens, and the live attenuated vaccine generated a greater immune response to OPS and the whole bacterial cells. Regardless of the candidate vaccine evaluated, the protection of mice was associated with a dampened cytokine response within the lungs after exposure to aerosolized bacteria. Despite being delivered by two different platforms and generating distinct immune responses, two experimental vaccines, a capsule conjugate + Hcp1 subunit vaccine and the live B. pseudomallei 668 ΔilvI strain, provided significant protection and were down-selected for further investigation and advanced development.

Layered and integrated medical countermeasures against Burkholderia pseudomallei infections in C57BL/6 mice.

Burkholderia pseudomallei, the gram-negative bacterium that causes melioidosis, is notoriously difficult to treat with antibiotics. A significant effort has focused on identifying protective vaccine strategies to prevent melioidosis. However, when used as individual medical countermeasures both antibiotic treatments (therapeutics or post-exposure prophylaxes) and experimental vaccine strategies remain partially protective. Here we demonstrate that when used in combination, current vaccine strategies (recombinant protein subunits AhpC and/or Hcp1 plus capsular polysaccharide conjugated to CRM197 or the live attenuated vaccine strain B. pseudomallei 668 ΔilvI) and co-trimoxazole regimens can result in near uniform protection in a mouse model of melioidosis due to apparent synergy associated with distinct medical countermeasures. Our results demonstrated significant improvement when examining several suboptimal antibiotic regimens (e.g., 7-day antibiotic course started early after infection or 21-day antibiotic course with delayed initiation). Importantly, this combinatorial strategy worked similarly when either protein subunit or live attenuated vaccines were evaluated. Layered and integrated medical countermeasures will provide novel treatment options for melioidosis as well as diseases caused by other pathogens that are refractory to individual strategies, particularly in the case of engineered, emerging, or re-emerging bacterial biothreat agents.

Predictive and Experimental Immunogenicity of Burkholderia Collagen-like Protein 8-Derived Antigens.

Burkholderia pseudomallei is an infectious bacterium of clinical and biodefense concern, and is the causative agent of melioidosis. The mortality rate can reach up to 50% and affects 165,000 people per year; however, there is currently no vaccine available. In this study, we examine the antigen-specific immune response to a vaccine formulated with antigens derived from an outer membrane protein in B. pseudomallei, Bucl8. Here, we employed a number of bioinformatic tools to predict Bucl8-derived epitopes that are non-allergenic and non-toxic, but would elicit an immune response. From these data, we formulated a vaccine based on two extracellular components of Bucl8, the ß-barrel loops and extended collagen and non-collagen domains. Outbred CD-1 mice were immunized with vaccine formulations-composed of recombinant proteins or conjugated synthetic peptides with adjuvant-to assess the antigen-specific immune responses in mouse sera and lymphoid organs. We found that mice vaccinated with either Bucl8-derived components generated a robust TH2-skewed antibody response when antigen was combined with the adjuvant AddaVax, while the TH1 response was limited. Mice immunized with synthetic loop peptides had a stronger, more consistent antibody response than recombinant protein antigens, based on higher IgG titers and recognition of bacteria. We then compared peptide-based vaccines in an established C57BL/6 inbred mouse model and observed a similar TH2-skewed response. The resulting formulations will be applied in future studies examining the protection of Bucl8-derived vaccines.

Plague Prevention and Therapy: Perspectives on Current and Future Strategies.

Plague, caused by the bacterial pathogen Yersinia pestis, is a vector-borne disease that has caused millions of human deaths over several centuries. Presently, human plague infections continue throughout the world. Transmission from one host to another relies mainly on infected flea bites, which can cause enlarged lymph nodes called buboes, followed by septicemic dissemination of the pathogen. Additionally, droplet inhalation after close contact with infected mammals can result in primary pneumonic plague. Here, we review research advances in the areas of vaccines and therapeutics for plague in context of Y. pestis virulence factors and disease pathogenesis. Plague continues to be both a public health threat and a biodefense concern and we highlight research that is important for infection mitigation and disease treatment.

Impact of Toll-Like Receptor-Specific Agonists on the Host Immune Response to the Yersinia pestis Plague rF1V Vaccine.

Relatively recent advances in plague vaccinology have produced the recombinant fusion protein F1-V plague vaccine. This vaccine has been shown to readily protect mice from both bubonic and pneumonic plague. The protection afforded by this vaccine is solely based upon the immune response elicited by the F1 or V epitopes expressed on the F1-V fusion protein. Accordingly, questions remain surrounding its efficacy against infection with non-encapsulated (F1-negative) strains. In an attempt to further optimize the F1-V elicited immune response and address efficacy concerns, we examined the inclusion of multiple toll-like receptor agonists into vaccine regimens. We examined the resulting immune responses and also any protection afforded to mice that were exposed to aerosolized Yersinia pestis. Our data demonstrate that it is possible to further augment the F1-V vaccine strategy in order to optimize and augment vaccine efficacy.

Development, Phenotypic Characterization and Genomic Analysis of a Francisella tularensis Panel for Tularemia Vaccine Testing.

Francisella tularensis is one of several biothreat agents for which a licensed vaccine is needed to protect against this pathogen. To aid in the development of a vaccine protective against pneumonic tularemia, we generated and characterized a panel of F. tularensis isolates that can be used as challenge strains to assess vaccine efficacy. Our panel consists of both historical and contemporary isolates derived from clinical and environmental sources, including human, tick, and rabbit isolates. Whole genome sequencing was performed to assess the genetic diversity in comparison to the reference genome F. tularensis Schu S4. Average nucleotide identity analysis showed >99% genomic similarity across the strains in our panel, and pan-genome analysis revealed a core genome of 1,707 genes, and an accessory genome of 233 genes. Three of the strains in our panel, FRAN254 (tick-derived), FRAN255 (a type B strain), and FRAN256 (a human isolate) exhibited variation from the other strains. Moreover, we identified several unique mutations within the Francisella Pathogenicity Island across multiple strains in our panel, revealing unexpected diversity in this region. Notably, FRAN031 (Scherm) completely lacked the second pathogenicity island but retained virulence in mice. In contrast, FRAN037 (Coll) was attenuated in a murine pneumonic tularemia model and had mutations in pdpB and iglA which likely led to attenuation. All of the strains, except FRAN037, retained full virulence, indicating their effectiveness as challenge strains for future vaccine testing. Overall, we provide a well-characterized panel of virulent F. tularensis strains that can be utilized in ongoing efforts to develop an effective vaccine against pneumonic tularemia to ensure protection is achieved across a range F. tularensis strains.

In Vivo Activity of Repurposed Amodiaquine as a Host-Targeting Therapy for the Treatment of Anthrax.

Anthrax is caused by Bacillus anthracis and can result in nearly 100% mortality due in part to anthrax toxin. Antimalarial amodiaquine (AQ) acts as a host-oriented inhibitor of anthrax toxin endocytosis. Here, we determined the pharmacokinetics and safety of AQ in mice, rabbits, and humans as well as the efficacy in the fly, mouse, and rabbit models of anthrax infection. In the therapeutic-intervention studies, AQ nearly doubled the survival of mice infected subcutaneously with a B. anthracis dose lethal to 60% of the animals (LD60). In rabbits challenged with 200 LD50 of aerosolized B. anthracis, AQ as a monotherapy delayed death, doubled the survival rate of infected animals that received a suboptimal amount of antibacterial levofloxacin, and reduced bacteremia and toxemia in tissues. Surprisingly, the anthrax efficacy of AQ relies on an additional host macrophage-directed antibacterial mechanism, which was validated in the toxin-independent Drosophila model of Bacillus infection. Lastly, a systematic literature review of the safety and pharmacokinetics of AQ in humans from over 2 000 published articles revealed that AQ is likely safe when taken as prescribed, and its pharmacokinetics predicts anthrax efficacy in humans. Our results support the future examination of AQ as adjunctive therapy for the prophylactic anthrax treatment.

Proteomic Analysis of Non-human Primate Peripheral Blood Mononuclear Cells During Burkholderia mallei Infection Reveals a Role of Ezrin in Glanders Pathogenesis.

Burkholderia mallei, the causative agent of glanders, is a gram-negative intracellular bacterium. Depending on different routes of infection, the disease is manifested by pneumonia, septicemia, and chronic infections of the skin. B. mallei poses a serious biological threat due to its ability to infect via aerosol route, resistance to multiple antibiotics and to date there are no US Food and Drug Administration (FDA) approved vaccines available. Induction of innate immunity, inflammatory cytokines and chemokines following B. mallei infection, have been observed in in vitro and small rodent models; however, a global characterization of host responses has never been systematically investigated using a non-human primate (NHP) model. Here, using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we identified alterations in expression levels of host proteins in peripheral blood mononuclear cells (PBMCs) originating from naïve rhesus macaques (Macaca mulatta), African green monkeys (Chlorocebus sabaeus), and cynomolgus macaques (Macaca fascicularis) exposed to aerosolized B. mallei. Gene ontology (GO) analysis identified several statistically significant overrepresented biological annotations including complement and coagulation cascade, nucleoside metabolic process, vesicle-mediated transport, intracellular signal transduction and cytoskeletal protein binding. By integrating an LC-MS/MS derived proteomics dataset with a previously published B. mallei host-pathogen interaction dataset, a statistically significant predictive protein-protein interaction (PPI) network was constructed. Pharmacological perturbation of one component of the PPI network, specifically ezrin, reduced B. mallei mediated interleukin-1ß (IL-1ß). On the contrary, the expression of IL-1ß receptor antagonist (IL-1Ra) was upregulated upon pretreatment with the ezrin inhibitor. Taken together, inflammasome activation as demonstrated by IL-1ß production and the homeostasis of inflammatory response is critical during the pathogenesis of glanders. Furthermore, the topology of the network reflects the underlying molecular mechanism of B. mallei infections in the NHP model.

Comparison of three non-human primate aerosol models for glanders, caused by Burkholderia mallei.

Burkholderia mallei is a gram-negative obligate animal pathogen that causes glanders, a highly contagious and potentially fatal disease of solipeds including horses, mules, and donkeys. Humans are also susceptible, and exposure can result in a wide range of clinical forms, i.e., subclinical infection, chronic forms with remission and exacerbation, or acute and potentially lethal septicemia and/or pneumonia. Due to intrinsic antibiotic resistance and the ability of the organisms to survive intracellularly, current treatment regimens are protracted and complicated; and no vaccine is available. As a consequence of these issues, and since B. mallei is infectious by the aerosol route, B. mallei is regarded as a major potential biothreat agent. To develop optimal medical countermeasures and diagnostic tests, well characterized animal models of human glanders are needed. The goal of this study was to perform a head-to-head comparison of models employing three commonly used nonhuman primate (NHP) species, the African green monkey (AGM), Rhesus macaque, and the Cynomolgus macaque. The natural history of infection and in vitro clinical, histopathological, immunochemical, and bacteriological parameters were examined. The AGMs were the most susceptible NHP to B. mallei; five of six expired within 14 days. Although none of the Rhesus or Cynomolgus macaques succumbed, the Rhesus monkeys exhibited abnormal signs and clinical findings associated with B. mallei infection; and the latter may be useful for modeling chronic B. mallei infection. Based on the disease progression observations, gross and histochemical pathology, and humoral and cellular immune response findings, the AGM appears to be the optimal model of acute, lethal glanders infection. AGM models of infection by B. pseudomallei, the etiologic agent of melioidosis, have been characterized recently. Thus, the selection of the AGM species provides the research community with a single NHP model for investigations on acute, severe, inhalational melioidosis and glanders.

Thinking Outside the Bug: Targeting Outer Membrane Proteins for Burkholderia Vaccines.

Increasing antimicrobial resistance due to misuse and overuse of antimicrobials, as well as a lack of new and innovative antibiotics in development has become an alarming global threat. Preventative therapeutics, like vaccines, are combative measures that aim to stop infections at the source, thereby decreasing the overall use of antibiotics. Infections due to Gram-negative pathogens pose a significant treatment challenge because of substantial multidrug resistance that is acquired and spread throughout the bacterial population. Burkholderia spp. are Gram-negative intrinsically resistant bacteria that are responsible for environmental and nosocomial infections. The Burkholderia cepacia complex are respiratory pathogens that primarily infect immunocompromised and cystic fibrosis patients, and are acquired through contaminated products and equipment, or via patient-to-patient transmission. The Burkholderia pseudomallei complex causes percutaneous wound, cardiovascular, and respiratory infections. Transmission occurs through direct exposure to contaminated water, water-vapors, or soil, leading to the human disease melioidosis, or the equine disease glanders. Currently there is no licensed vaccine against any Burkholderia pathogen. This review will discuss Burkholderia vaccine candidates derived from outer membrane proteins, OmpA, OmpW, Omp85, and Bucl8, encompassing their structures, conservation, and vaccine formulation.

Protection Elicited by Attenuated Live Yersinia pestis Vaccine Strains against Lethal Infection with Virulent Y. pestis.

The etiologic agent of plague, Yersinia pestis, is a globally distributed pathogen which poses both a natural and adversarial threat. Due largely to the rapid course and high mortality of pneumonic plague, vaccines are greatly needed. Two-component protein vaccines have been unreliable and potentially vulnerable to vaccine resistance. We evaluated the safety and efficacy of eight live Y. pestis strains derived from virulent strains CO92 or KIM6+ and mutated in one or more virulence-associated gene(s) or cured of plasmid pPst. Stringent, single-dose vaccination allowed down-selection of the two safest and most protective vaccine candidates, CO92 mutants pgm- pPst- and ΔyscN. Both completely protected BALB/c mice against subcutaneous and aerosol challenge with Y. pestis. Strain CD-1 outbred mice were more resistant to bubonic (but not pneumonic) plague than BALB/c mice, but the vaccines elicited partial protection of CD-1 mice against aerosol challenge, while providing full protection against subcutaneous challenge. A ΔyscN mutant of the nonencapsulated C12 strain was expected to display antigens previously concealed by the capsule. C12 ΔyscN elicited negligible titers to F1 but comparable antibody levels to whole killed bacteria, as did CO92 ΔyscN. Although one dose of C12 ΔyscN was not protective, vaccination with two doses of either CO92 ΔyscN, or a combination of the ΔyscN mutants of C12 and CO92, protected optimally against lethal bubonic or pneumonic plague. Protection against encapsulated Y. pestis required inclusion of F1 in the vaccine and was associated with high anti-F1 titers.

Comparative virulence of three different strains of Burkholderia pseudomallei in an aerosol non-human primate model.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a major cause of sepsis and mortality in endemic regions of Southeast Asia and Northern Australia. B. pseudomallei is a potential bioterrorism agent due to its high infectivity, especially via inhalation, and its inherent resistance to antimicrobials. There is currently no vaccine for melioidosis and antibiotic treatment can fail due to innate drug resistance, delayed diagnosis and treatment, or insufficient duration of treatment. A well-characterized animal model that mimics human melioidosis is needed for the development of new medical countermeasures. This study first characterized the disease progression of melioidosis in the African green monkey (AGM) and rhesus macaque (RM) for non-human primate model down-selection. All AGMs developed acute lethal disease similar to that described in human acute infection following exposure to aerosolized B. pseudomallei strain HBPUB10134a. Only 20% of RMs succumbed to acute disease. Disease progression, immune response and pathology of two other strains of B. pseudomallei, K96243 and MSHR5855, were also compared using AGMs. These three B. pseudomallei strains represent a highly virulent strain from Thailand (HBPUB101034a), a highly virulent strains from Australia (MSHR5855), and a commonly used laboratory strains originating from Thailand (K96243). Animals were observed for clinical signs of infection and blood samples were analyzed for cytokine responses, blood chemistry and leukocyte changes in order to characterize bacterial infection. AGMs experienced fever after exposure to aerosolized B. pseudomallei at the onset of acute disease. Inflammation, abscesses and/or pyogranulomas were observed in lung with all three strains of B. pseudomallei. Inflammation, abscesses and/or pyogranulomas were observed in lymph nodes, spleen, liver and/or kidney with B. pseudomallei, HBPUB10134a and K96243. Additionally, the Australian strain MSHR5855 induced brain lesions in one AGM similar to clinical cases of melioidosis seen in Australia. Elevated serum levels of IL-1ß, IL-1 receptor antagonist, IL-6, MCP-1, G-CSF, HGF, IFNγ, MIG, I-TAC, and MIP-1ß at terminal end points can be significantly correlated with non-survivors with B. pseudomallei infection in AGM. The AGM model represents an acute model of B. pseudomallei infection for all three strains from two geographical locations and will be useful for efficacy testing of vaccines and therapeutics against melioidosis. In summary, a dysregulated immune response leading to excessive persistent inflammation and inflammatory cell death is the key driver of acute melioidosis. Early intervention in these pathways will be necessary to counter B. pseudomallei and mitigate the pathological consequences of melioidosis.

Biological Validation of a Chemical Effluent Decontamination System.

Introduction: Failure of an existing effluent decontamination system (EDS) prompted the consideration of commercial off-the-shelf solutions for decontamination of containment laboratory waste. A bleach-based chemical EDS was purchased to serve as an interim solution. Methods: Studies were conducted in the laboratory to validate inactivation of Bacillus spores with bleach in complex matrices containing organic simulants including fetal bovine serum, humic acid, and animal room sanitation effluent. Results: These studies demonstrated effective decontamination of >106 spores at a free chlorine concentration of ≥5700 parts per million with a 2-hour contact time. Translation of these results to biological validation of the bleach-based chemical EDS required some modifications to the system and its operation. Discussion: The chemical EDS was validated for the treatment of biosafety levels 3 and 4 waste effluent using laboratory-prepared spore packets along with commercial biological indicators; however, several issues and lessons learned identified during the process of onboarding are also discussed, including bleach product source, method of validation, dechlorination, and treated waste disposal.

Clindamycin Protects Nonhuman Primates Against Inhalational Anthrax But Does Not Enhance Reduction of Circulating Toxin Levels When Combined With Ciprofloxacin.

BACKGROUND: Inhalational anthrax is rare and clinical experience limited. Expert guidelines recommend treatment with combination antibiotics including protein synthesis-inhibitors to decrease toxin production and increase survival, although evidence is lacking. METHODS: Rhesus macaques exposed to an aerosol of Bacillus anthracis spores were treated with ciprofloxacin, clindamycin, or ciprofloxacin + clindamycin after becoming bacteremic. Circulating anthrax lethal factor and protective antigen were quantitated pretreatment and 1.5 and 12 hours after beginning antibiotics. RESULTS: In the clindamycin group, 8 of 11 (73%) survived demonstrating its efficacy for the first time in inhalational anthrax, compared to 9 of 9 (100%) with ciprofloxacin, and 8 of 11 (73%) with ciprofloxacin + clindamycin. These differences were not statistically significant. There were no significant differences between groups in lethal factor or protective antigen levels from pretreatment to 12 hours after starting antibiotics. Animals that died after clindamycin had a greater incidence of meningitis compared to those given ciprofloxacin or ciprofloxacin + clindamycin, but numbers of animals were very low and no definitive conclusion could be reached. CONCLUSION: Treatment of inhalational anthrax with clindamycin was as effective as ciprofloxacin in the nonhuman primate. Addition of clindamycin to ciprofloxacin did not enhance reduction of circulating toxin levels.