We report the draft whole-genome sequence of the nonproteolytic Bacillus anthracis V770-NP1-R strain. Compared to those of other B. anthracis strains, the genome exhibits unique mutations in multiple targets potentially affecting proteolytic functions. One of these mutations is a deletion that disrupts the NprR quorum-sensing regulator of the NprA protease.
Recently we described an unbiased bacterial whole-genome immunoinformatic analysis aimed at selection of potential CTL epitopes located in "hotspots" of predicted MHC-I binders. Applying this approach to the proteome of the facultative intra-cellular pathogen Francisella tularensis resulted in identification of 170 novel CTL epitopes, several of which were shown to elicit highly robust T cell responses. Here we demonstrate that by DNA immunization using a short DNA fragment expressing six of the most prominent identified CTL epitopes a potent and specific CD8+ T cell responses is being induced, to all encoded epitopes, a response not observed in control mice immunized with the DNA vector alone Moreover, this CTL-specific mediated immune response prevented disease development, allowed for a rapid clearance of the bacterial infection and provided complete protection against lethal challenge (10LD50) with F. tularensis holarctica Live Vaccine Strain (LVS) (a total to 30 of 30 immunized mice survived the challenge while all control DNA vector immunized mice succumbed). Furthermore, and in accordance with these results, CD8 deficient mice could not be protected from lethal challenge after immunization with the CTL-polyepitope. Vaccination with the DNA poly-epitope construct could even protect mice (8/10) against the more demanding pulmonary lethal challenge of LVS. Our approach provides a proof-of-principle for selecting and generating a multi-epitpoe CD8 T cell-stimulating vaccine against a model intracellular bacterium.
Plague, initiated by Yersinia pestis infection, is a rapidly progressing disease with a high mortality rate if not quickly treated. The existence of antibiotic-resistant Y. pestis strains emphasizes the need for the development of novel countermeasures against plague. We previously reported the generation of a recombinant Y. pestis strain (Kim53?J+P) that over-expresses Y. enterocolitica YopP. When this strain was administered subcutaneously to mice, it elicited a fast and effective protective immune response in models of bubonic, pneumonic and septicemic plague. In the present study, we further characterized the immune response induced by the Kim53?J+P recombinant strain. Using a panel of mouse strains defective in specific immune functions, we observed the induction of a prompt protective innate immune response that was interferon-? dependent. Moreover, inoculation of mice with Y. pestis Kim53?J+P elicited a rapid protective response against secondary infection by other bacterial pathogens, including the enteropathogen Y. enterocolitica and the respiratory pathogen Francisella tularensis. Thus, the development of new therapies to enhance the innate immune response may provide an initial critical delay in disease progression following the exposure to highly virulent bacterial pathogens, extending the time window for successful treatment.
We demonstrate that disruption of the htrA (high temperature requirement A) gene in either the virulent Bacillus anthracis Vollum (pXO1(+) , pXO2(+) ), or in the ?Vollum (pXO1(-), pXO2(-), nontoxinogenic and noncapsular) strains, affect significantly the ability of the resulting mutants to withstand heat, oxidative, ethanol and osmotic stress. The ?htrA mutants manifest altered secretion of several proteins, as well as complete silencing of the abundant extracellular starvation-associated neutral protease A (NprA). Vollum?htrA bacteria exhibit delayed proliferation in a macrophage infection assay, and despite their ability to synthesize the major B. anthracis toxins LT (lethal toxin) and ET (oedema toxin) as well as the capsule, show a decrease of over six orders of magnitude in virulence (lethal dose 50% = 3 × 10(8) spores, in the guinea pig model of anthrax), as compared with the parental wild-type strain. This unprecedented extent of loss of virulence in B. anthracis, as a consequence of deletion of a single gene, as well as all other phenotypic defects associated with htrA mutation, are restored in their corresponding trans-complemented strains. It is suggested that the loss of virulence is due to increased susceptibility of the ?htrA bacteria to stress insults encountered in the host. On a practical note, it is demonstrated that the attenuated Vollum ?htrA is highly efficacious in protecting guinea pigs against a lethal anthrax challenge.
Plague, which is initiated by Yersinia pestis infection, is a fatal disease that progresses rapidly and leads to high mortality rates if not treated. Antibiotics are an effective plague therapy, but antibiotic-resistant Y. pestis strains have been reported and therefore alternative countermeasures are needed. In the present study, we assessed the potential of an F1 plus LcrV-based vaccine to provide protection shortly pre- or post-exposure to a lethal Y. pestis infection. Mice vaccinated up to one day before or even several hours after subcutaneous challenge were effectively protected. Mice immunized one or three days pre-challenge were protected even though their anti-F1 and anti-LcrV titers were below detection levels at the day of challenge. Moreover, using B-cell deficient ?MT mice, we found that rapidly induced protective immunity requires the integrity of the humoral immune system. Analysis of the individual contributions of vaccine components to protection revealed that rF1 is responsible for the observed rapid antibody-mediated immunity. Applying anti-F1 passive therapy in the mouse model of bubonic plague demonstrated that anti-F1 F(ab)(2) can delay mortality, but it cannot provide long-lasting protection, as do intact anti-F1 molecules. Fc-dependent immune components, such as the complement system and (to a lesser extent) neutrophils, were found to contribute to mouse survival. Interestingly, T cells but not B cells were found to be essential for the recovery of infected animals following passive anti-F1 mediated therapy. These data extend our understanding of the immune mechanisms required for the development of a rapid and effective post-exposure therapy against plague.
The lethal anthrax disease is caused by spores of the gram-positive Bacillus anthracis, a member of the cereus group of bacilli. Although the disease is very rare in the Western world, development of anthrax countermeasures gains increasing attention due to the potential use of B. anthracis spores as a bio-terror weapon. Protective antigen (PA), the non-toxic subunit of the bacterial secreted exotoxin, fulfills the role of recognizing a specific receptor and mediating the entry of the toxin into the host target cells. PA elicits a protective immune response and represents the basis for all current anthrax vaccines. Anti-PA neutralizing antibodies are useful correlates for protection and for vaccine efficacy evaluation. Post exposure anti-toxemic and anti-bacteremic prophylactic treatment of anthrax requires prolonged antibiotic administration. Shorter efficient postexposure treatments may require active or passive immunization, in addition to antibiotics. Although anthrax is acknowledged as a toxinogenic disease, additional factors, other than the bacterial toxin, may be involved in the virulence of B. anthracis and may be needed for the long-lasting protection conferred by PA immunization. The search for such novel factors is the focus of several high throughput genomic and proteomic studies that are already leading to identification of novel targets for therapeutics, for vaccine candidates, as well as biomarkers for detection and diagnosis.
The cellular arm of the immune response plays a central role in the defense against intracellular pathogens, such as F. tularensis. To date, whole genome immunoinformatic analyses were limited either to relatively small genomes (e.g. viral) or to preselected subsets of proteins in complex pathogens. Here we present, for the first time, an unbiased bacterial global immunoinformatic screen of the 1740 proteins of F. tularensis subs. holarctica (LVS), aiming at identification of immunogenic peptides eliciting a CTL response. The very large number of predicted MHC class I binders (about 100,000, IC(50) of 1000 nM or less) required the design of a strategy for further down selection of CTL candidates. The approach developed focused on mapping clusters rich in overlapping predicted epitopes, and ranking these "hotspot" regions according to the density of putative binding epitopes. Limited by the experimental load, we selected to screen a library of 1240 putative MHC binders derived from 104 top-ranking highly dense clusters. Peptides were tested for their ability to stimulate IFN? secretion from splenocytes isolated from LVS vaccinated C57BL/6 mice. The majority of the clusters contained one or more CTL responder peptides and altogether 127 novel epitopes were identified, of which 82 are non-redundant. Accordingly, the level of success in identification of positive CTL responders was 17-25 fold higher than that found for a randomly selected library of 500 predicted MHC binders (IC(50) of 500 nM or less). Most proteins (ca. 2/3) harboring the highly dense hotspots are membrane-associated. The approach for enrichment of true positive CTL epitopes described in this study, which allowed for over 50% increase in the dataset of known T-cell epitopes of F. tularensis, could be applied in immunoinformatic analyses of many other complex pathogen genomes.
Two alternative promoter trap libraries, based on the green fluorescence protein (gfp) reporter and on the chloramphenicol acetyltransferase (cat) cassette, were constructed for isolation of potent Francisella tularensis promoters. Of the 26,000 F. tularensis strain LVS gfp library clones, only 3 exhibited visible fluorescence following UV illumination and all appeared to carry the bacterioferritin promoter (Pbfr). Out of a total of 2,000 chloramphenicol-resistant LVS clones isolated from the cat promoter library, we arbitrarily selected 40 for further analysis. Over 80% of these clones carry unique F. tularensis DNA sequences which appear to drive a wide range of protein expression, as determined by specific chloramphenicol acetyltransferase (CAT) Western dot blot and enzymatic assays. The DNA sequence information for the 33 unique and novel F. tularensis promoters reported here, along with the results of in silico and primer extension analyses, suggest that F. tularensis possesses classical Escherichia coli ?(70)-related promoter motifs. These motifs include the -10 (TATAAT) and -35 [TTGA(C/T)A] domains and an AT-rich region upstream from -35, reminiscent of but distinct from the E. coli upstream region that is termed the UP element. The most efficient promoter identified (Pbfr) appears to be about 10 times more potent than the F. tularensis groEL promoter and is probably among the strongest promoters in F. tularensis. The battery of promoters identified in this work will be useful, among other things, for genetic manipulation in the background of F. tularensis intended to gain better understanding of the mechanisms involved in pathogenesis and virulence, as well as for vaccine development studies.
Francisella tularensis is an intercellular bacterium often causing fatal disease when inhaled. Previous reports have underlined the role of cell-mediated immunity and IFNgamma in the host response to Francisella tularensis infection.
We have shown previously that conjugation of polyethylene glycol (PEG) chains to recombinant human acetylcholinesterase (rHuAChE) results in the extension of its residence time in the circulation of mice and monkeys [1,2]. By profiling the pharmacokinetic behavior of an array of well-defined hypolysine human mutant AChE molecules following PEGylation, we now determine that the duration of these enzyme forms in the circulation of rhesus macaques correlates with their number of appended PEG moieties, and is influenced by the actual location of the PEG chains at the molecule surface, as well. These findings, which concur with those we have previously established in mice, indicate that a common set of rules dictates the circulatory fate of PEGylated HuAChEs in rodents and non-human primates. In addition to its effect on circulatory residence, PEGylation reduces the ability of the rHuAChE bioscavenger to elicit an immune response in the heterologous mouse animal system. Thus, an inverse relationship between anti-AChE antibody production and PEG loading was observed following repeated administration of the different PEGylated hypolysine human AChEs to mice. We note however, that in rhesus macaques, the essentially homologous (human) AChE does not induce specific anti-AChE antibodies after repeated administration of high doses of the enzyme in its PEGylated form, and even in its non-PEGylated form. Taken together, these findings indicate that PEG acts by veiling enzyme-related epitopes, which would otherwise interact with host circulatory elimination pathways and immune system. The barring of such interactions by obstructive PEGs, confers the enzyme molecule with both extended circulatory residence and mitigated immunogenic properties. Further modulation by incorporation of the F338A mutation into the PEGylated hypolysine rHuAChE enzyme mold, resulted in the generation of an OP-bioscavenger that displayed reduced aging rates and could effectively protect mice against repeated exposure to CW agents. This selected 4-PEG F338A-AChE can serve as a paradigm for new generation OP-bioscavengers, specifically tailored for prophylactic treatment against toxic OP-agents.
Bacillus anthracis is a Gram-positive, spore-forming bacterium representing the etiological cause of anthrax, a rare lethal disease of animals and humans. Development of anthrax countermeasures has gained increasing attention owing to the potential use of B. anthracis spores as a bioterror weapon. The various forms of infection by B. anthracis are characterized both by toxemia and septicemia, both of which are the result of spore entry into the host followed by their germination into rapidly multiplying, toxin-producing bacilli. Following the publication of the bacterial genome, proteomic studies were carried out to determine the protein composition of the spore and identify exposed vegetative (membrane-located or secreted) proteins. These studies included comparison of strains differing in their virulence, cultured under different conditions and, in some cases, were complemented by serological inspection, which addressed expression during infection of proteomically identified proteins and their immunogenicity. The proteomic approach emerged as a valuable strategy for the generation of a pool of potential B. anthracis protein targets for further evaluation in detection, diagnostics, therapy and prophylaxis, and contributed to the elucidation of some aspects of the pathogenesis of the disease.
A search for bacterium-specific biomarkers in peripheral blood following infection with Bacillus anthracis was carried out with rabbits, using a battery of specific antibodies generated by DNA vaccination against 10 preselected highly immunogenic bacterial antigens which were identified previously by a genomic/proteomic/serologic screen of the B. anthracis secretome. Detection of infection biomarkers in the circulation of infected rabbits could be achieved only after removal of highly abundant serum proteins by chromatography using a random-ligand affinity column. Besides the toxin component protective antigen, the following three secreted proteins were detected in the circulation of infected animals: the chaperone and protease HtrA (BA3660), an NlpC/P60 endopeptidase (BA1952), and a protein of unknown function harboring two SH3 (Src homology 3) domains (BA0796). The three proteins could be detected in plasma samples from infected animals exhibiting 10(3) to 10(5) CFU/ml blood and also in standard blood cultures at 3 to 6 h post-bacterial inoculation at a bacteremic level as low as 10(3) CFU/ml. Furthermore, the three biomarkers appear to be present only in the secretome of B. anthracis, not in those of the related pathogens B. thuringiensis and B. cereus. To the best of our knowledge, this is the first report of direct detection of B. anthracis-specific proteins, other than the toxin components, in the circulation of infected animals.
Yersinia pestis is the causative agent of plague. Previously we have isolated an attenuated Y. pestis transposon insertion mutant in which the pcm gene was disrupted. In the present study, we investigated the expression and the role of pcm locus genes in Y. pestis pathogenesis using a set of isogenic surE, pcm, nlpD and rpoS mutants of the fully virulent Kimberley53 strain. We show that in Y. pestis, nlpD expression is controlled from elements residing within the upstream genes surE and pcm. The NlpD lipoprotein is the only factor encoded from the pcm locus that is essential for Y. pestis virulence. A chromosomal deletion of the nlpD gene sequence resulted in a drastic reduction in virulence to an LD(50) of at least 10(7) cfu for subcutaneous and airway routes of infection. The mutant was unable to colonize mouse organs following infection. The filamented morphology of the nlpD mutant indicates that NlpD is involved in cell separation; however, deletion of nlpD did not affect in vitro growth rate. Trans-complementation experiments with the Y. pestis nlpD gene restored virulence and all other phenotypic defects. Finally, we demonstrated that subcutaneous administration of the nlpD mutant could protect animals against bubonic and primary pneumonic plague. Taken together, these results demonstrate that Y. pestis NlpD is a novel virulence factor essential for the development of bubonic and pneumonic plague. Further, the nlpD mutant is superior to the EV76 prototype live vaccine strain in immunogenicity and in conferring effective protective immunity. Thus it could serve as a basis for a very potent live vaccine against bubonic and pneumonic plague.
An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica Oratio8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 10(7)-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD(50) of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the virulence strategies of Y. pestis in nature.
Decades after the cessation of smallpox vaccination, the potential of the deliberate release of pathogenic orthopoxviruses has forced a reconsideration of using these extremely efficient human vaccines. Scenarios of sudden biothreats have prompted demand for rapidly protective vaccination. However, the feasibility of short-term vaccination (i.e., vaccination shortly before exposure) with vaccinia virus (VACV) is uncertain.
The role of the functional architecture of the HuAChE (human acetylcholinesterase) in reactivity toward the carbamates pyridostigmine, rivastigmine and several analogues of physostigmine, that are currently used or considered for use as drugs for Alzheimers disease, was analysed using over 20 mutants of residues that constitute the interaction subsites in the active centre. Both steps of the HuAChE carbamylation reaction, formation of the Michaelis complex as well as the nucleophilic process, are sensitive to accommodation of the ligand by the enzyme. For certain carbamate/HuAChE combinations, the mode of inhibition shifted from a covalent to a noncovalent type, according to the balance between dissociation and covalent reaction rates. Whereas the charged moieties of pyridostigmine and rivastigmine contribute significantly to the stability of the corresponding HuAChE complexes, no such effect was observed for physostigmine and its analogues, phenserine and cymserine. Moreover, physostigmine-like ligands carrying oxygen instead of nitrogen at position -1 of the tricyclic moiety (physovenine and tetrahydrofurobenzofuran analogues) displayed comparable structure-function characteristics toward the various HuAChE enzymes. The essential role of the HuAChE hydrophobic pocket, comprising mostly residues Trp(86) and Tyr(337), in accommodating (-)-physostigmine and in conferring approximately 300-fold stereoselectivity toward physostigmines, was elucidated through examination of the reactivity of selected HuAChE mutations toward enantiomeric pairs of different physostigmine analogues. The present study demonstrates that certain charged and uncharged ligands, like analogues of physostigmine and physovenine, seem to be accommodated by the enzyme mostly through hydrophobic interactions.
The therapeutic potential of human vaccinia immunoglobulin (VIG) in orthopoxvirus infection was examined using two mouse models for human poxvirus, based on Ectromelia virus and Vaccinia Western Reserve (WR) respiratory infections. Despite the relatively fast clearance of human VIG from mice circulation, a single VIG injection protected immune-competent mice against both infections. Full protection against lethal Ectromelia virus infection was achieved by VIG injection up to one day post-exposure, and even injection of VIG two or three days post-infection conferred solid protection (60-80%). Nevertheless, VIG failed to protect VACV-WR challenged immune-deficient mice, even though repeated injections prolonged SCID mice survival. These results suggest the involvement of host immunity in protection. VIG provides the initial protective time-window allowing induction of the adaptive response required to achieve complete protection. Additionally, VIG can be administered in conjunction with active Vaccinia-Lister vaccination. Vaccine efficiency is not impaired, providing a non-prohibitive VIG dose is used. Thus, VIG can be used as a prophylactic measure against post-vaccinal complications but could also serve for post-exposure treatment against smallpox.
Bacterial infection of the lungs triggers a swift innate immune response that involves the production of cytokines and chemokines that promote recruitment of immune cells from the bone marrow (BM) into the infected tissue and limit the ability of the pathogen to replicate. Recent in vivo studies of pneumonic plague in animal models indicate that the pulmonary pro-inflammatory response to airway infection with Yersinia pestis is substantially delayed in comparison to other pathogens. Consequently, uncontrolled proliferation of the pathogen in the lungs is observed, followed by dissemination to internal organs and death. While the lack of an adequate early immune response in the lung is well described, the response of BM-derived cells is poorly understood. In this study, we show that intranasal (i.n.) infection of mice with a fully virulent Y. pestis strain is sensed early by the BM compartment, resulting in a reduction in CXCR4 levels on BM neutrophils and their subsequent release into the blood 12 hours (h) post infection. In addition, increased levels of BM-derived hematopoietic stem and progenitor cells (HSPC) were detected in the blood early after infection. Mobilization of both immature and mature cells was accompanied by the reduction of BM SDF-1 (CXCL-12) levels and the reciprocal elevation of SDF-1 in the blood 24 h post infection. RT-PCR analysis of RNA collected from total BM cells revealed an early induction of myeloid-associated genes, suggesting a prompt commitment to myeloid lineage differentiation. These findings indicate that lung infection by Y. pestis is sensed by BM cells early after infection, although bacterial colonization of the BM occurs at late disease stages, and point on a potential cross-talk between the lung and the BM at early stages of pneumonic plague.
Protective antigen (PA), a key component of anthrax toxin, mediates the entry of lethal factor (LF) or edema factor (EF) through a membranal pore into target cells. We have previously reported the isolation and chimerization of cAb29, an anti-PA monoclonal antibody that effectively neutralizes anthrax toxin in an unknown mechanism. The aim of this study was to elucidate the neutralizing mechanism of this antibody in vitro and to test its ability to confer post-exposure protection against anthrax in vivo. By systematic evaluation of the steps taking place during the PA-based intoxication process, we found that cAb29 did not interfere with the initial steps of intoxication, namely its ability to bind to the anthrax receptor, the consecutive proteolytic cleavage to PA(63), oligomerization, prepore formation, or LF binding. However, the binding of cAb29 to the prepore prevented its pH-triggered transition to the transmembranal pore, thus preventing the last step of intoxication, i.e. the translocation of LF/EF into the cell. Epitope mapping, using a phage display peptide library, revealed that cAb29 binds the 2?(1) loop in domain 2 of PA, a loop that undergoes major conformational changes during pore formation. In vivo, we found that 100% of anthrax-infected rabbits survived when treated with cAb29 12 h after exposure. In conclusion, these experiments demonstrate that cAb29 exerts its potent neutralizing activity in a unique manner by blocking the prepore-to-pore conversion process.
This study examines the efficacy, bacterial load, and humoral response of extensively delayed ciprofloxacin or doxycycline treatments following airway exposure of mice to Francisella tularensis subsp. holarctica (strain LVS) or to the highly virulent F. tularensis subsp. tularensis (strain SchuS4). A delay in onset of both antibiotic treatments allowed the rescue of all LVS-infected animals. However, for animals infected with SchuS4, only ciprofloxacin was efficacious and prolongation of treatment rescued all animals.
In an event of a smallpox outbreak in humans, the window for efficacious treatment by vaccination with vaccinia viruses (VACV) is believed to be limited to the first few days post-exposure (p.e.). We recently demonstrated in a mouse model for human smallpox, that active immunization 2-3 days p.e. with either VACV-Lister or modified VACV Ankara (MVA) vaccines, can rescue animals from lethal challenge of ectromelia virus (ECTV), the causative agent of mousepox. The present study was carried out in order to determine whether a single dose of the anti-viral cidofovir (CDV), administered at different times and doses p.e. either alone or in conjunction with active vaccination, can rescue ECTV infected mice.
The virulence of Bacillus anthracis, the causative agent of anthrax, stems from its antiphagocytic capsule, encoded by pXO2, and the tripartite toxins encoded by pXO1. The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play major roles in pathogenicity. We tested this assumption by a systematic study of mutants with combined deletions of the pag, lef, and cya genes, encoding protective antigen (PA), lethal factor (LF), and edema factor (EF), respectively. The resulting seven mutants (single, double, and triple) were evaluated following subcutaneous (s.c.) and intranasal (i.n.) inoculation in rabbits and guinea pigs. In the rabbit model, virulence is completely dependent on the presence of PA. Any mutant bearing a pag deletion behaved like a pXO1-cured mutant, exhibiting complete loss of virulence with attenuation indices of over 2,500,000 or 1,250 in the s.c. or i.n. route of infection, respectively. In marked contrast, in guinea pigs, deletion of pag or even of all three toxin components resulted in relatively moderate attenuation, whereas the pXO1-cured bacteria showed complete attenuation. The results indicate that a pXO1-encoded factor(s), other than the toxins, has a major contribution to the virulence mechanism of B. anthracis in the guinea pig model. These unexpected toxin-dependent and toxin-independent manifestations of pathogenicity in different animal models emphasize the importance and need for a comprehensive evaluation of B. anthracis virulence in general and in particular for the design of relevant next-generation anthrax vaccines.
Deciphering the cellular immunome of a bacterial pathogen is challenging due to the enormous number of putative peptidic determinants. State-of-the-art prediction methods developed in recent years enable to significantly reduce the number of peptides to be screened, yet the number of remaining candidates for experimental evaluation is still in the range of ten-thousands, even for a limited coverage of MHC alleles. We have recently established a resource-efficient approach for down selection of candidates and enrichment of true positives, based on selection of predicted MHC binders located in high density "hotspots" of putative epitopes. This cluster-based approach was applied to an unbiased, whole genome search of Francisella tularensis CTL epitopes and was shown to yield a 17-25 fold higher level of responders as compared to randomly selected predicted epitopes tested in Kb/Db C57BL/6 mice. In the present study, we further evaluate the cluster-based approach (down to a lower density range) and compare this approach to the classical affinity-based approach by testing putative CTL epitopes with predicted IC(50) values of <10 nM. We demonstrate that while the percent of responders achieved by both approaches is similar, the profile of responders is different, and the predicted binding affinity of most responders in the cluster-based approach is relatively low (geometric mean of 170 nM), rendering the two approaches complimentary. The cluster-based approach is further validated in BALB/c F. tularensis immunized mice belonging to another allelic restriction (Kd/Dd) group. To date, the cluster-based approach yielded over 200 novel F. tularensis peptides eliciting a cellular response, all were verified as MHC class I binders, thereby substantially increasing the F. tularensis dataset of known CTL epitopes. The generality and power of the high density cluster-based approach suggest that it can be a valuable tool for identification of novel CTLs in proteomes of other bacterial pathogens.
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