In vivo and in vitro immune responses against Francisella tularensis vaccines are comparable among Fischer 344 rat substrains.

Identifying suitable animal models and standardizing preclinical methods are important for the generation, characterization, and development of new vaccines, including those against Francisella tularensis. Non-human primates represent an important animal model to evaluate tularemia vaccine efficacy, and the use of correlates of vaccine-induced protection may facilitate bridging immune responses from non-human primates to people. However, among small animals, Fischer 344 rats represent a valuable resource for initial studies to evaluate immune responses, to identify correlates of protection, and to screen novel vaccines. In this study, we performed a comparative analysis of three Fischer rat substrains to determine potential differences in immune responses, to evaluate methods used to quantify potential correlates of protection, and to evaluate protection after vaccination. To this end, we took advantage of data previously generated using one of the rat substrains by evaluating two live vaccines, LVS and F. tularensis SchuS4-ΔclpB (ΔclpB). We compared immune responses after primary vaccination, adaptive immune responses upon re-stimulation of leukocytes in vitro, and sensitivity to aerosol challenge. Despite some detectable differences, the results highlight the similarity of immune responses to tularemia vaccines and challenge outcomes between the three substrains, indicating that all offer acceptable and comparable approaches as animal models to study Francisella infection and immunity.

In vitro antiviral activity of stabilized chlorine dioxide containing oral care products.

OBJECTIVE: To determine the in vitro antiviral activity of oral care products containing stabilized chlorine dioxide toward infectious viruses that harbor in the oral cavity. Specfically, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), SARS-CoV, human coronavirus (HCoV) 229E, influenza A (H3N2), rhinovirus type 14, adenovirus type 5, and herpes simplex virus (HSV) type 1 and 2 were examined. METHODS: Validated in vitro suspension virucidal assays were used. Test product was mixed with the test virus for 30, 60, or 120 s, neutralized with sodium thiosulfate, serially diluted in dilution medium in a 96-well plate and incubated in a carbon dioxide incubator for 7 days. The 50% Tissue Culture Infectious Dose per milliliter was determined. RESULTS: Two rinses, one oral spray and one fluoride toothpaste showed log reduction of severe acute respiratory syndrome coronavirus-2 ranging from 1.81 to 2.98 and of influenza A from 2.58 to 4.13, respectively, within 30 s of contact time; similar results were obtained at 60 s. Further, the Ultra Sensitive rinse showed 0.19, 0.75, 1.58, 1.75, 2.66, and 3.24 log reduction of severe acute respiratory syndrome coronavirus, human coronavirus 229E, rhinovirus type 14, adenovirus type 5, and herpes simplex virus type 1 and type 2, respectively, within 30 s of contact time. CONCLUSION: Stabilized chlorine dioxide containing CloSYS® oral care products reduced the viral load of multiple viruses within 30 s. The results warrant further investigation for potential in vivo applications.

Modern Development and Production of a New Live Attenuated Bacterial Vaccine, SCHU S4 ΔclpB, to Prevent Tularemia.

Inhalation of small numbers of Francisella tularensis subspecies tularensis (Ftt) in the form of small particle aerosols causes severe morbidity and mortality in people and many animal species. For this reason, Ftt was developed into a bona fide biological weapon by the USA, by the former USSR, and their respective allies during the previous century. Although such weapons were never deployed, the 9/11 attack quickly followed by the Amerithrax attack led the U.S. government to seek novel countermeasures against a select group of pathogens, including Ftt. Between 2005-2009, we pursued a novel live vaccine against Ftt by deleting putative virulence genes from a fully virulent strain of the pathogen, SCHU S4. These mutants were screened in a mouse model, in which the vaccine candidates were first administered intradermally (ID) to determine their degree of attenuation. Subsequently, mice that survived a high dose ID inoculation were challenged by aerosol or intranasally (IN) with virulent strains of Ftt. We used the current unlicensed live vaccine strain (LVS), first discovered over 70 years ago, as a comparator in the same model. After screening 60 mutants, we found only one, SCHU S4 ΔclpB, that outperformed LVS in the mouse ID vaccination-respiratory-challenge model. Currently, SCHU S4 ΔclpB has been manufactured under current good manufacturing practice conditions, and tested for safety and efficacy in mice, rats, and macaques. The steps necessary for advancing SCHU S4 ΔclpB to this late stage of development are detailed herein. These include developing a body of data supporting the attenuation of SCHU S4 ΔclpB to a degree sufficient for removal from the U.S. Select Agent list and for human use; optimizing SCHU S4 ΔclpB vaccine production, scale up, and long-term storage; and developing appropriate quality control testing approaches.

Genomic characterization of Burkholderia pseudomallei isolates selected for medical countermeasures testing: comparative genomics associated with differential virulence.

Burkholderia pseudomallei is the causative agent of melioidosis and a potential bioterrorism agent. In the development of medical countermeasures against B. pseudomallei infection, the US Food and Drug Administration (FDA) animal Rule recommends using well-characterized strains in animal challenge studies. In this study, whole genome sequence data were generated for 6 B. pseudomallei isolates previously identified as candidates for animal challenge studies; an additional 5 isolates were sequenced that were associated with human inhalational melioidosis. A core genome single nucleotide polymorphism (SNP) phylogeny inferred from a concatenated SNP alignment from the 11 isolates sequenced in this study and a diverse global collection of isolates demonstrated the diversity of the proposed Animal Rule isolates. To understand the genomic composition of each isolate, a large-scale blast score ratio (LS-BSR) analysis was performed on the entire pan-genome; this demonstrated the variable composition of genes across the panel and also helped to identify genes unique to individual isolates. In addition, a set of ~550 genes associated with pathogenesis in B. pseudomallei were screened against the 11 sequenced genomes with LS-BSR. Differential gene distribution for 54 virulence-associated genes was observed between genomes and three of these genes were correlated with differential virulence observed in animal challenge studies using BALB/c mice. Differentially conserved genes and SNPs associated with disease severity were identified and could be the basis for future studies investigating the pathogenesis of B. pseudomallei. Overall, the genetic characterization of the 11 proposed Animal Rule isolates provides context for future studies involving B. pseudomallei pathogenesis, differential virulence, and efficacy to therapeutics.

Glanders: an overview of infection in humans.

Glanders is a highly contagious and often fatal zoonotic disease, primarily of solipds. In the developed world, glanders has been eradicated. However, prior use of B. mallei as a biological weapon and its high mortality in inhalation animal studies has affirmed B. mallei as a biodefense concern. This threat requires the development of new glanders medical countermeasures (MCMs), as there is a lack of an effective vaccine and lengthy courses of multiple antibiotics needed to eradicate B. mallei. Here, we present a literature review of human glanders in which we discuss the clinical epidemiology and risk factors, potential routes of exposure, symptoms, the incubation period, and specific diagnostics. This review focuses on pulmonary glanders, as this is the most likely outcome of a biological weapons attack. Additionally, we outline current treatment regimens and propose a clinical definition of human pulmonary glanders infection.

Complete Genome Sequence of the Encephalomyelitic Burkholderia pseudomallei Strain MSHR305.

We describe the complete genome sequence of Burkholderia pseudomallei MSHR305, a clinical isolate taken from a fatal encephalomyelitis case, a rare form of melioidosis. This sequence will be used for comparisons to identify the genes that are involved in neurological cases.

Genome Sequence of Burkholderia pseudomallei NCTC 13392.

Here, we describe the draft genome sequence of Burkholderia pseudomallei NCTC 13392. This isolate has been distributed as K96243, but distinct genomic differences have been identified. The genomic sequence of this isolate will provide the genomic context for previously conducted functional studies.

Efficacy of post exposure administration of doxycycline in a murine model of inhalational melioidosis.

Burkholderia pseudomallei is the causative agent of melioidosis. Treatment of melioidosis is suboptimal and developing improved melioidosis therapies requires animal models. In this report, we exposed male BALB/c mice to various amounts of aerosolized B. pseudomallei 1026b to determine lethality. After establishing a median lethal dose (LD(50)) of 2,772 colony forming units (cfu)/animal, we tested the ability of doxycycline administered 6 hours after exposure to a uniformly lethal dose of ~20 LD(50) to prevent death and eliminate bacteria from the lung and spleens. Tissue bacterial burdens were examined by PCR analysis. We found that 100% of mice treated with doxycycline survived and B. pseudomallei DNA was not amplified from the lungs or spleens of most surviving mice. We conclude the BALB/c mouse is a useful model of melioidosis. Furthermore, the data generated in this mouse model indicate that doxycycline is likely to be effective in post-exposure prophylaxis of melioidosis.

An objective approach for Burkholderia pseudomallei strain selection as challenge material for medical countermeasures efficacy testing.

Burkholderia pseudomallei is the causative agent of melioidosis, a rare disease of biodefense concern with high mortality and extreme difficulty in treatment. No human vaccines are available that protect against B. pseudomallei infection, and with the current limitations of antibiotic treatment, the development of new preventative and therapeutic interventions is crucial. Although clinical trials could be used to test the efficacy of new medical countermeasures (MCMs), the high mortality rates associated with melioidosis raises significant ethical issues concerning treating individuals with new compounds with unknown efficacies. The US Food and Drug Administration (FDA) has formulated a set of guidelines for the licensure of new MCMs to treat diseases in which it would be unethical to test the efficacy of these drugs in humans. The FDA "Animal Rule" 21 CFR 314 calls for consistent, well-characterized B. pseudomallei strains to be used as challenge material in animal models. In order to facilitate the efficacy testing of new MCMs for melioidosis using animal models, we intend to develop a well-characterized panel of strains for use. This panel will comprise of strains that were isolated from human cases, have a low passage history, are virulent in animal models, and are well-characterized phenotypically and genotypically. We have reviewed published and unpublished data on various B. pseudomallei strains to establish an objective method for selecting the strains to be included in the panel of B. pseudomallei strains with attention to five categories: animal infection models, genetic characterization, clinical and passage history, and availability of the strain to the research community. We identified 109 strains with data in at least one of the five categories, scored each strain based on the gathered data and identified six strains as candidate for a B. pseudomallei strain panel.

Disrupting the luxS quorum sensing gene does not significantly affect Bacillus anthracis virulence in mice or guinea pigs.

Many bacterial species use secreted quorum-sensing autoinducer molecules to regulate cell density- and growth phase-dependent gene expression, including virulence factor production, as sufficient environmental autoinducer concentrations are achieved. Bacillus anthracis, the causative agent of anthrax, contains a functional autoinducer (AI-2) system, which appears to regulate virulence gene expression. To determine if the AI-2 system is necessary for disease, we constructed a LuxS AI-2 synthase-deficient mutant in the virulent Ames strain of B. anthracis. We found that growth of the LuxS-deficient mutant was inhibited and sporulation was delayed when compared with the parental strain. However, spores of the Ames luxS mutant remained fully virulent in both mice and guinea pigs.

Exogenous Yersinia pestis quorum sensing molecules N-octanoyl-homoserine lactone and N-(3-oxooctanoyl)-homoserine lactone regulate the LcrV virulence factor.

LcrV is a key Yersinia pestis antigen, immune regulator, and component of the type III secretion system (T3SS). Researchers have shown that N-acyl-homoserine lactones (AHLs) can down-regulate the expression of the LcrV homolog, PcrV, in Pseudomonas aeruginosa. Using ELISA, western blot, DNA microarray analysis, and real time PCR we demonstrate that the addition of AHL molecules N-octanoyl-homoserine lactone (C8) or N-(3-oxooctanoyl)-homoserine lactone (oxo-C8) to Y. pestis cultures down-regulates LcrV protein expression. DNA microarray analysis shows 10 additional T3SS genes are consistently down-regulated by C8 or oxo-C8. This is the first report demonstrating that AHLs regulate Y. pestis virulence factor expression.

Perturbation of leukocyte function-associated antigen-1/intercellular adhesion molecule-1 results in differential outcomes in cardiac vs islet allograft survival.

BACKGROUND: Various studies indicate the requirements for tolerance induction may vary between different transplanted tissues and organs. Consequently, we compared the efficacy of anti-leukocyte function-associated antigen-1 (LFA-1)/anti-intercellular adhesion molecule-1 (ICAM-1) monoclonal antibody therapy for facilitating cardiac vs islet long-term allograft acceptance in mice. METHODS: BALB/c (H-2d) mouse cardiac or islet allografts were transplanted into recipient CBA/J (H-2k) mice. Monoclonal anti-body therapy with anti-LFA-1, anti-ICAM-1, the combination, or control rat immunoglobulin (Ig) was administered intraperitoneally on Days 0 to 5. Cardiac allograft function was assessed by palpation and islet graft function by blood glucose monitoring. Mixed lymphocyte assays were performed to assess proliferation of CD4 and CD8 T-cells under conditions of stimulator-cell ICAM-1 and/or LFA-1 deficiency. RESULTS: Anti-ICAM-1 therapy resulted in a modest prolongation of cardiac allografts but in pronounced survival of islet allografts. Anti-LFA-1 therapy promoted significant long-term survival of both cardiac and islet allografts. Surprisingly, combined anti-LFA-1/anti-ICAM-1 therapy abrogated long-term islet, but not cardiac, allograft acceptance relative to either monotherapy. Mixed lymphocyte reactions demonstrated complete blockade of CD4 and CD8 T-cell proliferation under conditions of ICAM-1 deficiency alone or in combination with anti-LFA-1 therapy. CONCLUSION: These results indicate that optimal therapies for some allografts (vascularized-heart) may not translate to other types of allografts (cellular-islet). Thus, the type of transplant represents an independent variable for optimizing strategies to promote indefinite allograft acceptance. Complete inhibition of CD4 and CD8 T-cell proliferation during ICAM-1/LFA-1 blockade suggests a threshold signal may be dependent upon ICAM-1/LFA-1 for regulatory tolerance to occur and that this signal may be lost under conditions of minimal graft cellular mass.

CD4-dependent generation of dominant transplantation tolerance induced by simultaneous perturbation of CD154 and LFA-1 pathways.

CD154 and LFA-1 (CD11a) represent conceptually distinct pathways of receptor/ligand interactions (costimulation and adhesion/homing, respectively) that have been effectively targeted to induce long-term allograft acceptance and tolerance. In the current study, we determined the relative efficacy and nature of tolerance induced by mAbs specific for these pathways. In vitro analysis indicated that simultaneous targeting of CD154 and LFA-1 resulted in profound inhibition of alloreactivity, suggesting that combined anti-CD154/anti-LFA-1 therapy could be highly effective in vivo. Thus, we evaluated combining mAb therapies targeting CD154 and LFA-1 for inducing transplantation tolerance to pancreatic islet allografts. Monotherapy with either anti-CD154 or anti-LFA-1 was partially effective for inducing long-term allograft survival, whereas the combination resulted in uniform allograft acceptance in high-responder C57BL/6 recipients. This combined therapy was not lymphocyte depleting and did not require the long-term deletion of donor-reactive T lymphocytes to maintain allograft survival. Importantly, combined anti-CD154/anti-LFA therapy uniquely resulted in "dominant" transplantation tolerance. Therefore, simultaneous perturbation of CD154 and LFA-1 molecules can result in profound tolerance induction not accomplished through individual monotherapy approaches. Furthermore, results show that such regulatory tolerance can coexist with the presence of robust anti-donor reactivity, suggesting that active tolerance does not require a corresponding deletion of donor-reactive T cells. Interestingly, although the induction of this regulatory state was highly CD4 dependent, the adoptive transfer of tolerance was less CD4 dependent in vivo.