Clusters versus affinity-based approaches in F. tularensis whole genome search of CTL epitopes

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.     

Protective Immunity against Lethal F. tularensis holarctica LVS Provided by Vaccination with Selected Novel CD8+ T Cell Epitopes

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.     

Immunoinformatic evaluation of multiple epitope ensembles as vaccine candidates: E coli 536

Epitope prediction is becoming a key tool for vaccine discovery. Prospective analysis of bacterial and viral genomes can identify antigenic epitopes encoded within individual genes that may act as effective vaccines against specific pathogens. Since B-cell epitope prediction remains unreliable, we concentrate on T-cell epitopes, peptides which bind with high affinity to Major Histacompatibility Complexes (MHC). In this report, we evaluate the veracity of identified T-cell epitope ensembles, as generated by a cascade of predictive algorithms (SignalP, Vaxijen, MHCPred, IDEB, EpiJen), as a candidate vaccine against the model pathogen uropathogenic gram negative bacteria Escherichia coli (E-coli) strain 536 (O6:K15:H31). An immunoinformatic approach was used to identify 23 epitopes within the E-coli proteome. These epitopes constitute the most promiscuous antigenic sequences that bind across more than one HLA allele with high affinity (IC50 < 50nM). The reliability of software programmes used, polymorphic nature of genes encoding MHC and what this means for population coverage of this potential vaccine are discussed.     

Characterization of Francisella tularensis outer membrane proteins

Francisella tularensis is a gram-negative coccobacillus that is capable of causing severe, fatal disease in a number of mammalian species, including humans. Little is known about the proteins that are surface exposed on the outer membrane (OM) of F. tularensis, yet identification of such proteins is potentially fundamental to understanding the initial infection process, intracellular survival, virulence, immune evasion and, ultimately, vaccine development. To facilitate the identification of putative F. tularensis outer membrane proteins (OMPs), the genomes of both the type A strain (Schu S4) and type B strain (LVS) were subjected to six bioinformatic analyses for OMP signatures. Compilation of the bioinformatic predictions highlighted 16 putative OMPs, which were cloned and expressed for the generation of polyclonal antisera. Total membranes were extracted from both Schu S4 and LVS by spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation, which separated OMs from cytoplasmic (inner) membrane and other cellular compartments. Validation of OM separation and enrichment was confirmed by probing sucrose gradient fractions with antibodies to putative OMPs and inner membrane proteins. F. tularensis OMs typically migrated in sucrose gradients between densities of 1.17 and 1.20 g/ml, which differed from densities typically observed for other gram-negative bacteria (1.21 to 1.24 g/ml). Finally, the identities of immunogenic proteins were determined by separation on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This is the first report of a direct method for F. tularensis OM isolation that, in combination with computational predictions, offers a more comprehensive approach for the characterization of F. tularensis OMPs.     

Larval exposure to Francisella tularensis LVS affects fitness of the mosquito Culex quinquefasciatus

Francisella tularensis is an environmental bacterium capable of infecting a wide spectrum of species from mammals and birds to reptiles. It has been demonstrated that F. tularensis can invade and survive within protozoa, but an association with aquatic insects has not been thoroughly investigated. We examined the interaction of F. tularensis LVS biofilms and Culex quinquefasciatus larvae to determine the effects on larvae and adults. Our results demonstrate that F. tularensis LVS can form and persist as biofilms in natural water and that the mosquito larvae of C. quinquefasciatus readily feed on biofilm and planktonic forms of F. tularensis LVS. Larvae raised in both bacteria-only cultures suffered significant delays in pupation. Adults resulting from larvae continuously exposed to the bacteria had significantly reduced wing lengths in males and fecundity of both sexes. The bacteria may be exerting these effects through localization and persistence within the midgut and Malpighian tubule cells of the larvae. The study of oral acquisition of pathogens by insect larvae can significantly contribute to the study of environmental persistence of pathogens. We show that oral uptake of F. tularensis LVS by C. quinquefasciatus larvae results in not only larval effects but also has effects on adult mosquitoes. These effects are important in understanding both the ecology of tularemia as well as bacterial interactions with aquatic invertebrates.     

Uptake of serum-opsonized Francisella tularensis by macrophages can be mediated by class A scavenger receptors

The bacterium Francisella tularensis is highly infective, and this is one of the chief attributes that has led to its development as a bioweapon. Establishment of infection requires efficient uptake of F. tularensis by host macrophages, which provide a safe in vivo environment for F. tularensis replication. Little is known, however, about the cellular entry mechanisms employed by this organism. This report shows that efficient uptake of F. tularensis live vaccine strain (LVS) by macrophages is dependent on a heat-sensitive serum component and is mediated in part by types I and II class A scavenger receptors (SRA), demonstrating for the first time that SRA can act as a receptor for opsonized pathogens. Specifically, uptake of serum-opsonized LVS was partially blocked by general scavenger receptor inhibitors [fucoidan and poly(I)] and was largely inhibited by a specific function-blocking antibody against SRA. A role for SRA in LVS binding was confirmed by showing that ectopic expression of SRA in human embryonic kidney cells conferred the capacity for robust serum-dependent LVS binding. Finally, SRA-/- macrophages ingested significantly fewer LVS than did macrophages from wild-type mice. These findings support a novel role for SRA in innate immunity and suggest a potential therapeutic approach for modulating F. tularensis infection, namely, blocking SRA as a means of hindering F. tularensis access to its intracellular niche.     

Chromosome rearrangement and diversification of Francisella tularensis revealed by the type B (OSU18) genome sequence

The gamma-proteobacterium Francisella tularensis is one of the most infectious human pathogens, and the highly virulent organism F. tularensis subsp. tularensis (type A) and less virulent organism F. tularensis subsp. holarctica (type B) are most commonly associated with significant disease in humans and animals. Here we report the complete genome sequence and annotation for a low-passage type B strain (OSU18) isolated from a dead beaver found near Red Rock, Okla., in 1978. A comparison of the F. tularensis subsp. holarctica sequence with that of F. tularensis subsp. tularensis strain Schu4 (P. Larsson et al., Nat. Genet. 37:153-159, 2005) highlighted genetic differences that may underlie different pathogenicity phenotypes and the evolutionary relationship between type A and type B strains. Despite extensive DNA sequence identity, the most significant difference between type A and type B isolates is the striking amount of genomic rearrangement that exists between the strains. All but two rearrangements can be attributed to homologous recombination occurring between two prominent insertion elements, ISFtu1 and ISFtu2. Numerous pseudogenes have been found in the genomes and are likely contributors to the difference in virulence between the strains. In contrast, no rearrangements have been observed between the OSU18 genome and the genome of the type B live vaccine strain (LVS), and only 448 polymorphisms have been found within non-transposase-coding sequences whose homologs are intact in OSU18. Nonconservative differences between the two strains likely include the LVS attenuating mutation(s).     

Francisella novicida LPS has greater immunobiological activity in mice than F. tularensis LPS,and contributes to F. novicida murine pathogenesis

To further understand the role of LPS in the pathogenesis of Francisella infection, we characterized murine infection with F. novicida, and compared immunobiological activities of F. novicida LPS and the LPS from F. tularensis live vaccine strain (LVS). F. novicida had a lower intradermal LD(50) in BALB/cByJ mice than F. tularensis LVS, and mice given a lethal F. novicida dose intraperitoneally died faster than those given the same lethal F. tularensis LVS dose. However, the pattern of in vivo dissemination was similar, and in vitro growth of both bacteria in bone marrow-derived macrophages was comparable. F. novicida LPS stimulated very modest in vitro proliferation of mouse splenocytes at high doses, but F. tularensis LVS LPS did not. Murine bone marrow macrophages treated in vitro with F. novicida LPS produced IL12 and TNF-alpha, but did not produce detectable interferon-gamma, IL10, or nitric oxide; in contrast, murine macrophages treated with F. tularensis LVS LPS produced none of these mediators. In contrast to clear differences in stimulation of proliferation and especially cytokines, both types of purified LPS stimulated early protection against lethal challenge of mice with F. tularensis LVS, but not against lethal challenge with F. novicida. Thus, although LPS recognition may not be a major factor in engendering protection, the ability of F. novicida LPS to stimulate the production of proinflammatory cytokines including TNF-alpha likely contributes to the increased virulence for mice of F. novicida compared to F. tularensis LVS.     

Identification of new HER2/neu-derived peptide epitopes that can elicit specific CTL against autologous and allogeneic carcinomas and melanomas.

Twenty-two new HLA-A2.1-binding peptides derived from the protooncogene HER2/neu were identified and analyzed for their capacity to elicit peptide and tumor-specific CTL responses. We used peptide-pulsed autologous DC from the ascites of patients with ovarian carcinomas to induce CTL. Of the 22 tested new HER2/neu-derived epitopes that could bind HLA-A2 with high (IC50 < 50 nM) or intermediate (50 nM < IC50 < 500 nM) affinity, we report the recognition by CTL of at least four novel epitopes, including HER2(9435), HER2(9665), HER2(9689), and HER2(10952), and confirm that of the known HER2 (9369) epitope. These epitopes were able to elicit CTL that specifically killed peptide-sensitized target cells and, most importantly, a HER2/neu-transfected cell line and the autologous tumor cells. We also confirm that HER2/neu is overexpressed in several melanoma lines, and as a new finding, report that some of these lines are sensitive to CTL induced by the HER2 (9369), HER2(9435), and HER2(9689) epitopes. Finally, CTL clones specific for HER2 (9369), HER2(9435), and HER2(9689) epitopes were isolated from tumor-specific CTL lines, further demonstrating the immunodominance of these epitopes. These findings broaden the potential application of HER2/neu-based immunotherapy.     

Galleria mellonella as a model host to study infection by the Francisella tularensis live vaccine strain

We used the killing of Galleria mellonella (Lepidoptera: Pyralidae; the greater wax moth) caterpillar by the live vaccine strain (LVS) of Francisella tularensis to develop an invertebrate host system that can be used to study F. tularensis infection and the in vivo effects of antibacterial compounds on F. tularensis LVS. After injection into the insect hemocoel, F. tularensis LVS, killed caterpillars despite the association of LVS with hemocytes. The rate of killing depended on the number of bacteria injected. Antibiotic therapy with ciprofloxacin, levofloxacin or streptomycin administered before or after inoculation prolonged survival and decreased the tissue burden of F. tularensis in the hemocoel. Delayed drug treatment reduced the efficacy of antibacterials and especially streptomycin. The G. mellonella-F. tularensis LVS system may facilitate the in vivo study of F. tularensis, efficacy with antibacterial agents.     

Antigen processing by proteasomes: insights into the molecular basis of crypticity

Eight to eleven amino acid residues are the sizes of predominant peptides found to be associated with MHC class I molecules. Proteasomes have been implicated in antigen processing and generation of such peptides. Advanced methodologies in peptide elution together with sequence determination have led to the characterisation of MHC class I binding motifs. More recently, screening of random peptide phage display libraries and synthetic combinatorial peptide libraries have also been successfully used. This has led to the development and use of predictive algorithms to screen antigens for potential CTL epitopes. Not all predicted epitopes will be generated in vivo and the emerging picture suggests differential presentation of predicted CTL epitopes ranging from cryptic to immunodominant. The scope of this review is to discuss antigen processing by proteasomes, and to put forward a hypothesis that the molecular basis of immunogenicity can be a function of proteasomal processing. This may explain how pathogens and tumours are able to escape immunosurveillance by altering sequences required by proteasomes for epitope generation. Abbreviations: CTL – cytotoxic T lymphocytes; DRiPs – defective ribosomal products; ER – endoplasmic reticulum; Hsps – heat shock proteins; LMP – low molecular weight peptide; MHC – major histocompatibility complex; TAP – transporter associated with antigen processing.     

<Emphasis Type="Italic">Francisella tularensis</Emphasis> strain LVS resides in MHC II-positive autophagic vacuoles in Macrophages

The Francisella tularensis strain LVS phagosome disintegrates during the first few hours after bacterial entry and microbes are released to the cytosol. Within 12 h both rapid multiplication of microbes and a steep increase of apoptosis of infected macrophages occur. We searched for signals involved in the death of macrophages and detected molecules associated with the autophagy machinery cathepsin D, PTEN, p53 and LC3, whose levels or modification were influenced by ongoing in vitro tularemic infection. The sequestration of cytoplasmic F. tularensis LVS into autophagosomes was confirmed by co-localization of the LVS strain containing vacuoles with LC3 (an autophagosomal marker). We also demonstrated the presence of MHC II antigens in these autophagosomes, indicating that they might act as a source of endogenous tularemic antigens for presentation to CD4+ T lymphocytes.     

The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages

Intracellular killing of Francisella tularensis by macrophages depends on interferon-gamma (IFN-gamma)-induced activation of the cells. The importance of inducible nitric oxide synthase (iNOS) or NADPH phagocyte oxidase (phox) for the cidal activity was studied. Murine IFN-gamma-activated peritoneal exudate cells (PEC) produced nitric oxide (NO), measured as nitrite plus nitrate, and superoxide. When PEC were infected with the live vaccine strain, LVS, of F. tularensis, the number of viable bacteria was at least 1000-fold lower in the presence than in the absence of IFN-gamma after 48 h of incubation. PEC from iNOS-gene-deficient (iNOS-/-) mice killed F. tularensis LVS less effectively than did PEC from wild-type mice. PEC from phox gene-deficient (p47phox-/-) mice were capable of killing the bacteria, but killing was less efficient, although still significant, in the presence of NG-monomethyl-L-arginine (NMMLA), an inhibitor of iNOS. A decomposition catalyst of ONOO-, FeTPPS, completely reversed the IFN-gamma-induced killing of F. tularensis LVS. Under host cell-free conditions, F. tularensis LVS was exposed to S-nitroso-acetyl-penicillamine (SNAP), which generates NO, or 3-morpholinosydnonimine hydrochloride (SIN-1), which generates NO and superoxide, leading to formation of ONOO-. During 6 h of incubation, SNAP caused no killing of F. tularensis LVS, whereas effective killing occurred in the presence of equimolar concentrations of SIN-1. The results suggest that mechanisms dependent on iNOS and to a minor degree, phox, contribute to the IFN-gamma-induced macrophage killing of F. tularensis LVS. ONOO- is likely to be a major mediator of the killing.     

Use of transposon-transposase complexes to create stable insertion mutant strains of Francisella tularensis LVS

Francisella tularensis is a highly virulent zoonotic bacterial pathogen capable of infecting numerous different mammalian species, including humans. Elucidation of the pathogenic mechanisms of F. tularensis has been hampered by a lack of tools to genetically manipulate this organism. Herein we describe the use of transposome complexes to create insertion mutations in the chromosome of the F. tularensis live vaccine strain (LVS). A Tn5-derived transposon encoding kanamycin resistance and lacking a transposase gene was complexed with transposase enzyme and transformed directly into F. tularensis LVS by electroporation. An insertion frequency of 2.6 x 10(-8) +/- 0.87 x 10(-8) per cell was consistently achieved using this method. There are 178 described Tn5 consensus target sites distributed throughout the F. tularensis genome. Twenty-two of 26 transposon insertions analyzed were within known or predicted open reading frames, but none of these insertions was associated with the Tn5 target site. Analysis of the insertions of sequentially passed strains indicated that the transposons were maintained stably at the initial insertion site after more than 270 generations. Therefore, transformation by electroporation of Tn5-based transposon-transposase complexes provided an efficient mechanism for generating random, stable chromosomal insertion mutations in F. tularensis.     

Francisella tularensis selectively induces proinflammatory changes in endothelial cells

Naturally acquired infections with Francisella tularensis, the bacterial agent of tularemia, occur infrequently in humans. However, the high infectivity and lethality of the organism in humans raise concerns that it might be exploited as a weapon of bioterrorism. Despite this potential for illicit use, the pathogenesis of tularemia is not well understood. To examine how F. tularensis interacts with cells of its mammalian hosts, we tested the ability of a live vaccine strain (LVS) to induce proinflammatory changes in cultured HUVEC. Living F. tularensis LVS induced HUVEC to express the adhesion molecules VCAM-1 and ICAM-1, but not E-selectin, and to secrete the chemokine CXCL8, but not CCL2. Stimulation of HUVEC by the living bacteria was partially suppressed by polymyxin B, an inhibitor of LPS, but did not require serum, suggesting that F. tularensis LVS does not stimulate endothelium through the serum-dependent pathway that is typically used by LPS from enteric bacteria. In contrast to the living organisms, suspensions of killed F. tularensis LVS acquired the ability to increase endothelial expression of both E-selectin and CCL2. Up-regulation of E-selectin and CCL2 by the killed bacteria was not inhibited by polymyxin B. Exposure of HUVEC to either live or killed F. tularensis LVS for 24 h promoted the transendothelial migration of subsequently added neutrophils. These data indicate that multiple components of F. tularensis LVS induce proinflammatory changes in endothelial cells in an atypical manner that may contribute to the exceptional infectivity and virulence of this pathogen.     

Peripheral human γδ T cells control growth of both avirulent and highly virulent strains of Francisella tularensis in vitro

In this paper we evaluate the role of human γδ T cells in control of Francisella tularensis infection. Using an in vitro model of infection, a reduction in bacterial numbers was detected in the presence of human γδ T cells for both attenuated LVS and virulent SCHU S4 strains of F. tularensis. Antibody neutralisation of IFN-γ caused an increase in survival of F. tularensis LVS suggesting that γδ T cell-mediated control of F. tularensis infection is partially mediated by IFN-γ.     

A variety of novel lipid A structures obtained from Francisella tularensis live vaccine strain

F. tularensis is a Gram-negative coccobacillus that causes tularemia. Its LPS has nominal biological activity. Currently, there is controversy regarding the structure of the lipid A obtained from F. tularensis live vaccine strain (LVS). Therefore, to resolve this controversy, the purification and structural identification of this LPS was crucial. To achieve this, LPS from F. tularensis LVS was acid hydrolyzed to obtain crude lipid A that was methylated and purified by HPLC and the fractions were analyzed by MALDI-TOF MS. The structure of the major lipid A species was composed of a glucosamine disaccharide backbone substituted with four fatty acyl groups and a phosphate (1-position) with a molecular mass of 1505. The major lipid A component contained 18:0[3-O(16:0)] in the distal subunit and two 18:0(3-OH) fatty acyl chains at the 2- or 3-positions of the reducing subunit. Additional variations in the lipid A species include: heterogeneity in fatty acyl groups, a phosphate or a phosphoryl galactosamine at the 1-position, and a hexose at the 4' or 6' position, some of which have not been previously described for F. tularensis LVS. This analysis revealed that lipid A from F. tularensis LVS is far more complex than originally believed.     

Inhibition of Francisella tularensis LVS infection of macrophages results in a reduced inflammatory response: evaluation of a therapeutic strategy for intracellular bacteria

Francisella tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. A flow cytometric assay was developed to measure bacterial uptake, using a fluorescein isothiocyanate-labelled anti-F. tularensis lipopolysaccharide antibody in conjunction with antibodies to cell surface markers, in order to determine the specific cell phenotypes that were positive for the bacteria. Several phagocytic inhibitors were evaluated in macrophage cell lines and a lung homogenate assay to determine whether the uptake of F. tularensis strain LVS could be altered. Our data show that cytochalasin B, LY294002, wortmannin, nocodazole, MG132 and XVA143 inhibitors reduced LVS uptake by >50% in these assays without having significant cytotoxic effects. Furthermore, a reduction in the inflammatory cytokines monocyte chemoattractant protein-1, interleukin-6 and tumour necrosis factor-α was found in the supernatant of lung tissue infected with LVS when the inhibitory compounds were present. Similarly, there was an alteration in bacterial uptake and a reduction in the inflammatory cytokine response following the administration of wortmannin to LVS-infected mice. Although wortmannin treatment alone did not correlate with the enhanced survival of LVS-infected mice, these inhibitors may have utility in combination therapeutic approaches or against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.