Patent Grant
9713637
Priority is claimed as a 371 of international application number PCT/IL2012/050254, filed on Jul. 18, 2012; which claims priority to Israeli patent application serial number 214246, filed on Jul. 21, 2011.
The present invention relates to infectious diseases. In particular, the present invention relates to vaccine compositions for the prophylaxis of Bacillus cereus sensu lato strain, such as Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis. In one particular aspect the invention relates to the prophylaxis of anthrax.
The gram-positive spore-forming obligate-pathogen Bacillus anthracis (also referred to as B. anthracis) represents the etiological agent of anthrax, a currently rare disease in humans, yet potentially associated with intentional bioterror use. In the most severe respiratory form, B. anthracis infection is initiated by inhalation of spores which germinate into fast dividing vegetative cells that secrete toxins and virulence factors during growth in the host, resulting in massive bacteremia and consequently generalized systemic failure and death.
The lethality of anthrax has been attributed to two main aspects of B. anthracis pathogenesis: the activity of the bacterial exotoxins and the remarkable proliferous nature of the bacteria in the host. B. anthracis secretes two exotoxins, Lethal Toxin (LT) and Edema Toxin (ET) composed of binary combinations of the three proteins Protective Antigen (PA), Lethal Factor (LF) and Edema Factor (EF). PA, the common subunit of both toxins, is not harmful by itself, yet it plays the essential role of binding to a receptor on the surface of host target cells and mediating the intracellular translocation of the lethal subunits of the toxin complex—LF (a zinc protease which together with PA forms the exotoxin LT) and EF (an adenylate cyclase which together with PA constitutes the exotoxin ET). PA elicits a protective immune response and therefore its administration represents the basis for all preventive anthrax countermeasures (Chitlaru et al., 2011) both for pre and post-exposure prophylaxis. The three components of the toxin, are encoded by genes located on pXO1, one of the two virulence plasmids naturally harbored by B. anthracis. A second well-established virulence factor is represented by a poly D-glutamate anti-phagocytic capsule synthesized by enzymes encoded by genes located on the second native plasmid pXO2. The virulence of B. anthracis is governed by regulatory factors which affect the synthesis of the virulence determinants and which coordinate cross-talk circuits linking chromosome and plasmid located genes.
Anthrax is acknowledged as a toxinogenic disease, owing to the lethality of its major toxins. Yet, during infection, B. anthracis secretes a large number of proteins, many of which bear biological functions indicative of a role in the onset and progression of the disease.
For all living organisms, quality control of protein synthesis is a vital activity. One central player in the context of protein quality control is represented by the HtrA (High Temperature Requirement A) family of serine proteases, which are structurally and functionally conserved across a wide range of evolutionary distinct phylogenetic classes both in prokaryots and eukaryots. HtrA proteins exhibit the dual biological activities of chaperones and proteases, and as such, bacterial HtrA has a known function in the turnover of damaged or malfolded proteins that may deleteriously accumulate, particularly under stress conditions. The HtrA family of proteins (often referred as Deg or Do serine protease) was originally identified in E. coli as essential for the response to heat shock, and subsequently shown to consist of the three chaperons DegP, DegQ and DegS, active in the correct folding and/or in the proteolytic processing and maturation of some proteins, as well as in the degradation of abnormal exported polypeptides. HtrA proteins, exhibit a characteristic structure, composed of an N-terminal trypsin-like serine protease domain and at least one C-terminal PDZ domain which recognizes substrates and activates the protease function. In E. coli and B. subtillis, the HtrA family of proteases are important for the survival of the bacteria under different stress regimens. In addition, in Gram positive bacteria, the HtrA chaperones/proteases are closely associated with the SecA membrane-translocation machinery. For example, in Streptococcus piogenes, HtrA is localized at the ExportA membranal complex dedicated to the biogenesis of secreted proteins. In some cases, HtrA was invoked as being directly involved in the proteolytic processing or secretion of specific virulence-associated proteins such as SpeB and Hemolysin in Streptococcus pyogenes, Pertussis toxin S1 and possibly Adhesin P1 of Streptococcus mutans. Furthermore, attenuated Salmonella enterica mutated in the htrA gene are effective live vaccines as well as possible vectors for delivery of recombinant heterologous antigens, compatible with human use.
The search for bacterial proteins fulfilling functions which sustain the outstanding ability of B. anthracis to expand in the host represented the objective of extensive genomic-proteomic-serologic screens of B. anthracis surface and secreted proteomes (reviewed in Chitlaru et al., 2009, and in Shaferman et al., 2010). Based on these studies, a list of immunogenic proteins could be assembled, many of which representing potential novel B. anthracis virulence factors (Chitlaru et al., 2007; Gat et al. 2006) for subsequent studies addressing their ability to serve as the basis for future improved vaccines, therapeutic intervention and diagnostics. HtrA was distinguished in these studies (i) by its assignment to a regulon which comprises secreted proteins exhibiting a pattern of expression similar to that of the toxin (elevated expression in the presence of CO2), (ii) by its high immunogenicity which indicates in-vivo expression in the host, and (iii) by the observation that significant amounts of HtrA can be detected in the circulation of infected animals relatively early in infection (WO2010/109451; Sela Abramovich et al., 2009). By examining the phenotype associated with disruption of the htrA gene, the present invention provides evidence that HtrA is directly or indirectly involved in the export of some bacterial secreted factors (other than the bacterial toxin), is required for the resistance of B. anthracis to stress conditions, it is unexpectedly essential for manifestation of B. anthracis virulence, and most importantly B. anthracis strains in which htrA gene was disrupted represent efficacious and safe immunization means for providing protection against B. anthracis infection.
It is an object of the present invention to provide an isolated Bacillus anthracis (B. anthracis) strain in which the htrA gene of said B. anthracis is silenced. Thus, encompassed by the invention is such a strain in which the htrA gene is mutated by the removal of said gene.
It is another object of the invention to provide an isolated Bacillus anthracis (B. anthracis) strain in which the htrA gene of said B. anthracis is silenced, which can be used for the preparation of a vaccine.
It is yet another object of the invention to provide an isolated B. anthracis strain in which the htrA gene of said B. anthracis is disrupted, which can be used as a platform for expression and protective immunization with homologous and heterologous bacterial antigens
In one aspect the invention relates to an isolated Bacillus cereus sensu lato strain, selected from Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis, in which the htrA gene or any part thereof is silenced. One strain of particular interest is the Bacillus anthracis (B. anthracis).
Accordingly, in one aspect the invention relates to an isolated B. anthracis strain in which the htrA gene or any part thereof is mutated by removal of the nucleotide sequence set forth in SEQ ID NO: 27 from the genome of said B. anthracis strain. Thus in one aspect the invention relates to a B. anthracis mutant strain having a disrupted, partially disrupted or mutated htrA gene.
In another aspect the invention relates to a vaccine comprising the isolated strain of Bacillus cereus sensu lato strain. An illustrative example of such vaccine is that which comprises the abovementioned B. anthracis mutant strain.
The vaccines according to the invention are useful for the prophylaxis of anthrax infection in a mammalian subject in need thereof.
The invention further encompasses the use of an isolated B. anthracis strain, expressing a homologous and/or a heterologous DNA integrated therein, for the preparation of a vaccine.
Similarly, the invention encompasses the use of an isolated B. anthracis strain, expressing a homologous and/or a heterologous antigen, for the preparation of a vaccine.
The invention is further directed to an isolated B. anthracis strain that is suitable as a backbone for the preparation of a vaccine against a heterologous infectious pathogen.
In another aspect the invention is directed to an isolated B. anthracis or a B. anthracis mutant strain, for use in the prophylaxis of anthrax, particularly for use in the preparation of a vaccine for the prophylaxis of anthrax.
All the above and other characteristics and advantages of the invention will be further understood through the following illustrative and non-limitative description of embodiments thereof, with reference to the appended drawings. In the drawings the same numerals are sometimes used to indicate the same elements in different drawings.
Survival rates of guinea pigs inoculated sub-cutaneously with spores of the strains Vollum (grey circles and curve), VΔhtrA (black squares and curve), VΔhtrA/HtrA (grey diamonds/dotted curve) and Sterne (pXO1+, pXO2−; open triangles/thin curve). The LD50 values determined for the parental and mutated strains are indicated. Each experimental group included at least 5 animals. Animals were monitored 3 weeks post inoculation. The mean time to death (MTD) recorded for succumbed animals was 2-3 days and did not vary in the various groups.
Cells of the J774.1 macrophage line were infected either with wild type Vollum (white bars), VollumΔhtrA (black bars), or with VΔhtrA/HtrA complemented strain spores (grey bars). Samples were withdrawn at the indicated times, and evaluated by (
a The disappearance from the secretome of spots 1 (EA1), 2 (Sap), 5 (HtrA itself, marked in italics) and 13 (NprA) is extensively discussed in the hereinbelow.
b Spots 3 and 4 represent proteins which contain SLH (S-layer homology) domains. Their absence from the mutated strain may indicate that in addition to Sap and EA1, the abundance of other SLH-containing proteins is affected, as well.
c The spots representing the full-size protein LytE are indicated. The abundance of this protein is not altered upon htrA disruption, yet the two isoforms representing N and C terminal domains (spots 8 and 12, respectively) could be detected only in the parental (htrA+) strain secretome. This HtrA dependent appearance of LytE sub-fragments strongly argues in favor of an involvement (direct or indirect) of HtrA in proteolytic processing of at least some bacterial proteins.
d TasA and VanW are extracellular proteins whose secretion may depend on HtrA.
e Proteins BMP, MntA, YpuA and PrsA, which are more abundant in the htrA mutated strain are all exported polypeptides containing N-terminal signal sequences. BMP, MntA and PrsA are lipoproteins exhibiting the characteristic anchorage cysteine containing N-terminal domain. PrsA is a chaperone with a potential function in the secretion of the toxin. MntA is a demonstrated virulence determinant of B. anthracis.
Coomassie blue and Western blot analysis of Vollum, VΔhtrA and VΔhtrA/HtrA cell pellets or secreted proteins collected from BHI rich media (left panels 1, 2, 3, 4, 5, 6) or NBY low nutrient media (right panels 7, 8, 9, 10, 11, 12). Upper panels: Coomassie blue stained gels; middle and lower panels: corresponding Western blots, probed with anti SLH-domain antibodies (panels 3 and 4), anti-NprA antibodies (panels 9 and 10) and anti-HtrA antibodies (panels 5, 6, 11, 12).
The invention will now be described with reference to specific examples and materials.
The htrA Locus of Bacillus anthracis, Philogenetic Similarity with B. cereus Group Orthologues, Genomic Organization and htrA Targeted Disruption
Phylogenetically, B. anthracis belongs to the group of spore-forming soil bacilli known as Bacillus cereus sensu lato which also includes B. cereus, B. mycoides, B. pseudomycoides and B. thuringiensis. Despite the high degree of physiological and morphologic similarity, as well as extensive chromosomal-DNA sequence homology (for example, Rasko et al.), only B. anthracis is highly pathogenic, while human infections by B. cereus and B. thuringiensis are rare and in most of the cases, not life threatening. Members of the Bacillus cereus group may be regarded as one strain among which, B. anthracis is discerned by the presence of the pXO1 and pXO2 virulence plasmids (encoding 140 and 80 genes, respectively) which confer its unique ability to produce the anthrax-causing toxin. The htrA gene, whose disruption in B. anthracis represents the basis of the present invention, is remarkably conserved among all members of the Bacillus cereus sensu lato group. For example, HtrA from B. anthracis (locus tag BA3660), from B. thuringiensis (NCBI locus tags in the Al Hakam strain BALH_324 and in the Kunkukian strain, BR9727_3357), from B. cereus (NCBI locus tags in the E33L strain BCZK3307 and in strain G9241 strain BCE_G9241_3552) exhibit complete conservation—100% identity in the amino acid sequence.
As depicted in
Throughout this specification HtrA designates the protein encoded by the gene BA3660 in all the genomes of B. anthracis strains in the NCBI data base. This locus tag refers to the “Ames ancestor” genome which is by consensus used for designation of B. anthracis genes.
The B. anthracis HtrA protein exhibits 100% identity with its respective paralogues from all members of the B. cereus sensu lato group: e.g. B. cereus and B. thuringiensis.
To gain an insight into the possible role of B. anthracis HtrA, the inventors have first generated an htrA mutated B. anthracis strain in the background of the avirulent ΔVollum (pXO1−/pXO2−) strain (designated ΔV, see list of strains used in this study in Table 1). Disruption of the htrA gene (indicated herein as ΔhtrA), resulted in the complete abrogation of HtrA expression (
Proteomic Examination of ΔVΔhtrA Secreteomes
In some gram positive bacteria (for example B. subtilis, and Streptococcus pyogenes) HtrA appears to be an integral part of the secretion apparatus and therefore, the bacterial secretome profile may be expected to change due to the htrA gene-disruption. Accordingly, we conducted a proteomic comparison (by high resolution 2D electrophoresis and subsequent mass-spectrometric identification of differentially expressed proteins) of the secretomes of the B. anthracis ΔV and its HtrA disrupted derivative from cultures grown in rich-media (FAG media,
Sap and EA1 (encoded by the two contiguous chromosomal genes sap and eag) represent major components of the proteinous make-up of the cells and bacterial secretome in vitro, and are expressed in vivo during infection. The physiological role of the B. anthracis S-layer in the bacterial expansion in the host is still not fully understood, yet S-layer association (mediated by S layer homology [SLH] domains) may represent a prevalent anchorage modality of exposed proteins on the surface of B. anthracis. The observation that Sap and EA1 are completely absent from the supernatant of the rich-medium culture of ΔVΔhtrA strain (
An additional protein most strikingly down-regulated in the ΔVΔhtrA cells, is the secreted protease NprA (see Western blot analysis in
Disruption of the htrA Gene in the Fully Virulent Vollum Strain and In-Vivo Assessment of the Importance of HtrA for Infection
To directly assess the importance of HtrA in B. anthracis virulence, an htrA disrupted strain as well as a trans-complemented strain expressing HtrA from a transfected plasmid, were generated in the background of the parental fully virulent Vollum strain containing the native virulence plasmids pXO1 and pXO2. Under in vitro optimal culturing conditions, the VΔhtrA and the corresponding HtrA transcomplemented strains exhibited growth profiles indistinguishable from the parental Vollum strain. Experimental animals were infected sub-coutaneously (SC) with increasing doses of spores of the parental virulent Vollum, the VΔhtrA, and the transcomplemented VΔhtrA/HtrA strains, and their survival was monitored for 14 days (
In view of the effect of the htrA disruption on the levels of S-layer proteins and NprA, observed in the ΔVΔhtrA strain, expression of these proteins was examined also in the VollumΔhtrA strain (
HtrA Mutated B. Anthracis Bacteria Exhibit Increased Sensitivity to Stress
To shed some light on the phenotypic characteristics which may be responsible for the remarkable attenuation of the htrA disrupted strain, and considering the known involvement of proteins belonging to the HtrA family in stress response in other bacteria, the inventors have examined the ability of the B. anthracis htrA disrupted strains to withstand a variety of stress regimens, compared to the parental virulent WT strains (
Most notably, in all instances, the reduced growth rate under stress conditions could be fully restored to a wild-type level by expression of HtrA in the trans-complemented strain (
Altered Ability of VollumΔhtrA Bacteria to Propagate in Cultured Macrophages
The interaction of B. anthracis spores with phagocytic cells is critical in anthrax pathogenesis representing the initial step of infection, required for germination of the spores into toxin producing bacilli and systemic dissemination of the bacteria. Furthermore, the intra-macrophage milieu exerts severe environmental insults, in particular oxidative stress, upon invading micro-organisms, representing a primary anti-bacterial sentinel mechanism. Therefore, in view of the results reported above, the ability of the htrA mutated strain to propagate in phagocytic cells was addressed in the J774.1 murine-macrophage infection assay. This assay monitors the expansion of the bacteria (by viable counting) and lysis of the macrophages (which may be quantified by release into the medium of the intracellular enzyme lactate dehydrogenase [LDH]) following internalization of spores. Typically, B. anthracis bacilli can be detected in the supernatant of infected J774.1 cells 4 hours post infection (multiplicity of infection [MOI] 1:5), concomitant with the onset of macrophage lysis. The data depicted in
The Vollum ΔhtrA Attenuated Spores can Induce Effective Protective Immunity
Given the remarkable attenuation of virulence promoted by htrA-disruption, the inventors have examined whether administration of VΔhtrA spores may confer protective anthrax immunity. To address this issue, the immune status of guinea pigs immunized with ΔhtrA spores was addressed by determining the anti PA humoral response and by a subsequent challenge with fully virulent spores (
As depicted in
The results are consistent with the observation (
Disruption of the htrA Gene in the Non-Capsular Sterne Strain and In-Vivo Assessment of Virulence of the Sterne ΔhtrA Strain
The B. anthracis Sterne strain containing only the native virulence plasmids pXO1, was developed more than 60 years ago as a vaccine strain, yet due its toxicity (evidenced by animal experimentation), it does not meet the safety requirements mandated for human use and therefore is accepted in the Western world for veterinary purposes only. Following realization of the importance of HtrA in B. anthracis virulence and that HtrA is essential for the full manifestation of B. anthracis pathogenicity by a mechanism that does not involve the anthrax exotoxins, an htrA disrupted strain was generated by the inventors also in the background of the Sterne strain, according to the same approach employed for the generation of the htrA disrupted strain in the background of the Vollum strain. The HtrA locus tag in the B. anthracis Sterne genome in the NCBI data base is BAS3395. Experimental animals were infected sub-coutaneously (SC) with increasing doses of spores of the parental virulent Sterne and the SterneΔhtrA strain and their survival was monitored (
Further, the inventors examined whether administration of SterneΔhtrA spores may confer protective anthrax immunity. To address this issue, the immune status of guinea pigs immunized with ΔhtrA Sterne spores was addressed by determining the anti PA humoral response and by a subsequent challenge with fully virulent spores (
Use of htrA Disrupted Strains as a Platform for Expression and Protective Immunization with Homologous and Heterologous Bacterial Antigens
Having realized that (i) disruption of the htrA gene provides means for generation of B. anthracis strains exhibiting protective immunization abilities as well as improved safety, and that (ii) this mutation can be successfully introduced in the Vollum, ΔVollum and Sterne strains resulting in similar phenotypes, the inventors provide evidence that the htrA mutation represents also a more general approach for safety improvement in additional B. anthracis derived vaccine strains suitable for prophylaxis of anthrax as well as for other pathogens. Indeed, the inventors have developed in the past a platform strain of B. anthracis based on the ATCC14185 non-encapsulated and non-proteolytic strain, which was further attenuated by curing the plasmid pXO1 which encodes the binary toxin. This strain, designated Δ14185, was used successfully as a platform for expression of recombinant versions of PA, providing long-lasting immunity in guinea-pigs following its administration as spores (Cohen et al., 2000; Gat et al., 2003; Mendelson et al., 2005), see
Disruption of the htrA Gene Affects the Levels of the Extracellular NprA Protease and the S-Layer Proteins Sap and EA1
Proteomic analysis of the secretome revealed two major differences between the ΔhtrA mutants and the corresponding Vollum or ΔV parental strains: (i) the complete absence of the extra-cellular protease NprA and (ii) alterations in the expression of the S-layer proteins Sap and EA1 (
Two additional proteins are profoundly affected by the HtrA null mutation; these are the S-layer proteins Sap and EA1. Strikingly, Sap and EA1 are absent both from the surface and the secreted fraction of the htrA disrupted strain (
HtrA is Essential for Manifestation of B. Anthracis Virulence Possibly Due to its Role in Resilience to Stress
A major observation of the present invention is the dramatic decrease in virulence caused by disruption of the htrA gene (
In Bacillus subtillis there are 3 monocistronic genes encoding HtrA homologous proteins (ykdA, yvtA and yyxA). HtrA—the product of ykdA and HtrB-encoded by yvtA, are highly similar proteins activated by the CssRS two-component system which senses accumulation of misfolded proteins at the membrane cell interface (Noone et al., 2001); furthermore, HtrA and HtrB may mutually compensate each other in mutated B. subtilis strains. In B. anthracis, a paralogue htrB gene is located on the yycGF operon (see
The role of HtrA in the virulence of other pathogens was suggested to reflect one or a combination of the following phenomena: (i) involvement in the processing/secretion of virulence factors; (ii) interaction with some host elements, as was recently described for Helicobacter pylori HtrA and other bacterial proteases which digest host tissue or immune effectors; (iii) HtrA has an important role in stress response, which in turn is responsible for the survival of the bacteria during infection (see examples in Streptococci, Yersinia, Salmonella typhimurium). In the case of B. anthracis, one cannot rule out that HtrA is necessary during infection for proteolysis of host proteins. Such a role is compatible with the fact that, in addition of its membranal localization that would be required for its function in secretion and processing of mal-folded polypeptides, HtrA is one of the most abundant extracellular protease secreted by B. anthracis under conditions mimicking infection (Chitlaru et al., 2006, 2010) as well as early during infection (Sela Abramovich et al., 2009). In this context, it is worth noting the HtrA-dependent effects on proteolysis of the putative LytE extracellular protein (see
It is important to stress that the extent of virulence attenuation associated with htrA disruption is significantly higher than that promoted by disruption of any other reported B. anthracis virulence factors assessed in murine or guinea pig models of anthrax. In view of its essential nature, HtrA represents the basis for the development of novel therapeutic interventions to countermeasure B. anthracis infection.
In certain embodiments, provided is the use of a B. anthracis mutant deficient in HtrA expression in a vaccine. The vaccine according to the present invention is suitable and effective both for pre- and post-exposure prophylaxis.
It should be noted that any reference herein to the disruption of the HtrA gene refers to a full or partial disruption of the gene by the removal of the entire HtrA gene or any part thereof or any mutation therein by any known recombinant techniques such as, for example, homologous recombination, transposon mutagenesis, and others, wherein deletions, insertions or (point) mutations are introduced in the genome.
Furthermore, the present invention demonstrates that the HtrA mutation represents a means by which an improved live attenuated vaccine strain for B. anthracis prevention can be developed.
A specific embodiment of the present invention provides the use of attenuated B. anthracis cells in the expression of various antigens, including heterologous antigens. As shown in
Bacterial Strains, Media, Growth Conditions and Stress Treatments
Bacterial strains, plasmids, and primers used herein are listed in Table 1 and 2 below. The B. anthracis strains used are the fully virulent Vollum strain (pXO1+; pXO2+, denoted V), its derivative—the attenuated strains Vollum (pXO1−; pXO2−, denoted ΔV), and ΔhtrA strains generated in either V or ΔV (Table 1), as well as their corresponding trans-complementation strains (designated VΔhtrA/HtrA and ΔVΔhtrA/HtrA, respectively). Cells were cultured either in FAG media (Cohen et al., 2000), Brain-Heart Infusion (BHI, DIFCO/Becton Dickinson, MD, USA) or NBY low-nutrient content media (0.8% [w/vol] Nutrient Broth [Difco], 0.3% Yeast extract [Difco] and 0.5% Glucose) for up to 24 hours at 37° C. with vigorous agitation. For identification of CO2-induced proteins, cells were grown at 37° C. in NBY supplemented with 0.9% NaHCO3 in hermetically-sealed filled-up flasks, without agitation (referred hereafter as NBY—CO2). NBY—CO2 media promotes very efficient toxin production and capsule synthesis (which can be visualized by negative staining using India ink [Becton Dickinson, MD, USA]). Spores were prepared in Schaeffer's sporulation media at 34° C. for 72 hours vigorously shaken, as described in Cohen et al., 2000. In all cases, cultures were initiated with a fresh starter inoculum diluted to a final OD of 0.1 in the respective media. For examination of the ability of the cells to tolerate various stress regimens, the cells were allowed to grow under optimal conditions for 2.5-3 hours (typically the time needed to enter logarithmic phase), then split into BHI cultures containing the indicated final concentrations of H2O2, EtOH or NaCl, or pre-heated at the indicated temperatures. The growth rate μ (expressed in hr−1) was calculated as 60×[ln(OD2/OD1)]/(t2−t1) where OD1 and OD2 are the optical densities measured at two time-points (t1 and t2 expressed in minutes) along the logarithmic growth phase.
Escherichia coli strains (Table 1) were used for plasmid construction. Antibiotic concentrations used for selection in Luria-Bertani (LB, Difco) agar/broth were: for E. coli strains, ampicillin (Ap, 100 μg ml−1), for B. anthracis strains, kanamycin (Km, 10 μg ml−1), chloramphenicol (Cm, 7.5 μg ml−1) and erythromycin (Em, 5 μg ml−1).
Two-Dimensional Electrophoresis (2-DE) Separation and MS Identification of Secreted Proteins
Secreted proteins were collected from the indicated media and subjected to proteomic 2-DE analysis essentially as described in Chitlaru et al., 2006. In brief, secreted proteins were collected from filtered culture media by overnight precipitation in 10% ice-cold TCA and resuspended in a IEF (isoelectric focusing) solution composed of 8M urea, 4% (w/v) 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 40 mM Tris, 2% Dithiothreitol (DTT) and 0.2 (w/v) Bio-Lyte 3/10 (BioRad). 200 μg total protein were resolved first by IEF on ready-made 17 cm, immobilized pH gradient (IPG) strips (Immobiline DryStrips, Pharmacia), applied to a Protean IEF cell (BioRad). Electrophoretic separation on the second dimension was performed on 12.5% SDS-PAGE on an Ettan DALT II System (Pharmacia). Gels were stained with G-250 Coomassie blue (Bio-safe, BioRad) and spots detected and analyzed by scanning on a GS-800 Calibrated Densitometer assisted by the PDQuest 2-D Software (BioRad). Protein spots were cut from the 2-DE gels and subjected to in-gel overnight digestion with 6.25 μg ml−1 trypsin (Promega). Peptides were eluted with 1% TFA followed by 50% CH3CN, dried and resuspended in 10 μl of 25% CH3CN, 0.1% TFA. Two μl were mixed with an equal volume of a α-cyano matrix (Sigma) and applied to a MALDI-TOF (Matrix Assisted Laser Disorption Ionization-Time of Flight) target. Mass spectra were acquired on a TofSpec 2E apparatus (Micromass) in positive ion reflectron mode. Spectra were compared to theoretical tryptic digest fragments of the genomic sequence of the B. anthracis Ames ancestor genome. Identification of proteins was based on a peptide-coverage of more than 30% and peptide mass deviation between observed and calculated values of less than 100 ppm. Identity of all proteins listed in
Plasmid and Strain Construction
Plasmids and oligonucleotide primers used in this study are summarized in Tables 1 and 2.
The oligonucleotide primers were designed according to the genomic sequence of B. anthracis Ames strain, and prepared using the Expedite synthesizer (Applied Biosystems).
Genomic DNA (containing the chromosomal DNA and the native plasmids, pXO1 and pXO2) was extracted from the Vollum strain, as described in Gat et al., 2008.
PCR amplifications were performed using the Taq (Qiagen) or Expand High Fidelity (Roche) systems.
DNA sequences were determined with the ABI rhodamine termination reaction kit (ABI310 Genetic Analyzer, Applied Biosystems).
For generation of specific anti-HtrA antibodies via DNA immunization, the plasmid pCI-htrA was constructed. Primers HTRA7 (forward) and HTRA8C (reverse) were used to amplify the htrA gene, excluding the 175-bp predicted leader sequence. The PCR product was digested with KpnI and NotI, and cloned in the eukaryotic expression vector pCI, carrying both the T7 promoter for in vitro expression and the eukaryotic cytomegalovirus promoter, for in vivo expression. Large-scale production of pCI-htrA for DNA immunization by gene gun was performed by alkali lysis followed by CsCl gradient centrifugation.
The vector used for disruption of the htrA gene by allelic replacement, pEO-htrA (Table 1), was constructed as follows:
(i) A KmR gene from pDG782 was inserted as a blunt-ended StuI-SmaI 1.5 kb cassette into the StuI site of pEO.
(ii) Into the vector from step i, a NotI-SpeI 526-bp restriction fragment of the htrA 5′ end derived by PCR using HTRA1 (forward) and HTRA2C (reverse) primers, was cloned.
(iii) Finally, a SalI-AscI 512-bp restriction fragment of the htrA 3′ end-derived PCR product (HTRA3 and HTRA4C) was introduced.
For the complementation plasmid pASC-HtrA, the htrA complete gene was cloned as a SnaBI/BamHI-digest of PCR product (HTRA5 and HTRA6C primers), replacing the pagA gene in the previously described vector pASC-α (Cohen et al., 2000).
All plasmids transformed into the Vollum strain were first propagated in a methylation deficient E. coli strain GM2929 (Table 1). B. anthracis cells were electrotransformed as described in Cohen et al., 2000.
To disrupt the htrA gene by homologous recombination, an allelic exchange technique was performed as follows:
plasmid pEO-htrA was introduced into competent cells of the Vollum or ΔVollum strains and transformants were selected for KmR at 30° C. Integrants were recovered by growing transformants in LB broth at 30° C. for 1.5 h, shifting to 38° C. (non-permissive temperature) for 6 h, and then plating serial dilutions on LB plates containing Km, incubated at 38° C. for 12-16 h. Single colonies were selected, resuspended in 0.1 ml LB broth and spotted (5 μl) on LB plates containing Km or Em, then incubated at 42° C. for 12-16 h. Deletion mutants were isolated as KmR EmS clones. The deletion of the internal fragment of htrA and insertion of the KmR cassette into the chromosome were confirmed by PCR using flanking chromosomal primers and primers derived from the Km cassette (primers used: HTRA9/KANA2C and KANA1/HTRA12C for the up-stream and down-stream integration htrA/Km junctions, respectively).
Complementation was accomplished by transforming pHtrA into the htrA mutant strain. The expression of HtrA was verified by Western blot analysis. Correction of the htrA-disruption phenotype by transcomplementation with the plasmid-expressed HtrA, confirms the predicted non-polar nature of the gene-disruption procedure.
B. anthracis
E. coli
E. coli -eukaryotic shuttle vector for DNA immunization.
B. anthracis allelic replacement suicide vector. E. coli/
Bacillus shuttle vector. A derivative of pHV1249.
E. coli-Bacillus expression vector, carrying the pagA gene.
RNA Isolation and Real Time qRT-PCR (Quantitative Reverse Transcriptase PCR) Analyses
Total RNA was extracted from bacteria grown for 6 hours (early stationary), using the RiboPure™-Bacteria Kit (Ambion). Contaminating genomic DNA was removed by DNase treatment according to manufacturer's instructions. RNA was quantified spectrophotometrically, and its integrity was examined by Agarose gel electrophoresis. cDNA was generated with Omniscript™ reverse transcriptase (Qiagen), 10 mM of Random Primers 9 (BioLabs) and 500 ng of RNA. Decimal dilutions (1:10, 1:100) of the cDNA preparations were amplified in triplicated experimental groups using 500 nM gene specific primers, 5 mM Magnesium, 0.25 mM dNTP, Manufacturer-supplied PCR buffer, 100 nM Super ROX (BioSearch technologies), AmpliTaq Gold® DNA polymerase (Applied Biosystems) and EVA green (Biotium Inc).
Experiments were repeated three times for each gene analysed using the 7500 ABI Real Time PCR System (Applied Biosystems). Plasmid DNA containing the analysed gene or standard chromosomal spore DNA (10-10,000 copies/reaction) served as a reference for cDNA quantification. In all cases, control groups using the products of mock reverse transcriptase reactions (no reverse transcriptase), served for determining possible chromosomal DNA contaminations. The values determined in these groups, typically representing less than 5% of the total copies measured, were substracted from the final expression value.
Primers were selected using the Primer Express software (ABI). The following primer combinations were used (Table 2): EAI1/EAI2C for determining expression of eag; GRO1/GRO2C and GRO3/GRO4C for groEL; HTRA11/HTRA12C for htrA; HTRB1 and HTRB2C for htrB; NPRA1/NPRA2C for nprA; SAP1/SAP2C for sap.
SDS-PAGE, Western Blot Analyses, Antibodies, and ELISA
Regular 1-dimensional SDS-PAGE was carried out on 4-12% NuPAGE® Bis-Tris gels (Invitrogen) using Precision Plus Molecular weight markers (Bio-Rad). Western blots were generated using the Nitrocellulose Western iBlot Gel Transfer Semi-dry system (Invitrogen). Visualisation of immunoreactive bands was carried out by an ECL (electro-chemo-luminescence) reaction (Pierce SuperSignal® West Pico Chemiluminescent substrate kit, Thermo Scientific) mediated by peroxidase-conjugated secondary antibodies (Amersham) and detected by the FUJIFILM LAS-3000 detection system.
The following antibodies were used in this study:
(i) Specific anti-HtrA antibodies, obtained by DNA immunization with the pCI-htrA plasmid in female outbred ICR mice, using the Helios Gene Gun System (Bio-Rad), as described by Gat et al., 2007;
(ii) Antibodies recognizing the S-layer proteins Sap or EA1 were kindly provided by Dr. E. Elhanany from IIBR. These specific anti-SLH antibodies, were generated by vaccination of NZW female rabbits with a synthetic peptide representing an epitope from the SLH (S-layer homology) N-terminal domain present in Sap and EA1 proteins.
All other antibodies were previously described:
(iii) Mouse anti-NprA (Chitlaru et al., 2006);
(iv) Rabbit anti-PA antibodies (Gat et al., 2003);
(v) Anti LF; and
(vi) anti-EF mouse (Cohen et al., 2000).
Primary and secondary antibodies were used at 1:1000 and 1:5000 dilutions, respectively.
ELISA tests for quantification of PA in sera samples and detection of anti-PA antibodies in the serum of infected animals were carried out as described previously (Cohen et al., 2000).
Macrophage Infection Assay
Infection of macrophages was carried out as previously described (Gat et al., 2005; Weiss et al., 2009). In brief, murine macrophages of the J774.1 line, grown in DMEM supplemented with 10% fetal calf serum (37° C.; 5% CO2 atmosphere) were seeded at a concentration of 105 cells/well in a 24-wells plate, one day prior to infection. Cells were incubated for 1 hour in the presence of 5×105 B. anthracis spores/well, washed extensively with DMEM supplemented with 2.5 μg/ml gentamycin and incubated for 3 hours in the presence of 2.5 μg/ml gentamycin, washed again and layered with fresh media. Under these conditions, essentially all bacteria which are not internalized in the cells are removed. Supernatants from identically treated wells were harvested at various time points and subjected to viable counting of bacteria emerging from the infected macrophages. Macrophage lysis paralleling the multiplication of the bacteria in the culture was monitored by assaying the accumulated lactate dehydrogenase (LDH) released in the media from the damaged cells at the harvesting times, using a standard LDH-L kit (Thermo-Fisher Scientific, Middletown, Va., USA).
Infection of Guinea Pigs
Female Hartley guinea pigs (Charles River Laboratories), weighting 220 to 250 g, were infected with spore preparations of the mutant or trans-complemented strains and compared with the parental Vollum strain. Prior to infecting the animals, the spore preparations were heat-shocked (70° C., 20 min.) to kill residual vegetative bacteria and serially diluted 10-fold in saline, to produce spore suspensions within the range 102-109 per ml. A 0.1 ml spore dose volume was administered subcutaneously (s.c.) to each guinea pig. 5-10 guinea pigs per dose-strain were used. The remaining spore dose suspensions were plated for total viable counts (CFU ml−1). 24-48 hours post infection, animals were bled by cardiac puncture for determination of the levels of PA in peripheral circulation. The animals were observed daily for 21 days. Four weeks post-infection, surviving animals were bled by cardiac puncture for serological studies.
In immunization experiments, 6 weeks post-infection, animals were challenged s.c. with the indicated lethal dose of the parental B. anthracis Vollum strain (LD50=100 spores). The spore lethal dose required to kill 50% (LD50) of the animals was calculated by the method of Reed and Muench (Reed & Muench, 1938).
Animal experiments were approved by the IIBR committee for animal research (protocol GP-10-2010). Experimental animals were handled according to the National Research Council 1996 Guide for the Care and Use of Laboratory Animals and regulations of the IIBR Animal Use Committee.
Although embodiments of the invention have been described by way of illustration, it will be understood that the invention may be carried out with many variations, modifications, and adaptations, without exceeding the scope of the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 214246 | Jul 2011 | IL | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IL2012/050254 | 7/18/2012 | WO | 00 | 1/14/2014 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2013/011509 | 1/24/2013 | WO | A |
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| 6994997 | Poquet et al. | Feb 2006 | B1 |
| 7850970 | Shapiro | Dec 2010 | B2 |
| 8591899 | Shafferman | Nov 2013 | B2 |
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| 20050103712 | Voyce | May 2005 | A1 |
| 20080050774 | Berka et al. | Feb 2008 | A1 |
| 20090209007 | Meima et al. | Aug 2009 | A1 |
| 20100291246 | Brady | Nov 2010 | A1 |
| 20140314716 | Pomerantsev | Oct 2014 | A1 |
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| 2005092372 | Oct 2005 | WO |
| 2009022162 | Feb 2009 | WO |
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| Number | Date | Country | |
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| 20140170188 A1 | Jun 2014 | US |
