Orit Gat

Search for Potential Vaccine Candidate Open Reading Frames in the Bacillus anthracis Virulence Plasmid pXO1: In Silico and In Vitro Screening

ABSTRACT A genomic analysis of the Bacillus anthracis virulence plasmid pXO1, aimed at identifying potential vaccine candidates and virulence-related genes, was carried out. The 143 previously defined open reading frames (ORFs) (R. T. Okinaka, K. Cloud, O. Hampton, A. R. Hoffmaster, K. K. Hill, P. Keim, T. M. Koehler, G. Lamke, S. Kumano, J. Mahillon, D. Manter, Y. Martinez, D. Ricke, R. Svensson, and P. J. Jackson, J. Bacteriol. 181:6509-6515, 1999) were subjected to extensive sequence similarity searches (with the nonredundant and unfinished microbial genome databases), as well as motif, cellular location, and domain analyses. A comparative genomics analysis was conducted with the related genomes of Bacillus subtilis, Bacillus halodurans, and Bacillus cereus and the pBtoxis plasmid of Bacillus thuringiensis var. israeliensis. As a result, the percentage of ORFs with clues about their functions increased from ∼30% (as previously reported) to more than 60%. The bioinformatics analysis permitted identification of novel genes with putative relevance for pathogenesis and virulence. Based on our analyses, 11 putative proteins were chosen as targets for functional genomics studies. A rapid and efficient functional screening method was developed, in which PCR-amplified full-length linear DNA products of the selected ORFs were transcribed and directly translated in vitro and their immunogenicities were assessed on the basis of their reactivities with hyperimmune anti-B. anthracis antisera. Of the 11 ORFs selected for analysis, 9 were successfully expressed as full-length polypeptides, and 3 of these were found to be antigenic and to have immunogenic potential. The latter ORFs are currently being evaluated to determine their vaccine potential.

The solute-binding component of a putative Mn(II) ABC transporter (MntA) is a novel Bacillus anthracis virulence determinant

Here we describe the characterization of a lipoprotein previously proposed as a potential Bacillus anthracis virulence determinant and vaccine candidate. This protein, designated MntA, is the solute‐binding component of a manganese ion ATP‐binding cassette transporter. Coupled proteomic‐serological screen of a fully virulent wild‐type B. anthracis Vollum strain, confirmed that MntA is expressed both in vitro and during infection. Expression of MntA is shown to be independent of the virulence plasmids pXO1 and pXO2. An mntA deletion, generated by allelic replacement, results in complete loss of MntA expression and its phenotypic analysis revealed: (i) impaired growth in rich media, alleviated by manganese supplementation; (ii) increased sensitivity to oxidative stress; and (iii) delayed release from cultured macrophages. The ΔmntA mutant expresses the anthrax‐associated classical virulence factors, lethal toxin and capsule, in vitro as well as in vivo, and yet the mutation resulted in severe attenuation; a 104‐fold drop in LD50 in a guinea pig model. MntA expressed in trans allowed to restore, almost completely, the virulence of the ΔmntA B. anthracis strain. We propose that MntA is a novel B. anthracis virulence determinant essential for the development of anthrax disease, and that B. anthracisΔmntA strains have the potential to serve as platform for future live attenuated vaccines.

Interrelationship between Dendritic Cell Trafficking and Francisella tularensis Dissemination following Airway Infection

Francisella tularensis, the etiological agent of the inhalation tularemia, multiplies in a variety of cultured mammalian cells. Nevertheless, evidence for its in vivo intracellular residence is less conclusive. Dendritic cells (DC) that are adapted for engulfing bacteria and migration towards lymphatic organs could serve as potential targets for bacterial residence and trafficking. Here, we focus on the in vivo interactions of F. tularensis with DC following airway infection of mice. Lethal airway infection of mice with the live vaccine strain (LVS) results in trafficking of a CD11bhigh/CD11cmed/autofluorescencelow DC subset from the respiratory tract to the draining mediastinal lymph node (MdLN). Simultaneously, a rapid, massive bacterial colonization of the MdLN occurs, characterized by large bacterial foci formation. Analysis of bacteria in the MdLN revealed a major population of extracellular bacteria, which co-exists with a substantial fraction of intracellular bacteria. The intracellular bacteria are viable and reside in cells sorted for DC marker expression. Moreover, in vivo vital staining experiments indicate that most of these intracellular bacteria (∼75%) reside in cells that have migrated from the airways to the MdLN after infection. The correlation between DC and bacteria accumulation in the MdLN was further demonstrated by manipulating DC migration to the MdLN through two independent pathways. Impairment of DC migration to the MdLN, either by a sphingosine-1-phosphate receptor agonist (FTY720) or by the D prostanoid receptor 1 agonist (BW245C), resulted in reduced bacterial colonization of MdLN. Moreover, BW245C treatment delayed the onset of morbidity and the time to death of the infected mice. Taken together, these results suggest that DC can serve as an inhabitation niche for F. tularensis in the early stages of infection, and that DC trafficking plays a role in pathogen dissemination. This underscores the therapeutic potential of DC migration impairing drugs in tularemia treatment.

Oral Spore Vaccine Based on Live Attenuated Nontoxinogenic Bacillus anthracis Expressing Recombinant Mutant Protective Antigen

ABSTRACT An attenuated nontoxinogenic nonencapsulated Bacillus anthracis spore vaccine expressing high levels of recombinant mutant protective antigen (PA), which upon subcutaneous immunization provided protection against a lethal B. anthracis challenge, was found to have the potential to serve also as an oral vaccine. Guinea pigs immunized per os with the recombinant spore vaccine were primed to B. anthracis vegetative antigens as well as to PA, yet only a fraction of the animals (30% to 50%) mounted a humoral response to all of these antigens. Protective immunity provided by per os immunization correlated with a threshold level of PA neutralizing antibody titers and was long-lasting. Protection conferred by per os immunization was attained when the vaccine was administered in the sporogenic form, which, unlike the vegetative cells, survived passage through the gastrointestinal tract. A comparison of immunization of unirradiated spores with immunization of γ-irradiated spores demonstrated that germination and de novo synthesis of PA were prerequisites for mounting an immune protective response. Oral immunization of guinea pigs with attenuated B. anthracis spores resulted in a characteristic anti-PA immunoglobulin isotype profile (immunoglobulin [G1 IgG1] versus IgG2), as well as induction of specific anti-PA secretory IgA, indicating development of mucosal immunity.

Use of a Promoter Trap System in Bacillus anthracis and Bacillus subtilis for the Development of Recombinant Protective Antigen-Based Vaccines

ABSTRACT We have recently reported Bacillus anthracis attenuated live vaccine strains efficiently expressing recombinant protective antigen (rPA) and have shown a direct correlation between the level of rPA secreted by these cells and efficacy (S. Cohen, I. Mendelson, Z. Altboum, D. Kobiler, E. Elhanany, T. Bino, M. Leitner, I. Inbar, H. Rosenberg, Y. Gozes, R. Barak, M. Fisher, C. Kronman, B. Velan, and A. Shafferman, Infect. Immun. 68:4549-4558, 2000). To isolate more potent Bacillus promoters for a further increase in the production of rPA, we developed a promoter trap system based on various gfp reporter genes adapted for use in both Bacillus subtilis and B. anthracis backgrounds. Accordingly, a B. anthracis library of 6,000 clones harboring plasmids with chromosomal B. anthracis DNA fragments inserted upstream from gfpuv was constructed. Based on fluorescence intensity, 57 clones carrying potentially strong promoters were identified, some of which were DNA sequenced. The most potent B. anthracis promoter identified (Pntr; 271 bp) was 500 times more potent than the native pagA promoter and 70 times more potent than the α-amylase promoter (Pamy). This very potent promoter was tested along with the other promoters (which are three, six, and eight times more potent than Pamy) for the ability to drive expression of rPA in either B. subtilis or B. anthracis. The number of cell-associated pre-PA molecules in B. anthracis was found to correlate well with the strength of the promoter. However, there appeared to be an upper limit to the amount of mature PA secreted into the medium, which did not exceed that driven by Pamy. Furthermore, the rPA constructs fused to the very potent promoters proved to be deleterious to the bacterial hosts and consequently led to genetic instability of the PA expression plasmid. Immunization with attenuated B. anthracis expressing rPA under the control of promoters more potent than Pamy was less efficient in eliciting anti-PA antibodies than that attained with Pamy. The results are consistent with the notion that overexpression of PA leads to severe secretion stress and have practical implications for the design of second-generation rPA-based vaccines.

Isolation and chimerization of a highly neutralizing antibody conferring passive protection against lethal Bacillus anthracis infection.

Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD50 B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD50 of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.

In Vitro Screen of Bioinformatically Selected Bacillus anthracis Vaccine Candidates by Coupled Transcription, Translation, and Immunoprecipitation Analysis

The availability of the Bacillus anthracis genome sequence allowed for in silico selection of a few hundred open reading frames (ORFs) as putative vaccine candidates. To screen such a vast number of candidate ORFs, without resorting to laborious cloning and protein purification procedures, methods were developed for generation of PCR elements, compatible with in vitro transcription-translation and immunoprecipitation, as well as with their evaluation as DNA vaccines. Protocols will be provided for application of these methods to analyze the anti-B. anthracis antibody repertoire of hyperimmune sera or sera from convalescent and from DNA-vaccinated animals.

Reverse Vaccinology in Bacillus anthracis

In search for antigens which may form the basis for improved subunit or live attenuated B. anthracis vaccines, extensive genomic, proteomic and serologic analyses coupled with functional screens for surface exposed and/or secreted proteins, were carried out. The screens resulted in selection of over 50 promising novel in-vivo expressed immunogens, classified as S-Layer Homology (SLH) proteins, repeat proteins, hydrolytic enzymes and ABC transporters. DNA vaccination experiments established that most of these novel antigens are indeed able to elicit a strong humoral response. Yet, unlike the major B. anthracis immunogen Protective Antigen (PA), none of the selected immunogens could provide protection against a subsequent virulent B. anthracis strain challenge. When over-expressed in an attenuated non-toxinogenic and non-encapsulated B. anthracis platform strain, at least three of the novel antigens did confer partial protection against a lethal B. anthracis challenge.

Proteomic Studies of Bacillus anthracis Reveal In Vitro CO2-Modulation and Expression During Infection of Extracellular Proteases

A comparative proteomic study of secretomes of virulent and avirulent Bacillus anthracis strains in various culturing conditions, including those encountered in the host (high CO2/bicarbonate), enabled identification of approximately 70 proteins representing collectively more than 99% of the secretome. In-vivo expression of 50 proteins was established by 2-dimension Western-analysis using anti B. anthracis immune sera. Many of the abundant proteins harbor features characteristic of virulence determinants and exhibit different patterns of expression. In minimal medium, virulent and avirulent B. anthracis strains manifest similar protein signatures and the metalloprotease NprA, (previously suggested to act in the context of a starvation-induced mechanism), represents 90% of the total secretome. Under high CO2/bicarbonate, NprA is repressed (possibly by a mechanism which preserves toxin integrity), while other proteins, including the bacterial toxins, are induced. One of the immunogens observed to be induced under high CO2-tension, was HtrA. We investigated the phenotype associated with disruption of HtrA by biochemical and proteomic approaches. The HtrA- bacteria are severely affected in their ability to respond to stress and fail to secrete the most abundant extracellular protease NprA. Most surprisingly, HtrA- cells do not possess the characteristic S-layer. This unique phenotype may have important implications for the role of HtrA in manifestation of B. anthracis virulence. Furthermore, the data show that distinct CO2/bicarbonate responsive chromosome-and plasmid-encoded regulatory factors modulate the secretion of potential novel virulence factors, most of which are associated with extracellular proteolytic activities.

Effect of Disruption of mglA on the Virulence and Immunogenicity of the Francisella tularensis Live Vaccine Strain (LVS)

MglA (Macrophage Growth Locus A) is a pleiotropic transcription factor controlling the expression pattern of more than 100 genes in Francisella novicida, including all the ORFs located on the pathogenicity island. To further probe the role of MglA in the pathogenicity of the related strain Francisella tularensis LVS, we generated a mutant LVS strain in which the mglA locus was disrupted by insertion of a non-polar selectable marker cassette.