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.

Dysregulation of TNF-α and IFN-γ expression is a common host immune response in a chronically infected mouse model of melioidosis when comparing multiple human strains of Burkholderia pseudomallei.

BACKGROUND: Melioidosis is endemic in Southeast Asia and Northern Australia and is caused by the Gram-negative, facultative intracellular pathogen Burkholderia pseudomallei. Diagnosis of melioidosis is often difficult because of the protean clinical presentation of the disease, and it may mimic other diseases, such as tuberculosis. There are many different strains of B. pseudomallei that have been isolated from patients with melioidosis, but it was not clear if they could cause a similar disease in a chronic BALB/c murine model of melioidosis. Hence, we wanted to examine chronically infected mice exposed to different strains of B. pseudomallei to determine if there were differences in the host immune response to the pathogen. RESULTS: We identified common host immune responses exhibited in chronically infected BALB/c mice, although there was some heterogeneity in the host response in chronically infected mice after exposure to different strains of B. pseudomallei. They all displayed pyogranulomatous lesions in their spleens with a large influx of monocytes/macrophages, NK cells, and neutrophils identified by flow cytometry. Sera from chronically infected mice by ELISA exhibited elevated IgG titers to the pathogen, and we detected by Luminex micro-bead array technology a significant increase in the expression of inflammatory cytokines/chemokines, such as IFN-γ, IL-1α, IL-1ß, KC, and MIG. By immunohistochemical and in situ RNA hybridization analysis we found that the increased expression of proinflammatory cytokines (IL-1α, IL-1ß, TNF-α, IFN-γ) was confined primarily to the area with the pathogen within pyogranulomatous lesions. We also found that cultured splenocytes from chronically infected mice could express IFN-γ, TNF-α, and MIP-1α ex vivo without the need for additional exogenous stimulation. In addition by flow cytometry, we detected significant amounts of intracellular expression of TNF-α and IFN-γ without a protein transport blocker in monocytes/macrophages, NK cells, and neutrophils but not in CD4+ or CD8+ T cells in splenocytes from chronically infected mice. CONCLUSION: Taken together the common features we have identified in chronically infected mice when 10 different human clinical strains of B. pseudomallei were examined could serve as biomarkers when evaluating potential therapeutic agents in mice for the treatment of chronic melioidosis in humans.

The D-alanyl-d-alanine carboxypeptidase enzyme is essential for virulence in the Schu S4 strain of Francisella tularensis and a dacD mutant is able to provide protection against a pneumonic challenge.

Low molecular mass penicillin binding proteins (LMM PBP) are bacterial enzymes involved in the final steps of peptidoglycan biosynthesis. In Escherichia coli, most LMM PBP exhibit dd-carboxypeptidase activity, are not essential for growth in routine laboratory media, and contributions to virulent phenotypes remain largely unknown. The Francisella tularensis Schu S4 genome harbors the dacD gene (FTT_1029), which encodes a LMM PBP with homology to PBP6b of E. coli. Disruption of this locus in the fully virulent Schu S4 strain resulted in a mutant that could not grow in Chamberlain's Defined Medium and exhibited severe morphological defects. Further characterization studies demonstrated that the growth defects of the dacD mutant were pH-dependent, and could be partially restored by growth at neutral pH or fully restored by genetic complementation. Infection of murine macrophage-like cells showed that the Schu S4 dacD mutant is capable of intracellular replication. However, this mutant was attenuated in BALB/c mice following intranasal challenge (LD50 = 603 CFU) as compared to mice challenged with the parent (LD50 = 1 CFU) or complemented strain (LD50 = 1 CFU). Additionally, mice that survived infection with the dacD mutant showed significant protection against subsequent challenge with the parent strain. Collectively, these results indicate that the DacD protein of F. tularensis is essential for growth in low pH environments and virulence in vivo. These results also suggest that a PBP mutant could serve as the basis of a novel, live attenuated vaccine strain.

In vitro antibiotic susceptibilities of Yersinia pestis determined by broth microdilution following CLSI methods.

In vitro susceptibilities to 45 antibiotics were determined for 30 genetically and geographically diverse strains of Yersinia pestis by the broth microdilution method at two temperatures, 28°C and 35°C, following Clinical and Laboratory Standards Institute (CLSI) methods. The Y. pestis strains demonstrated susceptibility to aminoglycosides, quinolones, tetracyclines, ß-lactams, cephalosporins, and carbapenems. Only a 1-well shift was observed for the majority of antibiotics between the two temperatures. Establishing and comparing antibiotic susceptibilities of a diverse but specific set of Y. pestis strains by standardized methods and establishing population ranges and MIC50 and MIC90 values provide reference information for assessing new antibiotic agents and also provide a baseline for use in monitoring any future emergence of resistance.

Comparison of the early host immune response to two widely diverse virulent strains of Burkholderia pseudomallei that cause acute or chronic infections in BALB/c mice.

Burkholderia pseudomallei is the etiologic agent of melioidosis, which is endemic in Southeast Asia and Northern Australia. We previously found by the intraperitoneal (IP) route that we could discern differences in virulence in mice amongst different strains of B. pseudomallei. We report an early immune response study comparing two strains in our collection which represent the least, B. pseudomallei 1106a, and one of the most, HBPUB10134a, virulent strains in BALB/c mice. B. pseudomallei HBPUB10134a infected mouse spleens contained a 2-3 log higher bacterial burden than mice infected with B. pseudomallei 1106a 3 days post-infection (PI). More and higher amounts of inflammatory cytokines/chemokines were detected in sera and spleen extracts from B. pseudomallei HBPUB10134a than B. pseudomallei 1106a infected mice. The most prominent were IFNγ, IL-1α, IL-1ß, IL-6, IL-10, IP-10, and MIG. After 7 days PI, there was a decrease in bacterial burden in spleens from 1106a infected mice and a decrease in cytokines/chemokines in sera and spleen extracts from both sets of mice. By day 14 PI we saw an increase in monocytes/macrophages, NK cells, and granulocytes in spleens from both sets of mice. No B. pseudomallei HBPUB10134a infected mice survived after this time. In summary, B. pseudomallei HBPUB10134a was more virulent and induced host innate immune responses typical of a more acute-type infection than did B. pseudomallei 1106a which produced a more chronic infection in mice.

Development of real-time PCR assays for specific detection of hmsH, hmsF, hmsR, and irp2 located within the 102-kb pgm locus of Yersinia pestis.

Virulent isolates of three pathogenic Yersinia species (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) harbor a 102-kb chromosomal region which encodes elements critical for virulence. A 35-kb high pathogenicity island is contained in this region, is a known virulence determinant, contains irp1 and irp2 iron-regulating genes. An additional segment, the 68-kb high pathogenicity island, contains genetic elements responsible for conferring the Y. pestis pigmentation phenotype on Congo red agar at 28 °C. Collectively, these contiguous segments are referred to as the pigmentation (pgm) locus, the absence of which results in strain attenuation and exemption from CDC Select Agent status. In this study, we developed a set of four real-time PCR assays to detect the presence or absence of multiple virulence genes located within this region. Specifically, we designed TaqMan(®) PCR assays to individually detect three hemin storage genes (hmsH, hmsF, and hmsR) which are genetic elements that confer the pigmentation phenotype, as well as the iron-regulating status of 25 Y. pestis isolates (representing 23 different strains), thus establishing a molecular based assay capable of determining the pgm status of candidate Y. pestis isolates. Included in the validation process, was a comparison of these real-time PCR assays and newly developed conventional PCR assays targeting much larger areas of the 102-kb region (including one assay spanning hmsR and hmsF, one spanning hmsH and hsmF, one targeting hmsF, and one targeting irp2). There was high concordance between the conventional and real-time PCR assays for all Y. pestis strains tested. The results from the comparative analysis document the specificity and sensitivity of the real-time PCR assays and further solidify the ostensible benefits of real-time PCR over conventional PCR.

Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation.

Before the anthrax letter attacks of 2001, the developing field of microbial forensics relied on microbial genotyping schemes based on a small portion of a genome sequence. Amerithrax, the investigation into the anthrax letter attacks, applied high-resolution whole-genome sequencing and comparative genomics to identify key genetic features of the letters' Bacillus anthracis Ames strain. During systematic microbiological analysis of the spore material from the letters, we identified a number of morphological variants based on phenotypic characteristics and the ability to sporulate. The genomes of these morphological variants were sequenced and compared with that of the B. anthracis Ames ancestor, the progenitor of all B. anthracis Ames strains. Through comparative genomics, we identified four distinct loci with verifiable genetic mutations. Three of the four mutations could be directly linked to sporulation pathways in B. anthracis and more specifically to the regulation of the phosphorylation state of Spo0F, a key regulatory protein in the initiation of the sporulation cascade, thus linking phenotype to genotype. None of these variant genotypes were identified in single-colony environmental B. anthracis Ames isolates associated with the investigation. These genotypes were identified only in B. anthracis morphotypes isolated from the letters, indicating that the variants were not prevalent in the environment, not even the environments associated with the investigation. This study demonstrates the forensic value of systematic microbiological analysis combined with whole-genome sequencing and comparative genomics.

Shiga-Toxin-Producing Strains of Escherichia coli O104:H4 and a Strain of O157:H7, Which Can Cause Human Hemolytic Uremic Syndrome, Differ in Biofilm Formation in the Presence of CO2 and in Their Ability to Grow in a Novel Cell Culture Medium.

One pathogen that commonly causes gastrointestinal illnesses from the consumption of contaminated food is Escherichia coli O157:H7. In 2011 in Germany, however, there was a prominent outbreak of bloody diarrhea with a high incidence of hemolytic uremic syndrome (HUS) caused by an atypical, more virulent E. coli O104:H4 strain. To facilitate the identification of this lesser-known, atypical E. coli O104:H4 strain, we wanted to identify phenotypic differences between it and a strain of O157:H7 in different media and culture conditions. We found that E. coli O104:H4 strains produced considerably more biofilm than the strain of O157:H7 at 37 °C (p = 0.0470-0.0182) Biofilm production was significantly enhanced by the presence of 5% CO2 (p = 0.0348-0.0320). In our study on the innate immune response to the E. coli strains, we used HEK293 cells that express Toll-like receptors (TLRs) 2 or 4. We found that E. coli O104:H4 strains had the ability to grow in a novel HEK293 cell culture medium, while the E. coli O157:H7 strain could not. Thus, we uncovered previously unknown phenotypic properties of E. coli O104:H4 to further differentiate this pathogen from E. coli O157:H7.

Activation of Toll-Like Receptors by Live Gram-Negative Bacterial Pathogens Reveals Mitigation of TLR4 Responses and Activation of TLR5 by Flagella.

Successful bacterial pathogens have evolved to avoid activating an innate immune system in the host that responds to the pathogen through distinct Toll-like receptors (TLRs). The general class of biochemical components that activate TLRs has been studied extensively, but less is known about how TLRs interact with the class of compounds that are still associated with the live pathogen. Accordingly, we examined the activation of surface assembled TLR 2, 4, and 5 with live Tier 1 Gram-negative pathogens that included Yersinia pestis (plague), Burkholderia mallei (glanders), Burkholderia pseudomallei (melioidosis), and Francisella tularensis (tularemia). We found that Y. pestis CO92 grown at 28°C activated TLR2 and TLR4, but at 37°C the pathogen activated primarily TLR2. Although B. mallei and B. pseudomallei are genetically related, the former microorganism activated predominately TLR4, while the latter activated predominately TLR2. The capsule of wild-type B. pseudomallei 1026b was found to mitigate the activation of TLR2 and TLR4 when compared to a capsule mutant. Live F. tularensis (Ft) Schu S4 did not activate TLR2 or 4, although the less virulent Ft LVS and F. novicida activated only TLR2. B. pseudomallei purified flagellin or flagella attached to the microorganism activated TLR5. Activation of TLR5 was abolished by an antibody to TLR5, or a mutation of fliC, or elimination of the pathogen by filtration. In conclusion, we have uncovered new properties of the Gram-negative pathogens, and their interaction with TLRs of the host. Further studies are needed to include other microorganism to extend our observations with their interaction with TLRs, and to the possibility of leading to new efforts in therapeutics against these pathogens.

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.

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.

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.

Genome sequence of the deep-rooted Yersinia pestis strain Angola reveals new insights into the evolution and pangenome of the plague bacterium.

To gain insights into the origin and genome evolution of the plague bacterium Yersinia pestis, we have sequenced the deep-rooted strain Angola, a virulent Pestoides isolate. Its ancient nature makes this atypical isolate of particular importance in understanding the evolution of plague pathogenicity. Its chromosome features a unique genetic make-up intermediate between modern Y. pestis isolates and its evolutionary ancestor, Y. pseudotuberculosis. Our genotypic and phenotypic analyses led us to conclude that Angola belongs to one of the most ancient Y. pestis lineages thus far sequenced. The mobilome carries the first reported chimeric plasmid combining the two species-specific virulence plasmids. Genomic findings were validated in virulence assays demonstrating that its pathogenic potential is distinct from modern Y. pestis isolates. Human infection with this particular isolate would not be diagnosed by the standard clinical tests, as Angola lacks the plasmid-borne capsule, and a possible emergence of this genotype raises major public health concerns. To assess the genomic plasticity in Y. pestis, we investigated the global gene reservoir and estimated the pangenome at 4,844 unique protein-coding genes. As shown by the genomic analysis of this evolutionary key isolate, we found that the genomic plasticity within Y. pestis clearly was not as limited as previously thought, which is strengthened by the detection of the largest number of isolate-specific single-nucleotide polymorphisms (SNPs) currently reported in the species. This study identified numerous novel genetic signatures, some of which seem to be intimately associated with plague virulence. These markers are valuable in the development of a robust typing system critical for forensic, diagnostic, and epidemiological studies.

Laser Scanning Confocal Microscopy Was Used to Validate the Presence of Burkholderia Pseudomallei or B. Mallei in Formalin-Fixed Paraffin Embedded Tissues.

Burkholderia pseudomallei and B. mallei are Gram-negative, facultative intracellular bacteria that cause melioidosis and glanders, respectively. Currently, there are no vaccines for these two diseases. Animal models have been developed to evaluate vaccines and therapeutics. Tissues from infected animals, however, must be fixed in formalin and embedded in paraffin (FFPE) before analysis. A brownish staining material in infected tissues that represents the exopolysaccharide of the pathogen was seen by bright field microscopy but not the actual microorganism. Because of these results, FFPE tissue was examined by laser scanning confocal microscopy (LSCM) in an attempt to see the microorganism. Archival FFPE tissues were examined from ten mice, and five nonhuman primates after exposure to B. pseudomallei or B. mallei by LSCM. Additionally, a historical spleen biopsy from a human suspected of exposure to B. mallei was examined. B. pseudomallei was seen in many of the infected tissues from mice. Four out of five nonhuman primates were positive for the pathogen. In the human sample, B. mallei was seen in pyogranulomas in the spleen biopsy. Thus, the presence of the pathogen was validated by LSCM in murine, nonhuman primate, and human FFPE tissues.

The Impact of Age and Sex on Mouse Models of Melioidosis.

Mouse models have been used to generate critical data for many infectious diseases. In the case of Burkholderia pseudomallei, mouse models have been invaluable for bacterial pathogenesis studies as well as for testing novel medical countermeasures including both vaccines and therapeutics. Mouse models of melioidosis have also provided a possible way forward to better understand the chronicity associated with this infection, as it appears that BALB/c mice develop an acute infection with B. pseudomallei, whereas the C57BL/6 model is potentially more suggestive of a chronic infection. Several unanswered questions, however, persist around this model. In particular, little attention has been paid to the effect of age or sex on the disease outcome in these animal models. In this report, we determined the LD50 of the B. pseudomallei K96243 strain in both female and male BALB/c and C57BL/6 mice in three distinct age groups. Our data demonstrated a modest increase in susceptibility associated with sex in this model, and we documented important histopathological differences associated with the reproductive systems of each sex. There was a statistically significant inverse correlation between age and susceptibility. The older mice, in most cases, were more susceptible to the infection. Additionally, our retrospective analyses suggested that the impact of animal supplier on disease outcome in mice may be minimal. These observations were consistent regardless of whether the mice were injected with bacteria intraperitoneally or if they were exposed to aerosolized bacteria. All of these factors should be considered when designing experiments using mouse models of melioidosis.

Binding Sites of Anti-Lcr V Monoclonal Antibodies Are More Critical than the Avidities and Affinities for Passive Protection against Yersinia pestis Infection in a Bubonic Plague Model.

Plague is a zoonotic disease that is caused by Yersinia pestis. Monoclonal antibodies (mAbs) that bind to the V-antigen, a virulence factor that is produced by Y. pestis, can passively protect mice from plague. An analysis of protective mAbs that bind to V-antigen was made to assess binding sites, avidities, and affinities. Anti-V mAbs were screened for their efficacy in a murine model of plague. Antigen-binding sites of protective V mAbs were determined with a linear peptide library, V-antigen fragment, competitive binding, and surface plasmon resonance. The avidities to the V-antigen was determined by ELISA, and affinities of the mAbs to the V-antigen were determined by surface plasmon resonance. The most protective mAb 7.3 bound to a unique conformational site on the V-antigen, while a less protective mAb bound to a different conformational site located on the same V-antigen fragment as mAb 7.3. The avidity of mAb 7.3 for the V-antigen was neither the strongest overall nor did it have the highest affinity for the V-antigen. The binding site of the most protective mAb was critical in its ability to protect against a lethal plague challenge.

Deletion of Two Genes in Burkholderia pseudomallei MSHR668 That Target Essential Amino Acids Protect Acutely Infected BALB/c Mice and Promote Long Term Survival.

Melioidosis is an emerging disease that is caused by the facultative intracellular pathogen Burkholderia pseudomallei. It is intrinsically resistant to many antibiotics and host risk factors play a major role in susceptibility to infection. Currently, there is no human or animal vaccine against melioidosis. In this study, multiple B. pseudomallei MSHR668 deletion mutants were evaluated as live attenuated vaccines in the sensitive BALB/c mouse model of melioidosis. The most efficacious vaccines after an intraperitoneal challenge with 50-fold over the 50% median lethal dose (MLD50) with B. pseudomallei K96243 were 668 ΔhisF and 668 ΔilvI. Both vaccines completely protected mice in the acute phase of infection and showed significant protection (50% survivors) during the chronic phase of infection. The spleens of the survivors that were examined were sterile. Splenocytes from mice vaccinated with 668 ΔhisF and 668 ΔilvI expressed higher amounts of IFN-γ after stimulation with B. pseudomallei antigens than splenocytes from mice vaccinated with less protective candidates. Finally, we demonstrate that 668 ΔhisF is nonlethal in immunocompromised NOD/SCID mice. Our results show that 668 ΔhisF and 668 ΔilvI provide protective cell-mediated immune responses in the acute phase of infection and promote long term survival in the sensitive BALB/c mouse model of melioidosis.

Assays for the rapid and specific identification of North American Yersinia pestis and the common laboratory strain CO92.

We present TaqMan-minor groove binding (MGB) assays for an SNP that separates the Yersinia pestis strain CO92 from all other strains and for another SNP that separates North American strains from all other global strains.

An alternative approach to combination vaccines: intradermal administration of isolated components for control of anthrax, botulism, plague and staphylococcal toxic shock.

BACKGROUND: Combination vaccines reduce the total number of injections required for each component administered separately and generally provide the same level of disease protection. Yet, physical, chemical, and biological interactions between vaccine components are often detrimental to vaccine safety or efficacy. METHODS: As a possible alternative to combination vaccines, we used specially designed microneedles to inject rhesus macaques with four separate recombinant protein vaccines for anthrax, botulism, plague and staphylococcal toxic shock next to each other just below the surface of the skin, thus avoiding potentially incompatible vaccine mixtures. RESULTS: The intradermally-administered vaccines retained potent antibody responses and were well- tolerated by rhesus macaques. Based on tracking of the adjuvant, the vaccines were transported from the dermis to draining lymph nodes by antigen-presenting cells. Vaccinated primates were completely protected from an otherwise lethal aerosol challenge by Bacillus anthracis spores, botulinum neurotoxin A, or staphylococcal enterotoxin B. CONCLUSION: Our results demonstrated that the physical separation of vaccines both in the syringe and at the site of administration did not adversely affect the biological activity of each component.The vaccination method we describe may be scalable to include a greater number of antigens, while avoiding the physical and chemical incompatibilities encountered by combining multiple vaccines together in one product.