Live Attenuated B. pertussis as a Single-Dose Nasal Vaccine against Whooping Cough

Pertussis is still among the principal causes of death worldwide, and its incidence is increasing even in countries with high vaccine coverage. Although all age groups are susceptible, it is most severe in infants too young to be protected by currently available vaccines. To induce strong protective immunity in neonates, we have developed BPZE1, a live attenuated Bordetella pertussis strain to be given as a single-dose nasal vaccine in early life. BPZE1 was developed by the genetic inactivation or removal of three major toxins. In mice, BPZE1 was highly attenuated, yet able to colonize the respiratory tract and to induce strong protective immunity after a single nasal administration. Protection against B. pertussis was comparable to that induced by two injections of acellular vaccine (aPV) in adult mice, but was significantly better than two administrations of aPV in infant mice. Moreover, BPZE1 protected against Bordetella parapertussis infection, whereas aPV did not. BPZE1 is thus an attractive vaccine candidate to protect against whooping cough by nasal, needle-free administration early in life, possibly at birth

The missing link: Bordetella petrii is endowed with both the metabolic versatility of environmental bacteria and virulence traits of pathogenic Bordetellae

Gross R, Guzman CA, Sebaihia M, et al. The missing link: Bordetella petrii is endowed with both the metabolic versatility of environmental bacteria and virulence traits of pathogenic Bordetellae. BMC Genomics. 2008;9(1): 449.Background: Bordetella petrii is the only environmental species hitherto found among the otherwise host-restricted and pathogenic members of the genus Bordetella. Phylogenetically, it connects the pathogenic Bordetellae and environmental bacteria of the genera Achromobacter and Alcaligenes, which are opportunistic pathogens. B. petrii strains have been isolated from very different environmental niches, including river sediment, polluted soil, marine sponges and a grass root. Recently, clinical isolates associated with bone degenerative disease or cystic fibrosis have also been described. Results: In this manuscript we present the results of the analysis of the completely annotated genome sequence of the B. petrii strain DSMZ12804. B. petrii has a mosaic genome of 5,287,950 bp harboring numerous mobile genetic elements, including seven large genomic islands. Four of them are highly related to the clc element of Pseudomonas knackmussii B13, which encodes genes involved in the degradation of aromatics. Though being an environmental isolate, the sequenced B. petrii strain also encodes proteins related to virulence factors of the pathogenic Bordetellae, including the filamentous hemagglutinin, which is a major colonization factor of B. pertussis, and the master virulence regulator BvgAS. However, it lacks all known toxins of the pathogenic Bordetellae. Conclusion: The genomic analysis suggests that B. petrii represents an evolutionary link between free-living environmental bacteria and the host-restricted obligate pathogenic Bordetellae. Its remarkable metabolic versatility may enable B. petrii to thrive in very different ecological niches

Mutations in ampG and Lytic Transglycosylase Genes Affect the Net Release of Peptidoglycan Monomers from Vibrio fischeri▿ †

The light-organ symbiont Vibrio fischeri releases N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramylalanyl-γ-glutamyldiaminopimelylalanine, a disaccharide-tetrapeptide component of peptidoglycan that is referred to here as “PG monomer.” In contrast, most gram-negative bacteria recycle PG monomer efficiently, and it does not accumulate extracellularly. PG monomer can stimulate normal light-organ morphogenesis in the host squid Euprymna scolopes, resulting in regression of ciliated appendages similar to that triggered by infection with V. fischeri. We examined whether the net release of PG monomers by V. fischeri resulted from lytic transglycosylase activity or from defects in AmpG, the permease through which PG monomers enter the cytoplasm for recycling. An ampG mutant displayed a 100-fold increase in net PG monomer release, indicating that AmpG is functional. The ampG mutation also conferred the uncharacteristic ability to induce light-organ morphogenesis even when placed in a nonmotile flaJ mutant that cannot infect the light-organ crypts. We targeted five potential lytic transglycosylase genes singly and in specific combinations to assess their role in PG monomer release. Combinations of mutations in ltgA, ltgD, and ltgY decreased net PG monomer release, and a triple mutant lacking all three of these genes had little to no accumulation of PG monomers in culture supernatants. This mutant colonized the host as well as the wild type did; however, the mutant-infected squid were more prone to later superinfection by a second V. fischeri strain. We propose that the lack of PG monomer release by this mutant results in less regression of the infection-promoting ciliated appendages, leading to this propensity for superinfection

Comparison of radiolabeled isatin analogs for imaging apoptosis with positron emission tomography

INTRODUCTION: Caspase-3 is one of the executioner caspases activated as a result of apoptosis. Radiolabeled isatins bind to caspase-3 with high affinity and are potential tracers for use with positron emission tomography to image apoptosis. We compared the ability of 2 novel radiolabeled isatins, [(18)F]WC-IV-3 and [(11)C]WC-98, to detect caspase-3 activation in a rat model of cycloheximide-induced liver injury. METHODS: Male Sprague-Dawley rats were treated with cycloheximide and then imaged with microPET 3 hours later with [(18)F]WC-IV-3 and [(11)C]WC-98. Biodistribution studies were also performed simultaneously, with caspase-3 activation verified by fluorometric enzyme assay and Western blots. RESULTS: MicroPET imaging studies demonstrated similar behavior of both tracers but with a lower maximum peak with [(11)C]WC-98 than with [(18)F]WC-IV-3. Biodistribution studies demonstrated increased uptake of both tracers in the liver and spleen, but this was statistically significant only in the liver with both compounds. The level of [(18)F]WC-IV-3 uptake appeared to correlate roughly with rates of caspase-3 activation by the enzyme assay, but the magnitude of difference between treated and control groups was lower than that observed in previously published data with [(18)F]WC-II-89, another radiolabeled isatin analog. Activation was also confirmed in the liver and spleen but not in fat by Western blot. CONCLUSION: [(18)F]WC-IV-3 uptake appears to correlate with increased caspase-3 enzyme activity, but the dynamic range of uptake of these 2 tracers appears to be less than that seen with [(18)F]WC-II-89. Studies are ongoing to verify these results in other animal models of apoptosis

In Vivo Characterization of B. pertussis BPZE1

<div><p>(A) Lung colonization by BPSM (solid lines) and BPZE1 (dotted lines) of adult mice infected intranasally with 10⁶ CFU of BPZE1 or BPSM. The results are expressed as mean (± standard error) CFUs from three to four mice per group, and are representative of two separate experiments. The dashed line represents the limit of bacterial counts.</p><p>(B) Histological analysis of lungs from BPZE1 (upper panel) or BPSM-infected (middle panel) adult mice compared to controls given PBS (lower panel). One week after infection, the lungs were aseptically removed and fixed in formaldehyde. Sections were stained with hematoxylin and eosin, and examined by light microscopy.</p><p>(C) Susceptibility of BPZE1-infected mice to infection by M. tuberculosis. Balb/C mice were infected intranasally with 10⁶ CFU of BPZE1 (filled columns) or received PBS (open columns) and were intranasally infected one week later with 5 × 10⁴M. tuberculosis H37Rv. One week (left columns) and 5 wk (right columns) after M. tuberculosis infection, the M. tuberculosis CFUs present in the lungs were counted. The results are expressed as mean (± standard error) CFUs from four mice per group.</p></div

Phenotypic Characterization of B. pertussis BPZE1

<div><p>(A) Growth rates of BPSM (solid line) and BPZE1 (dotted line) in liquid culture.</p><p>(B) Electron micrographs representative of BPSM (left) and BPZE1 (right) grown in liquid medium for 24 h.</p><p>(C) In vitro adherence of BPSM (filled columns) and BPZE1 (open columns) to human pulmonary epithelial A549 cells (left) and murine macrophage-like J774 cells (right). The results are expressed as means (± standard error) of percentages of binding bacteria relative to the bacteria present in the inoculum from three different experiments.</p></div

Protection against <i>Bordetella</i> Infection

<p>Protection against B. pertussis in adult (A) and infant mice (B) and (C), or against B. parapertussis in infant mice (D). Mice immunized with BPZE1, aPV, or PBS (naive) were challenged with BPSM (A), (B), and (C), or B. parapertussis (D), and lung CFU counts were determined 3 h (open columns) or 7 d (filled columns) later. Results are expressed as mean (± standard error) CFUs from three to four mice per group and are representative of two separate experiments. (C) CFU counts 3 h after BPSM challenge in adult mice vaccinated with BPZE1 or aPV, compared to controls. The dashed lines in panels (A), (B), and (D) represent the limit of bacterial counts.</p

Immune Responses Induced in Infant Mice by BPZE1 or aPV Immunization

<p>(A) Anti-FHA, (B) anti-PTX, (C) anti-B. pertussis IgG heavy and light chain (H+L) titers, and (D) anti-FHA IgG1/IgG2a ratios before (open columns) or 1 wk after BPSM challenge (filled columns) in BPZE1 or aPV immunized 3-wk-old mice, compared to controls. (E) IFN-γ to IL-5 ratios produced by FHA-, PTX- or ConA-stimulated splenocytes from 8-wk-old mice vaccinated 2 mo before with BPZE1 (filled columns) or aPV (open columns), compared to controls (gray columns). Antibodies and cytokines were measured in individual mice, and the results are expressed as mean values (± standard error) for four mice per group tested in triplicate.</p