Toxin Production by Aeromonas sobria in Natural Environments: River Water vs. Seawater

Aeromonas are water-borne pathogens. They are halotolerant, which means that they can survive in environments whose salt content corresponds to that of seawater (3.0% NaCl). However, the presence of Aeromonas in seawater is extremely rare compared with that in river water. In this study, we tested the ability of Aeromonas sobria to produce toxins in river water and seawater. First, we cultured A. sobria on skim milk agar plates supplemented with either river water (SARW) or seawater (SASW). The bacteria grew on both plates. A clear zone around the bacteria was generated in SARW. However, such a zone was not observed in SASW, suggesting that proteases were not generated in SASW. Subsequently, we cultured A. sobria in a nutrient broth supplemented with either river water (NRW) or with seawater (NSW), and examined the protease activity of their culture supernatants. The protease activity of the culture supernatant from NSW was extremely low compared to that from NRW. The immunoblotting analysis showed that serine protease (ASP) was not produced by the culture in NSW. By contrast, aerolysin-like hemolysin was produced in all conditions examined in this study. This indicates that the salinity of water is deeply involved in the production of ASP by A. sobria.</p

An Improved Tobacco Mosaic Virus (TMV)-Conjugated Multiantigen Subunit Vaccine Against Respiratory Tularemia

Francisella tularensis, the causative agent of the fatal human disease known as tularemia is classified as a Category A Select Agent by the Centers for Disease Control. No licensed vaccine is currently available for prevention of tularemia in the United States. Previously, we published that a tri-antigen tobacco mosaic virus (TMV) vaccine confers 50% protection in immunized mice against respiratory tularemia caused by F. tularensis. In this study, we refined the TMV-vaccine formulation to improve the level of protection in immunized C57BL/6 mice against respiratory tularemia. We developed a tetra-antigen vaccine by conjugating OmpA, DnaK, Tul4, and SucB proteins of Francisella to TMV. CpG was also included in the vaccine formulation as an adjuvant. Primary intranasal (i.n.) immunization followed by two booster immunizations with the tetra-antigen TMV vaccine protected 100% mice against i.n. 10LD100 challenges dose of F. tularensis live vaccine strain (LVS). Mice receiving three immunization doses of tetra-antigen TMV vaccine showed only transient body weight loss, cleared the infection rapidly, and showed minimal histopathological lesions in lungs, liver, and spleen following a lethal respiratory challenge with F. tularensis LVS. Mice immunized with the tetra-antigen TMV vaccine also induced strong ex vivo recall responses and were protected against a lethal challenge as late as 163 days post-primary immunization. Three immunization with the tetra-antigen TMV vaccine also induced a stronger humoral immune response predominated by IgG1, IgG2b, and IgG2c antibodies than mice receiving only a single or two immunizations. Remarkably, a single dose protected 40% of mice, while two doses protected 80% of mice from lethal pathogen challenge. Immunization of Interferongamma (IFN-g)-deficient mice with the tetra-antigen TMV vaccine demonstrated an absolute requirement of IFN-g for the generation of protective immune response against a lethal respiratory challenge with F. tularensis LVS. Collectively, this study further demonstrates the feasibility of TMV as an efficient platform for the delivery of multiple F. tularensis antigens and that tetra-antigen TMV vaccine formulation provides complete protection, and induces long-lasting protective and memory immune responses against respiratory tularemia caused by F. tularensis LVS

Development of a Multivalent Subunit Vaccine against Tularemia Using Tobacco Mosaic Virus (TMV) Based Delivery System

Francisella tularensisis a facultative intracellular pathogen, and is the causative agent of a fatal human disease known as tularemia. F. tularensis is classified as a Category A Biothreat agent by the CDC based on its use in bioweapon programs by several countries in the past and its potential to be used as an agent of bioterrorism. No licensed vaccine is currently available for prevention of tularemia. In this study, we used a novel approach for development of a multivalent subunit vaccine against tularemia by using an efficient tobacco mosaic virus (TMV) based delivery platform. The multivalent subunit vaccine was formulated to contain a combination of F. tularensis protective antigens: OmpA-like protein (OmpA), chaperone protein DnaK and lipoprotein Tul4 from the highly virulent F. tularensisSchuS4 strain. Two different vaccine formulations and immunization schedules were used. The immunized mice were challenged with lethal (10xLD100) doses of F. tularensisLVS on day 28 of the primary immunization and observed daily for morbidity and mortality. Results from this study demonstrate that TMV can be used as a carrier for effective delivery of multiple F. tularensisantigens. TMV-conjugate vaccine formulations are safe and multiple doses can be administered without causing any adverse reactions in immunized mice. Immunization with TMV-conjugated F. tularensisproteins induced a strong humoral immune response and protected mice against respiratory challenges with very high doses of F. tularensis LVS. This study provides a proof-of-concept that TMV can serve as a suitable platform for simultaneous delivery of multiple protective antigens of F. tularensis. Refinement of vaccine formulations coupled with TMV-targeting strategies developed in this study will provide a platform for development of an effective tularemia subunit vaccine as well as a vaccination approach that may broadly be applicable to many other bacterial pathogens

Detection and Characterization of Human Group C Rotaviruses in Bangladesh

Group C rotaviruses were detected by reverse transcription-PCR in 14 (2.3%) of 611 group A rotavirus-negative stool specimens from the patients admitted to the International Centre for Diarrhoeal Disease Research, Bangladesh hospital, Dhaka, Bangladesh, during July to December 2003. The low rate of detection suggested that infection with group C rotaviruses was an uncommon cause of hospitalization due to gastroenteritis. In addition, coinfections with pathogenic enteric bacteria were frequently observed in group C rotavirus-infected patients. Nucleotide sequence comparison of the VP4, VP6, and VP7 genes revealed that the Bangladeshi group C rotaviruses were most similar to Nigerian group C rotavirus strains. Phylogenetic analysis suggested that all human group C rotaviruses, including the strains isolated in our study, clustered in a monophyletic branch, which was distantly related to the branch comprised of animal group C rotaviruses

Identification of a live attenuated vaccine candidate for tularemia prophylaxis.

Francisella tularensis is the causative agent of a fatal human disease, tularemia. F. tularensis was used in bioweapon programs in the past and is now classified as a category A select agent owing to its possible use in bioterror attacks. Despite over a century since its discovery, an effective vaccine is yet to be developed. In this study four transposon insertion mutants of F. tularensis live vaccine strain (LVS) in Na/H antiporter (FTL_0304), aromatic amino acid transporter (FTL_0291), outer membrane protein A (OmpA)-like family protein (FTL_0325) and a conserved hypothetical membrane protein gene (FTL_0057) were evaluated for their attenuation and protective efficacy against F. tularensis SchuS4 strain. All four mutants were 100-1000 fold attenuated for virulence in mice than parental F. tularensis. Except for the FTL_0304, single intranasal immunization with the other three mutants provided 100% protection in BALB/c mice against intranasal challenge with virulent F. tularensis SchuS4. Differences in the protective ability of the FTL_0325 and FTL_0304 mutant which failed to provide protection against SchuS4 were investigated further. The results indicated that an early pro-inflammatory response and persistence in host tissues established a protective immunity against F. tularensis SchuS4 in the FTL_0325 immunized mice. No differences were observed in the levels of serum IgG antibodies amongst the two vaccinated groups. Recall response studies demonstrated that splenocytes from the FTL_0325 mutant immunized mice induced significantly higher levels of IFN-γ and IL-17 cytokines than the FTL_0304 immunized counterparts indicating development of an effective memory response. Collectively, this study demonstrates that persistence of the vaccine strain together with its ability to induce an early pro-inflammatory innate immune response and strong memory responses can discriminate between successful and failed vaccinations against tularemia. This study describes a live attenuated vaccine which may prove to be an ideal vaccine candidate for prevention of respiratory tularemia

An Improved Tobacco Mosaic Virus (TMV)-Conjugated Multiantigen Subunit Vaccine Against Respiratory Tularemia

Francisella tularensis, the causative agent of the fatal human disease known as tularemia is classified as a Category A Select Agent by the Centers for Disease Control. No licensed vaccine is currently available for prevention of tularemia in the United States. Previously, we published that a tri-antigen tobacco mosaic virus (TMV) vaccine confers 50% protection in immunized mice against respiratory tularemia caused by F. tularensis. In this study, we refined the TMV-vaccine formulation to improve the level of protection in immunized C57BL/6 mice against respiratory tularemia. We developed a tetra-antigen vaccine by conjugating OmpA, DnaK, Tul4, and SucB proteins of Francisella to TMV. CpG was also included in the vaccine formulation as an adjuvant. Primary intranasal (i.n.) immunization followed by two booster immunizations with the tetra-antigen TMV vaccine protected 100% mice against i.n. 10LD100 challenges dose of F. tularensis live vaccine strain (LVS). Mice receiving three immunization doses of tetra-antigen TMV vaccine showed only transient body weight loss, cleared the infection rapidly, and showed minimal histopathological lesions in lungs, liver, and spleen following a lethal respiratory challenge with F. tularensis LVS. Mice immunized with the tetra-antigen TMV vaccine also induced strong ex vivo recall responses and were protected against a lethal challenge as late as 163 days post-primary immunization. Three immunization with the tetra-antigen TMV vaccine also induced a stronger humoral immune response predominated by IgG1, IgG2b, and IgG2c antibodies than mice receiving only a single or two immunizations. Remarkably, a single dose protected 40% of mice, while two doses protected 80% of mice from lethal pathogen challenge. Immunization of Interferon-gamma (IFN-γ)-deficient mice with the tetra-antigen TMV vaccine demonstrated an absolute requirement of IFN-γ for the generation of protective immune response against a lethal respiratory challenge with F. tularensis LVS. Collectively, this study further demonstrates the feasibility of TMV as an efficient platform for the delivery of multiple F. tularensis antigens and that tetra-antigen TMV vaccine formulation provides complete protection, and induces long-lasting protective and memory immune responses against respiratory tularemia caused by F. tularensis LVS

BB0844, an RpoS-Regulated Protein, Is Dispensable for Borrelia burgdorferi Infectivity and Maintenance in the Mouse-Tick Infectious Cycle▿

The genome of Borrelia burgdorferi, the causative agent of Lyme disease, is comprised of a large linear chromosome and numerous smaller linear and circular plasmids. B. burgdorferi exhibits substantial genomic variation, and previous studies revealed genotype-specific variation at the right chromosomal telomere. A correlation has also been established between genotype and invasiveness. The correlation between chromosome length and genotype and between genotype and invasiveness suggested that a gene(s) at the right chromosome telomere may be required for virulence. Of particular interest was bb0844, an RpoS-regulated gene at the right telomere, the expression of which is induced when the spirochete undergoes adaptation to the mammalian host. The structure of the right chromosomal telomere was examined in 53 B. burgdorferi clinical isolates of various genotypes. Four distinct patterns were observed for bb0844: (i) chromosomal localization, (ii) plasmid localization, (iii) presence on both chromosome and plasmid, and (iv) complete absence. These patterns correlated with the B. burgdorferi genotype. On the basis of available sequence data, we propose a mechanism for the genomic rearrangements that accounts for the variability in bb0844 genomic localization. To further explore the role of BB0844 in the spirochete life cycle, a bb0844 deletion mutant was constructed by allelic exchange, and the viability of wild-type and bb0844 deletion mutants was examined in an experimental mouse-tick infection model. The bb0844 mutant was fully infectious in C3H/HeJ mice by either needle inoculation or tick transmission with B. burgdorferi-infected Ixodes scapularis larvae. Naïve larval ticks acquired both wild-type and mutant spirochetes with equal efficiency from B. burgdorferi-infected mice. The results demonstrate that BB0844 is not required for spirochete viability, pathogenicity, or maintenance in the tick vector or the mammalian host. At present, a defined role for BB0844 in B. burgdorferi cannot be ascertained

Preclinical testing of a vaccine candidate against tularemia.

Tularemia is caused by a gram-negative, intracellular bacterial pathogen, Francisella tularensis (Ft). The history weaponization of Ft in the past has elevated concerns that it could be used as a bioweapon or an agent of bioterrorism. Since the discovery of Ft, three broad approaches adopted for tularemia vaccine development have included inactivated, live attenuated, or subunit vaccines. Shortcomings in each of these approaches have hampered the development of a suitable vaccine for prevention of tularemia. Recently, we reported an oxidant sensitive mutant of Ft LVS in putative EmrA1 (FTL_0687) secretion protein. The emrA1 mutant is highly sensitive to oxidants, attenuated for intramacrophage growth and virulence in mice. We reported that EmrA1 contributes to oxidant resistance by affecting the secretion of antioxidant enzymes SodB and KatG. This study investigated the vaccine potential of the emrA1 mutant in prevention of respiratory tularemia caused by Ft LVS and the virulent SchuS4 strain in C57BL/6 mice. We report that emrA1 mutant is safe and can be used at an intranasal (i. n.) immunization dose as high as 1x106 CFU without causing any adverse effects in immunized mice. The emrA1 mutant is cleared by vaccinated mice by day 14-21 post-immunization, induces minimal histopathological lesions in lungs, liver and spleen and a strong humoral immune response. The emrA1 mutant vaccinated mice are protected against 1000-10,000LD100 doses of i.n. Ft LVS challenge. Such a high degree of protection has not been reported earlier against respiratory challenge with Ft LVS using a single immunization dose with an attenuated mutant generated on Ft LVS background. The emrA1 mutant also provides partial protection against i.n. challenge with virulent Ft SchuS4 strain in vaccinated C57BL/6 mice. Collectively, our results further support the notion that antioxidants of Ft may serve as potential targets for development of effective vaccines for prevention of tularemia

Immunization with the <i>FTL_0325</i> mutant induces an early pro-inflammatory cytokine response in lungs.

<p>BALB/c mice (n = 4) were infected with 1×10⁷ CFU of the indicated mutants and wild type <i>F. tularensis</i> LVS. At the indicated times, the levels of pro-inflammatory cytokines were measured in lung homogenates using a Cytometric Bead Array assay. The data are representative of two independent experiments conducted and were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and <i>P</i> values were recorded. <i>**P</i><0.01; ***<i>P<</i>0.001. Ψ = Mice infected with 1×10⁷ CFU of <i>F. tularensis</i> LVS succumbed to infection by day 7 PI and hence were unavailable for comparison.</p