ARTICLE IN PRESS - EpiVax, Inc.epivax.com/wp-content/uploads/2013/09/JVAC_6891_23Jan07.pdf · Our...

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ARTICLE IN PRESSVAC 6891 1–13

Vaccine xxx (2007) xxx–xxx

Diversity of Francisella tularensis Schu4 antigens recognized by Tlymphocytes after natural infections in humans: Identification of

candidate epitopes for inclusion in a rationallydesigned tularemia vaccine

Julie A. McMurry a, Stephen H. Gregory b, Leonard Moise a,b,Daniel Rivera a,b, Soren Buus c, Anne S. De Groot a,b,∗

a EpiVax, Inc., Providence, RI 02903, USAb Brown University, Providence, RI 02912, USA

c University of Copenhagen, Denmark

bstract

The T lymphocyte antigens, which may have a role in protection against tularemia, were predicted by immunoinformatics analysis ofrancisella tularensis Schu4. Twenty-seven class II putative promiscuous epitopes and 125 putative class I supertype epitopes were chosen forynthesis; peptides were tested in vitro for their ability to bind HLA and to induce immune responses from PBMCs of 23 previously infected

EDubjects. While the immune responses of individual subjects showed heterogeneity, 95% of the subjects responded strongly to a pool of 27

romiscuous peptides; 25%, 33%, and 44% of subjects responded to pools of 25 A2, A24, and B7 peptides, respectively. These data can aidn the development of novel epitope-based and subunit tularemia vaccines.

2007 Published by Elsevier Ltd.

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Teywords: Francisella tularensis; Epitope; Vaccine; Immunoinformatics; H. Introduction

The intracellular Gram-negative bacterium Francisellaularensis is the causative agent of tularemia and is includedn the US government Category A list of bioterrorism agentsecause of the low dose required for infection, its virulencend its potential for aerosol dissemination [1].

.1. Correlates of immunity to tularemia

The fully annotated F. tularensis genome was published

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Please cite this article in press as: McMurry JA, et al., Diversity of Fraafter natural infections in humans: Identification of candidate epitopes(2007), doi:10.1016/j.vaccine.2007.01.039

n 2005; the proteome possibly contains 1603 ORFS, ofhich 523 are still categorized as “hypothetical proteins” and0% are unique to F. tularensis (FT) [2]. Although secreted

∗ Corresponding author at: Brown University, Providence, RI 02912, USA.el.: +1 401 272 2123; fax: +1 401 272 7652.

E-mail address: [email protected] (A.S. De Groot).

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264-410X/$ – see front matter © 2007 Published by Elsevier Ltd.oi:10.1016/j.vaccine.2007.01.039

ss II; T cell

roteins are generally associated with protective immuneesponses in other intracellular bacteria, no protein or groupf proteins has yet been shown to protect against the devel-pment of active tularensis infection.

Studies of human immune responses to F. tularensis pro-eins may uncover critical antigens for vaccine development.t is now well established that within the lung microen-ironment, pathogens and their hosts interact in complexays not observed in other organs. Thus, antigens that are

ecognized by individuals who have recovered from pneu-onic tularemia may be particularly relevant to the design

f vaccines aimed to protect against aerosol infection with F.ularensis.

F. tularensis is a facultative intracellular bacterium, thus it

ncisella tularensis Schu4 antigens recognized by T lymphocytesfor inclusion in a rationally designed tularemia vaccine, Vaccine

s not surprising that protective immunity in the mouse model 26s associated with both CD4+ and CD8+ T-cell response to 27roteins of F. tularensis [3–6]. CD4+ and CD8+ T cells also 28lay a critical role in the efficient resolution of secondary 29

dx.doi.org/10.1016/j.vaccine.2007.01.039mailto:[email protected]/10.1016/j.vaccine.2007.01.039mcmurryjNoteItalics are an appropriate modification. Line breaks are now suboptimal.To improve readability, "T" should be on the same line as "lymphocytes" and "rationally" and "designed" should be on the same line.

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ARTICLEJ.A. McMurry et al. /

nfections in mice [7]. Such studies have also indicated thathe induction of broad Type 1 cytokines including interferonamma (IFN-�) and IL-12 were strictly required for protec-ion, since mice deficient in IFN-�, IL-12 p35, or IL-12 p40ll succumbed to LVS doses that were sublethal for wild-typeice [8].The role of antibodies is currently disputed. Passive trans-

er of serum from immune mice to naı̈ve immunocompetentice confers protection from LVS challenge; however, trans-

er of serum from immune mice into athymic nu/nu mice didot confer protection, suggesting that clearance depends onhost T-cell response [9].

Thus in summary, a tularemia vaccine that stimulatesD4+ and CD8+ T cells to produce broad type I cytokines

hould effectively contain or prevent tularemia.Combining information gained from murine studies of

. tularensis with knowledge about critical antigens forther intracellular pathogens, researchers have hypothesizedhat proteins that are both (a) expressed/upregulated withinnfected macrophages and (b) potent T-cell activators may beritical components of an effective vaccine [10]. However,fforts to utilize single proteins such as the FT-associatedransmembrane proteins to produce improved vaccines have

et only marginal success [11,5].Our approach has been to use immunoinformatics tools,

hich provide a practical means for systematic identifica-ion of potential epitopes and antigens for tularemia vaccineevelopment.

.2. Goal of study

The purpose of this study was to identify epitopes fornclusion in a novel, broadly reactive epitope-based F.ularensis vaccine. To that end, we used in silico mappingools to identify 165 putative class I and II epitopes fromhe FT proteome. These epitopes were then synthesized andnalyzed for their abilities to: bind to MHC; activate lym-hocytes; and induce secretion of IFN-�, TNF�, IL-2, IL-4,L-5, IL-10, Mip1�, Mip1�, GMCSF, and FasL.

. Methods

.1. Source of protein sequences for analysis

We performed epitope screens of two sets of F. tularensisroteins. The first set (“Genome Scan 1/GS1”) was com-rised of F. tularensis open reading frames (ORFs) whoseroducts were predicted to be secreted according to the pres-nce of a secretion tag [12,13]. The second set of proteins“Genome Scan 2/GS2”) was comprised of 53 previouslytudied proteins.
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.1.1. Putative secreted proteins (GS1)For GS1, we chose ORFs that encoded proteins that were

utatively expressed and secreted. Each ORF identified in

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he Schu4 genome was analyzed as follows: the Phobius weberver [14] was used to identify signal peptides and trans-embrane segments in the input sequences. The LipoP web

erver [15] was used to identify and exclude proteins thatontain a lipoprotein attachment site; this tool works onlyith Gram-negative bacteria.We chose for analysis the resulting proteins that: (1)

ontained a signal sequence (according to Phobius); (2) con-ained no transmembrane domains (according to Phobius);nd (3) did not contain a lipoprotein attachment site (accord-ng to LipoP).

.1.2. Proteins confirmed to be expressed (GS2)To complement the GS1 set, in January 2005 we selected

rom the literature those expressed proteins with at least oneaccine-suitable characteristic: immunogenicity, secretion,pregulation, and/or implication in virulence. To identifyhese proteins, we started at the GenBank annotations of the20 proteins among which 15 publications were referenced.

PubMed search for tular* in any field yielded 1259 arti-les. Publications in or after the year 2000 comprised 265f the 1259 articles; each of these 265 underwent both titlend abstract review where available. Any article with at leastne the following words in the abstract underwent full texteview: microarray, T cell, B cell, immun*, antigen*, upreg-lated, pathogenic*, virulen*. Among the articles discardedere case studies, epidemiological studies, genetic descrip-

ions lacking functional analysis. Of the 265 publications, 36escribed proteins that were considered for analysis. Proteinsrom the PubMed search that were not readily identifiablen sequence, or those which were lacking from Schu4 wereiscarded. From the 15 papers referenced in GenBank androm the 36 papers referenced in PubMed, a total of 53 pro-eins were identified for analysis; the only characteristic thatll of these proteins had in common was that they had beenonfirmed to be expressed.

.2. Scoring the proteins using EpiMatrix

Using EpiMatrix [16] we screened all 147 GS1 putativeecreted proteins and all 53 GS2 known expressed proteinsor matches to a list of MHC class I and MHC class II bindingotifs; a shortlist was synthesized for in vitro and ex vivo

nalysis.

.2.1. Scoring the proteins using class II matricesSeventy-four class II epitope-mapping matrices were

eveloped at EpiVax using the pocket profile method17]. From among the 74 matrices, we selected a set of

“archetype”/supertype class II matrices correspondingo alleles that were both common and relatively distinctn their pocket preferences: DRB1*0101, DRB1*0301,


RB1*0401, DRB1*0701, DRB1*1101, DRB1*1301, 127RB1*1501. Taken together, these eight alleles are expected 128

o cover over 95% of any given human population [18]. We 129ave found that this archetype method allows for accurate 130

dx.doi.org/10.1016/j.vaccine.2007.01.039

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rediction of promiscuous class II epitopes without over-eighting one family of motifs.Every match to a class II motif was counted and scored

ccording to methods previously published [19]. A rankedist of putative promiscuous epitopes was then developed.

The proteins were scanned for epitope clusters using ourluster finding algorithm ClustiMer [20]. All sequences withcluster score of 15 or higher were considered. After aanual review to identify near duplicates, overlapping, and

ydrophobic sequences, the list was pared down and the topanking putative epitopes (as shown in Table 1) were selectedor synthesis. FopA had previously been shown to be anmmunogenic protein thus two putative promiscuous epitopesere selected as controls even though they were not among

he highest ranked.

.2.2. Scoring proteins using class I matricesThirty class I alleles were developed for use with Epi-

atrix; to balance population coverage and efficiency wehose to use five class I “supertype” alleles: A*0201, A*0301,*2402, B*0702, and B*4403. Epitopes restricted by theseve supertypes cover >90% of humans in five distinct humanopulation groups [21].

.3. Elimination of candidates with homology to humanequences

GS1 and GS2 sets of proteins selected for EpiMatrix anal-sis were first evaluated for homology with human proteinssing BLAST [22]. No significant homology was found athe protein level. In addition, the peptides selected for synthe-is were analyzed for regions of local similarity with humanequences again using BLAST. Peptides were either uniqueo Schu4 or shared significant homology primarily with othereptides in the tularensis complex. Peptides that shared 7/9mino acids identity with human sequences were eliminatedrom consideration prior to synthesis.

.3.1. Peptide synthesisPeptides corresponding to epitope selections were pre-

ared by 9-fluoronylmethoxycarbonyl (Fmoc) synthesis onn automated Rainen Symphony/Protein Technologies syn-hesizer (SynPep). The peptides were delivered >80% pures ascertained by HPLC, Mass Spec, and UV scan (insur-ng purity, mass and spectrum, respectively). Seven of the

ost promising peptides have since been re-synthesizedt New England Peptide and confirmatory studies arelanned.

.4. MHC I binding assay

MHC I binding assays were performed in collaboration
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ith Soren Buus laboratory according to protocols theyublished in 2002 [23]. Briefly, the pertinent HLA class Iolecule was incubated at an active concentration aroundnM together with 25 nM human beta2 microglobulin (b2m)

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nd an increasing concentration of the test peptide at 18 ◦C for8 h. The HLA molecules were then captured on an ELISAlate coated with the pan-specific anti-HLA antibody, W6/32,nd HLA–peptide complexes were detected with an anti-b2mpecific polyclonal serum conjugated with Horse Radish Per-xidase (Dako P0174) followed by a signal enhancer (Dakonvision). The plates were developed, and the colorimet-

ic reaction was read at 450 nm using a Victor2 MultilabelLISA reader. These readings were then converted to theoncentration of HLA–peptide complexes using a standardurve, and plotted against the concentration of test peptidesed in the assay. The concentration of peptide required toalf-saturate (EC50) the HLA was determined. At the limitedumber of distinct HLA concentrations used here, the EC50pproximates the equilibrium dissociation constant, KD. Theetection antibody used in these assays was conformationpecific and readily differentiated between HLA moleculesith and without bound peptide.

.4.1. Statistical analysis of MHC I binding assaysKD values were calculated by non-linear regression

nalysis using the SigmaPlot analysis program. Based onomparisons with known peptides, a KD of 500 nM or mores indicative of a weak or non-binding interaction.

.5. MHC II binding assays

Purified, soluble HLA class II peptide binding com-etition assays were performed using a europium labeledompetitor as previously described [24]. Fluorescence inhi-ition was measured on a Wallac Victor 3-V time-resolveduorometer.

.5.1. Statistical analysis of MHC II binding assaysBased on comparisons with known peptides, an IC50 of

50 �M or more is indicative of a weak or non-binding inter-ction. Although full IC50 curves are required to discriminateetween two comparable binders, a screen at 100 �M is a rea-onable estimate as to whether a peptide is a strong (75–100%nhibition), moderate (50–75% inhibition), weak (30–50%nhibition), or non-binder (0–30% inhibition).

.6. Study subject characteristics

Bloods were obtained from Martha’s Vineyard HospitalOak Bluffs, MA) and from the Island Health Clinic (Oakluffs, MA) in accordance with all federal guidelines and

nstitutional policies. A heparinized blood sample was drawnfter obtaining informed consent. A total of 23 subjects wereecruited for this study. The study group consisted of 18 mennd 5 women (age range 18–91 years) who previously hadulmonary tularemia (15 cases), ulceroglandular tularemia (2


ases), pharyngeal (1 case), or typhoidal tularemia (1 case). 227wo additional subjects were positively diagnosed long after 228

nfection—the form of which could thus not be determined. 229ubjects were only eligible for inclusion if their diagnosis had 230


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Please cite this article in press as: McMurry JA, et al., Diversity of Francisella tularensis Schu4 antigens recognized by T lymphocytesafter natural infections in humans: Identification of candidate epitopes for inclusion in a rationally designed tularemia vaccine, Vaccine(2007), doi:10.1016/j.vaccine.2007.01.039

ARTICLE IN PRESSJVAC 6891 1–134 J.A. McMurry et al. / Vaccine xxx (2007) xxx–xxx

Table 1Epitopes selected for class II, A2, and A3 in Genome Scans 1 and 2

Column 1: peptide ID, comprised of the Genome Scan number (GS1 or GS2), an abbreviated description for the parent protein, allele for which peptide ispredicted to bind, and a four-digit ID number; column 2: amino acid sequence; column 3: GenBank accession of parent protein; column 4: protein descriptionfrom GenBank.


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reviously been confirmed serologically. To date, only typeF. tularensis has been isolated from Martha’s Vineyard

pecimens [25,26].The time from the onset of tularemia to the first visit

o the health care center was 24–72 h for most subjects. Inther cases, when tularemia was not properly diagnosed andreated, symptoms persisted for days. Blood specimens forhe study were taken as early as 2 months or as late as 27ears following diagnosis of tularemia.

Hartford Hospital Transplant Immunology Laboratoryerformed class I and class II HLA typing on DNAxtracted from each sample. One Lambda Micro SSPTM Highesolution HLA class II kits were used according to manu-

acturer’s instructions: http://www.onelambda.com/pdffiles/roductinserts/dna pi EN.pdf.

To protect the confidentiality of individual study subjects,he clinical data shown in Table 4 are aggregated; gender andLA type are not shown.

.7. PBMC isolation

Bloods were drawn in Heparinized tubes at Martha’s Vine-ard hospital and were hand delivered to EpiVax on the dayf draw. PBMC were washed three times, pelleted, and resus-ended in R10 (RPMI supplemented with 2 mM l-glutamine,00 mg/ml penicillin/streptomycin, 50 mg/ml gentamycin,0% heat-inactivated HAB serum).

.8. Human PBMC ELISpot assay

The frequency of epitope-specific T lymphocytes wasetermined using Mabtech® IFN-specific ELISpot kits withtrict adherence to manufacturer’s instructions (http://www.abtech.com/elispot.asp). PBMC (1 × 107 well−1) were cul-

ured in 12-well plates with a pool of the peptides at 10 �g/ml;L-2 and IL-7 were added on day 2 and on every other day ofulture up to 3 days before assay. After 5–20 days of culture,ells were washed three times, transferred to ELISpot platest 200,000 cells/well, and incubated overnight with individ-al peptides in triplicate and with peptide pools in triplicate.o stimulus controls and mitogen (PHA) controls were also

ssessed.

.9. Statistical analysis of ELISpot assay

The data were considered to be positive if two criteria wereatisfied: (1) the average number of spots in the triplicate pep-ide wells was at least twice the average of the backgroundells, and (2) the average number of spots was at least 20

pots per 1 million cells over background (1 response overackground per 50,000 PBMC). The stringency of these crite-ia increases with the number of background spots. Virtually
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ll of the responses that were considered positive accord-ng to these criteria were also positive by the two-tailedon-parametric Mann–Whitney U-test. A response was con-idered positive if the number of spots in the peptide wells was

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tatistically different (at p < 0.05) from that of the backgroundells.

. Results

.1. Epitope selection

A total of 25 class II peptides (17 GS1 and 8 GS2) wereelected. Likewise for each of 5 class I alleles (A2, A3, A24,7, and B44), 25 peptides were selected (20 GS1 and 5S2) for a total of 125 class I peptides (Tables 1 and 2).cores for predicted epitopes based on these matrices con-istently fell within the range of scores of a set of epitopesublished in the literature. There were no published HLAigands or epitopes identified in the set. All selected putativepitopes, except for the following 11 had identical or closelyatched counterparts in LVS: II 3007, II 3024, II 3023,

I 3025, A2 3501, A2 3515, A24 3216, B7 3701, B7 3713,44 3411, B44 3424.

.1.1. Selection of class II epitopes for Genome Scan 1Our analysis of the 1603 ORFs identified 147 ORFs

ncoding putatively secreted proteins. Each of these 147roteins was analyzed for epitope content using the Epi-atrix system. Eight archetype class II alleles were

sed: DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701,RB1*0801, DRB1*1101, DRB1*1301, DRB1*1501. Ourroprietary cluster finding algorithm ClustiMer was then usedo scan the proteins for regions (≤25 residues long) whereutative class II epitopes densely overlapped. From approx-mately 40,000 sequences, this analysis yielded 252 uniqueandidate class II clusters with a score above 15.

The top scoring 102 selected clusters were blasted againsthe protein database at GenBank; 83 (80%) were discardedor having at least one 9-mer in which at least seven ofine amino acids were identical to those in a human protein.lusters lacking a significant motif match for DRB1*0101ere also discarded. High scoring clusters were weighted

ccording to their putative class I content and resorted.he top ranked 17 candidate peptide sequences selected forynthesis.

.1.2. Selection of class I epitopes for Genome Scan 1We also analyzed all 147 proteins for 9-mer and 10-mer

hat were putative class I epitopes. We selected the 20 topcoring 9-mer and 10-mer peptides for each of five alle-es: A*0201, A*0301, A*2402, B*0702, and B*4403. Sixf these 20 shared 7/9 identities with human sequences, nonef these 20 shared more than 7/9. Only one selected class Ipitope (B44 3404) was contained within a class II clusterreviously chosen.


.1.3. Selection of class II epitopes for Genome Scan 2 327Our analysis of the published literature identified 53 pro- 328

eins previously shown to be expressed. Each of these 53 329

dx.doi.org/10.1016/j.vaccine.2007.01.039http://www.onelambda.com/pdffiles/productinserts/dna_pi_EN.pdfhttp://www.mabtech.com/elispot.asp

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Table 2Epitopes selected for HLA*A24, HLA*B7, and HLA*B44 in Genome Scans 1 and 2

Column 1: peptide ID, comprised of the Genome Scan number (GS1 or GS2), an abbreviated description for the parent protein, allele for which peptide ispredicted to bind, and a four-digit ID number; column 2: amino acid sequence; column 3: GenBank accession of parent protein; column 4: protein descriptionfrom GenBank.

dx.doi.org/10.1016/j.vaccine.2007.01.039mcmurryjNoteTables 1&2 are grainy for printing and are not text searchable. I suggest either higher resolution or pasting text from the excel file provided previously and attached here as well.

Peptides

Peptide IDAA SequenceGenBank AccessionProtein Description

Class IIFOPA 102 (AKA Fopa-2)IGYNINKYFAVQYNQLVGRVFAGLGEGI-1332665immunogenic chaperone protein

FOPA 176 (AKA Fopa-4)SVDYRYIQTMAPSNISGAGI-1332665immunogenic chaperone protein

Genome Scan 1GS1_Secr_II_3001SLGYELWRRYNGFPINLNGI-56707198Secretion protein

GS1_Glyc_II_3002DQAWRYWLAYSYQQTGQKAKAGI-56707549soluble lytic murein transglycosylase

GS1_hemK_II_3003PLAYILGYKYFWNQKLYVTKDGI-56707339hemK protein homolog

GS1_Chitn_II_3004ADGKKYYLAIAVNGARSRIEAMGI-56707834chitinase family 18 protein

GS1_Epim_II_3005KEKYYKINTQLTYDLAKQGI-56708503UDP-glucose 4-epimerase

GS1_Toler_II_3006NEVTFYIKNGWLQQAKNTGNYSGI-56707609organic solvent tolerance protein

GS1_Hyp_II_3007NIIMVKVFVYNLNNNINAIKGI-56707430hypothetical membrane protein

GS1_Asp_II_3008IDSVDMTMAMRIKLAETVQKLGI-56707606Periplasmic L-asparaginase II precursor

GS1_Hyp_II_3009GAKLAVINWQRLNKAPLGGETGI-56707677hypothetical protein FTT0546

GS1_Mbrn_II_3010TGIIAVMNYRKLASMDYDVKGI-56708593hypothetical membrane protein

GS1_Mbrn_II_3011NQNMLANFANAQKYRADKDGI-56708618hypothetical membrane protein

GS1_Hyp_II_3012NEQYMQMFQVNRFTGYDRINNANQGI-56708545hypothetical protein FTT1507

GS1_TolC_II_3013QIDFNYNLRRDLYNQFGGRGI-56708730outer membrane protein tolC precursor

GS1_Hyp_II_3014STMGVSMYAPFGANNMRLQRNYGI-56708532hypothetical protein FTT1493c

GS1_FAD_II_3015GEQFAFRPNNKITIAMVGI-56708305FAD binding family protein

GS1_NADH_II_3016QTGQRSYNVVLMLQQHGYDAYNGI-56708472NADH oxidase

GS1_Hyp_II_3017ENGIWKVNRPNPGPVTIAGI-56708544hypothetical protein FTT1506

GS 2GS2_pilNBP_II_3018AESLQIVISQRLLKRKGGGRVAAYEVGI-56707266Type IV pili nucleotide-binding protein

GS2_1699_II_3019HKDFNFLLSPNQPILLDIQGI-56708708hypothetical protein FTT1699

GS2_1700_II_3020TSSITVSLINYVINSVVNPTYNKNGI-56708709hypothetical protein FTT1700

GS2_IcID_II_3021KVIEKNGIKYIYNQLSLSLEHSYGGI-56708720intracellular growth locus, subunit D

GS2_IcID_II_3022PEEFFVATRYYLFLKGI-56708720intracellular growth locus, subunit D

GS2_1715c_II_3023RGDVKSFIPLYLRISGKASSALFGI-56708724hypothetical protein FTT1715c

GS2_1715c_II_3024FSDMEIRVRFGLYSKQSASKLGI-56708724hypothetical protein FTT1715c

GS2_1715c_II_3025DRLVRGFYFLLRRMVKNRNTKVGSHNGI-56708724hypothetical protein FTT1715c

A2Genome Scan 1GS1_HAD_A2_3501KLGGYVSFVGI-56707745HAD superfamily protein

GS1_hyp_A2_3502KLLGQINLVGI-56707688hypothetical protein FTT0558

GS1_chtn_A2_3503VLPGVVVPIVGI-56707834chitinase family 18 protein

GS1_mbrn_A2_3504QLVGRVFAGLGI-56707711outer membrane associated protein

GS1_oxid_A2_3505LMWDNVGLVGI-56708507L-aspartate oxidase

GS1_hyp_A2_3506FLSPNQVTNVGI-56708019hypothetical protein FTT0918

GS1_hyp_A2_3507FMPKVNFEVGI-56708178hypothetical protein FTT1095c

GS1_sulf_A2_3508GTDGFPFKLGI-56707894Arylsulfatase

GS1_toler_A2_3509IMSSFEFQVGI-56707609organic solvent tolerance protein

GS1_pept_A2_3510QLWPEEIGVGI-56708248Gamma-glutamyltranspeptidase

GS1_hyp_A2_3511WLGNHGFEVGI-56707973hypothetical protein FTT0869

GS1_hyp_A2_3512KLLPEGYWVGI-56707626hypothetical protein FTT0484

GS1_hypm_A2_3513FLSNVGHYVGI-56707205hypothetical membrane protein

GS1_Hdrx_A2_3514KLAKKVLSRVGI-567082802-octaprenyl-6-methoxyphenyl hydroxylase

GS1_hypm_A2_3515GIDGNVTFTVGI-56708618hypothetical membrane protein

GS1_oxgn_A2_3516RLIGHISTLGI-56708279monooxygenase family protein

GS1_hypm_A2_3517KIDSTSFSVGI-56708770hypothetical membrane protein

GS1_pilin_A2_3518GLNLVVSSSVGI-56708227Type IV pilin multimeric outer membrane protein

GS1_hyp_A2_3519NLDTSPFFVGI-56707423hypothetical protein FTT026

GS1_AcpA_A2_3520ILDPKTGLVGI-56707380acid phosphatase (precursor)

GS 2GS2_pilGly_ A2_3521FLMPFMHYIVGI-56708006Type IV pili glycosylation protein

GS2_1709_ A2_3522KLWGLVDFVGI-56708718hypothetical protein FTT1709

GS2_pilGly_ A2_3523FLNKRIFSEVGI-56708006Type IV pili glycosylation protein

GS2_pilGly_ A2_3524ILLPWFVDLGI-56708006Type IV pili glycosylation protein

GS2_1709_ A2_3525WLGETFHGLGI-56708718hypothetical protein FTT1709

A3Genome Scan 1GS1_gluc_A3_3301KVFPFHFDLKGI-567075664-alpha-glucanotransferase

GS1_mbrn_A3_3302VLFPVPFIKGI-56708599outer membrane protein

GS1_FtsQ_A3_3303KVWPSTLVIYGI-56707351cell division protein FtsQ

GS1_pncl_A3_3304KLLGRVHAKGI-56708126D-alanyl-D-alanine carboxypeptidase (Penicillin binding protein) family protein

GS1_AcpA_A3_3305KLFNNANTLKGI-56707380acid phosphatase (precursor)

GS1_epim_A3_3306KVFGNLTYEKGI-56708503UDP-glucose 4-epimerase

GS1_phos_A3_3307DLFYKTLKDKGI-56707470undecaprenyl pyrophosphate synthetase

GS1_hyp_A3_3308KLFTYSIEAYGI-56707677hypothetical protein FTT0546

GS1_hypm_A3_3309KTFNRPVYYGI-56707276conserved membrane hypothetical protein

GS1_phos_A3_3310RTFGVAFYKGI-56708675Carbamoyl-phosphate synthase large chain

GS1_TolC_A3_3311VLFDWSKWKGI-56708730outer membrane protein tolC precursor

GS1_hydr_A3_3312KLYDVNSNVKGI-56708296choloylglycine hydrolase family protein

GS1_FtsQ_A3_3313ELFFKSYTKGI-56707351cell division protein FtsQ

GS1_mbrn_A3_3314KVGSVSLTGKGI-56708599outer membrane protein

GS1_hyp_A3_3315ILWGGVVKNYGI-56708642hypothetical protein FTT1625c

GS1_msrA_A3_3316KVWQNIDWKGI-56708785peptide methionine sulfoxide reductase msrA

GS1_FAD_A3_3317QVFWGSNYSKGI-56708305FAD binding family protein

GS1_hemK_A3_3318QLDKNTLKKGI-56707339hemK protein homolog

GS1_ligse_A3_3319WLYQNILKYGI-56707582UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-me so-diaminopimelate ligase

GS1_hyp_A3_3320AIFNSALYKGI-56707672hypothetical protein FTT0540c

GS 2GS2_1700_A3_3321ILYPKSIKYGI-56708709hypothetical protein FTT1700

GS2_1699_A3_3322ILYRKGLISKGI-56708708hypothetical protein FTT1699

GS2_1709_A3_3323KLLKSGLLNKGI-56708718hypothetical protein FTT1709

GS2_transA_A3_3324SLGQKVVYKGI-56707283Lipid A transport protein, ABC transporter,ATP-binding and membrane protein

GS2_1709_A3_3325SLDKIIHKTKGI-56708718hypothetical protein FTT1709

A24Genome Scan 1GS1_rdct_A24_3201KYWNNHRQGIGI-56707982peptide methionine sulfoxide reductase

GS1_hemK_A24_3202WYTNLDTDKFGI-56707339hemK protein homolog

GS1_chnt_A24_3203PYLKSFLSFGI-56707834chitinase family 18 protein

GS1_mbrn_A24_3204YYLDRGYLDFGI-56708599outer membrane protein

GS1_hyp_A24_3205TYPNSQQQTLGI-56707194hypothetical protein FTT0014c

GS1_hyp_A24_3206LYQPLLDLLGI-56707857hypothetical lipoprotein

GS1_mbrn_A24_3207IYNNYDVLFGI-56708599outer membrane protein

GS1_hyp_A24_3208LYSENGHFSWGI-56708577hypothetical protein FTT1549

GS1_lgse_A24_3209RYTDSEYFVIGI-56707582UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-me so-diaminopimelate ligase

GS1_hyp_A24_3210NYVDSAQLAFGI-56708571hypothetical protein FTT1539c

GS1_toler_A24_3211TYTSTLHPRLGI-56707609organic solvent tolerance protein

GS1_estr_A24_3212NYQKWTAPLGI-56707843glycerophosphoryl diester phosphodiesterase family protein

GS1_pncl_A24_3213PYLKQGIEFGI-56708118D-alanyl-D-alanine carboxypeptidase (Penicillin binding protein) family protein

GS1_hyp_A24_3214SYTFTQTFFGI-56707400hypothetical membrane protein

GS1_toler_A24_3215AYVKNGYFEIGI-56707609organic solvent tolerance protein

GS1_hyp_A24_3216IYSDKGLLFGI-56708577hypothetical protein FTT1549

GS1_asp_A24_3217NYNNLDDKFGI-56707606Periplasmic L-asparaginase II precursor

GS1_hyp_A24_3218SYSYLVYLIGI-56707520hypothetical protein FTT0369c

GS1_hyp_A24_3219SYDRWGIPIGI-56708127conserved hypothetical lipoprotein

GS1_lgse_A24_3220IFWQFHQLLGI-56707582UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-me so-diaminopimelate ligase

GS 2GS2_pil4_A24_3221TYLGIDLGFGI-56708230Type IV pili associated protein

GS2_1700_A24_3222SYNKTDDRWGI-56708709hypothetical protein FTT1700

GS2_1700_A24_3223EYINNQFPFGI-56708709hypothetical protein FTT1700

GS2_1709_A24_3224FYQLLIRLIGI-56708718hypothetical protein FTT1709

GS2_1709_A24_3225LYMTYLYCIGI-56708718hypothetical protein FTT1709

B7Genome Scan 1GS1_hypm_B7_3701VPMPNIIMVGI-56707430hypothetical membrane protein

GS1_mbrn_B7_3702APFANTYSALGI-56707711outer membrane associated protein

GS1_mbrn_B7_3703SPMGPLAVSFGI-56708599outer membrane protein

GS1_lactm_B7_3704FPICSVFKFLGI-56707736beta-lactamase

GS1_hyp_B7_3705APAFVVIADLGI-56707520hypothetical protein FTT0369c

GS1_hyp_B7_3706VPRKPLLSYIGI-56708127conserved hypothetical lipoprotein

GS1_asp_B7_3707SPRVVGNILGI-56707606Periplasmic L-asparaginase II precursor

GS1_lactm_B7_3708FPICSTYKFLGI-56707801Beta-lactamase class A

GS1_hypm_B7_3709IPMTLTASPLGI-56707205hypothetical membrane protein

GS1_glyc_B7_3710FPKAFINIVGI-56707549soluble lytic murein transglycosylase

GS1_solut_B7_3711DPHTFVSSVGI-56707372periplasmic solute binding family protein

GS1_hyp_B7_3712SPQANNLYLGI-56707802hypothetical protein FTT0682c

GS1_hyp_B7_3713SPMEHSKRIGI-56707738hypothetical protein FTT0613c

GS1_hyp_B7_3714FPATGQNIYMGI-56707423hypothetical protein FTT0267

GS1_chtn_B7_3715APAWFQASAGI-56707834chitinase family 18 protein

GS1_scrtn_B7_3716FPINLNSSQLGI-56707198Secretion protein

GS1_tolC_B7_3717FPQANLTGGIGI-56708730outer membrane protein tolC precursor

GS1_pncl_B7_3718APASLTKIMGI-56708118D-alanyl-D-alanine carboxypeptidase (Penicillin binding protein) family protein

GS1_hyp_B7_3719APYPNVVEMGI-56708630conserved hypothetical protein

GS1_chtn_B7_3720FPNINFSPEVGI-56707834chitinase family 18 protein

GS 2GS2_pilFib_B7_3721IPMYNNYILGI-56707387Type IV pili fiber building block protein

GS2_pilNBP_B7_3722LPRYNIATSVGI-56708209Type IV pili nucleotide binding protein, ABC transporter, ATP-binding protein

GS2_pyruv_B7_3723IPRTMLPLSLGI-56708523pyruvate dehydrogenase, E2 component

GS2_mglA_B7_3724TPNGNIPTLGI-56708335macrophage growth locus, subunit A

GS2_pilNBP_B7_3725MPVSRQISRMGI-56708209Type IV pili nucleotide binding protein, ABC transporter, ATP-binding protein

B44Genome Scan 1GS1_glut_B44_3401EEMKTVKGTYGI-56708248Gamma-glutamyltranspeptidase

GS1_phos_B44_3402GEVLGVIVQFGI-56708675Carbamoyl-phosphate synthase large chain

GS1_hyp_B44_3403EENGSSKKIYGI-56707245hypothetical protein FTT0066

GS1_secr_B44_3404YELWRRYNGFGI-56707198Secretion protein

GS1_mbrn_B44_3405FEVPSIWNLWGI-56708599outer membrane protein

GS1_phos_B44_3406AELVGRTERYGI-56708675Carbamoyl-phosphate synthase large chain

GS1_isom_B44_3407FEIDRKTREYGI-567086001-deoxy-D-xylulose 5-phosphate reductoisomerase

GS1_hyp_B44_3408DENGQVTKILGI-56708050hypothetical membrane protein

GS1_catal_B44_3409AENGNEQKFGI-56707839Peroxidase/catalase

GS1_glut_B44_3410AEDGIPVSYGI-56708248Gamma-glutamyltranspeptidase

GS1_HAD_B44_3411EENLTKEDIYGI-56707745HAD superfamily protein

GS1_mbrn_B44_3412KERPIIAGFGI-56708599outer membrane protein

GS1_hyp_B44_3413NEDDNVYTWGI-56708544hypothetical protein FTT1506

GS1_toler_B44_3414FEIQDIPVMYGI-56707609organic solvent tolerance protein

GS1_hyp_B44_3415QEIPNIVNKFGI-56708531hypothetical protein FTT1492c

GS1_gluc_B44_3416SEKTAINGEWGI-567075664-alpha-glucanotransferase

GS1_vitB12_B44_3417SEPLPVATTFGI-56708090Cobalamin (vitamin B12) synthesis protein/P47K family protein

GS1_hyp_B44_3418FEGDAQTWFGI-56707423hypothetical protein FTT0267

GS1_dhydr_B44_3419AELTADHIFGI-56708049Pyruvate/2-oxoglutarate dehydrogenase complex,dihydrolipoamide dehydrogenase component

GS1_hypm_B44_3420DENIGDFRFGI-56708638hypothetical membrane protein

GS 2GS2_pyruv_B44_3421GENLIIKKYGI-56708523pyruvate dehydrogenase, E2 component

GS2_threo_B44_3422EEIKQVYRQYGI-56707573threonine synthase

GS2_MFS_B44_3423AEIPVSVVYGI-56707837major facilitator superfamily (MFS) transport protein

GS2_ptran_B44_3424NEITTGEVVWGI-56707297oligopeptide transporter, subunit F, ABC transporter, ATP-binding protein

GS2_1715c_B44_3425EEIRSSQGFGI-56708724hypothetical protein FTT1715c

ELISpot

701702703704706707708709710711712713714715716717719720722723725726727

Most Recent Landscaping History on MVNoneNoneNoneNoneNoneNone6-10 yearsNone12 mo.12 mo.12 mo.12 mo.12 mo.12 mo.NoneNoneNoneNone12 mo.12 mo.12 mo.12 mo.12 mo.

Number of Years at Risk> 10> 10> 10> 10> 10 years> 10>10> 103-4>10> 10> 10> 10> 10> 10> 10> 10> 107 - 85 - 67 - 8< 0.57-8

Weeks per Year at RiskUnk++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

Age50-5415-2450-5445-4955-5940-4435-3945-4915-2440-4445-4955-5925-2945-4950-5460+55-5955-5915-2415-2425-2950-5450-54

Form of TularemiaPneuPneuPneuPneuPneuPneuUnkTyphPneuPneuUl/GlPneuPneuUnkPneuPneuPneuPneuTyphUl/GlTyphPharPneu

DiagnosisUnk200019991980’s20042000200120042004UnkUnk20001996Unk197819781978197820052005200520052005

701702703704706707708709710711712713714715716717719720722723725726727

FOPA 102n/sNTn/sn/s11211332583NTn/sn/sNTNTn/sn/sn/sNT32n/sNTn/sNTn/s

FOPA 176n/sNTn/s168293n/sn/sn/sNTn/sn/sNTNTn/sn/sn/sNTn/sNTn/sn/sNTn/s

GS1_Secr_II_3001NTn/sn/sn/sn/sn/sn/sn/sn/sn/sn/sn/sn/sNTn/sn/sn/sn/sn/sn/sn/sn/sn/s

GS1_Glyc_II_300261n/sn/sn/s32n/s32n/s219n/sn/s659n/s218n/sn/s166225n/sn/sn/sn/sn/s

GS1_hemK_II_3003NTn/sn/sn/s30n/s30028233n/sn/s225n/sNT2165516143540n/sn/sn/sn/sn/s

GS1_Chitn_II_300437n/sn/s1768123n/sn/s82673n/s670345n/s198n/sn/s1422103n/sn/sn/sn/s402

GS1_Epim_II_3005n/sn/sn/s179742n/sn/sn/s52n/sn/s145n/sn/s343n/s124373123n/sn/sn/sn/s

GS1_Toler_II_3006NTn/sn/sn/s115n/sNTNT117n/s165124n/sNT182n/s1583n/sn/sn/sn/sNTn/s

GS1_Hyp_II_3007NTn/sn/s207333n/sn/sn/sn/sn/sn/s171n/sNTn/sn/sn/s22n/sn/sn/sn/sn/s

GS1_Asp_II_3008NTn/s38n/s103n/s3732376n/sn/s275n/sNTn/sn/s787n/s160n/sn/sn/sn/s

GS1_Hyp_II_3009NTn/sn/sn/s45n/sn/sn/s88n/sn/s887n/sNTn/sn/sn/s42n/sn/sn/sn/sn/s

GS1_Mbrn_II_301064n/sn/sn/sn/sn/s382n/sn/s22n/s263n/sn/s267n/s136232n/sn/sn/sn/sn/s

GS1_Mbrn_II_3011NTn/sn/sn/s40NT508n/sNTNTNT104n/sNT133n/sn/s20n/sn/sn/sn/sn/s

GS1_Hyp_II_3012NTn/sn/sn/sn/sNTn/sn/sNTNTNTn/sn/sNTn/sn/sn/sn/sn/sn/sn/sn/s1895

GS1_TolC_II_3013NTn/sn/sn/sn/sNTn/sn/sNTNTNTNTn/sNTn/sn/sn/sn/sn/sn/sn/sn/sn/s

GS1_Hyp_II_3014NTNTn/sn/sn/sNTn/sn/sNTNTNTNTn/sNTn/sn/sn/sn/sn/sn/sn/sn/sn/s

GS1_FAD_II_3015NTNTn/sn/s72NT290n/sNTNTNTNTn/sNT1482n/sn/sn/sn/sn/sn/sn/sn/s

GS1_NADH_II_3016NTNTn/sn/sn/sNTn/sn/sNTNTNTNTn/sNT157n/sn/sn/sn/sn/sn/sn/sn/s

GS1_Hyp_II_3017NTNTn/sn/s45NT68n/sNTNTNTNTn/sNTn/sn/sn/sn/sn/sn/sn/sn/sn/s

GS2_pilNBP_II_3018NTNTn/sn/sn/sNTn/sn/sNTNTNTNTn/sNT170n/sn/sn/s75n/sn/sn/sn/s

GS2_1699_II_301963NTn/sn/sn/sn/sn/sn/sn/sn/sn/s37n/sn/sn/sn/sn/sn/sn/sn/sn/sn/s1867

GS2_1700_II_3020NTNTn/sn/sn/sn/sn/sn/s85n/sNT453n/sNT230n/sNTn/sn/sn/sn/sn/sNT

GS2_IcID_II_3021223NTn/s1823n/s38270n/sn/sn/s19056n/s71148n/sNT32n/sn/sn/s25NT

GS2_IcID_II_3022167NTn/sn/s23n/sn/sn/sn/sn/sn/s107n/s26n/sn/sNTn/sn/sn/sn/sn/sNT

GS2_1715c_II_30231056NTn/sn/s98n/sn/sn/s86n/sn/s71n/sn/sn/sn/sNTn/s242n/s25n/sn/s

GS2_1715c_II_3024NTNTn/sn/s30n/sn/sn/s156n/sn/sn/sn/sNTn/sn/sNT28n/sn/s40n/sn/s

GS2_1715c_II_3025NTNTn/sn/s1205113170n/s162n/sn/s565n/sNTn/sn/sNT78n/sn/sn/sn/sn/s

Class II Pool1148250NT1686NT1152963475976NT8031160NT381633553385860565n/s50581867

PHA759723049651126331553242409532374291019331602852438469072801289220183048107343178319822952313

Average significant response239n/s3818291328820918120422342280n/s128528516135644150n/s33251388

1013272727192626171918192510272719272626272424

70151731041113160410171240213

Percent of peptides recognized70%0%4%19%63%16%38%15%65%5%17%84%0%40%37%4%37%44%15%0%7%4%13%

PneuUnkUl/GlTyphPhar

70170471071371671972072251316934210725

GS1_HAD_A2_3501n/s260n/sn/sn/sn/sn/sn/s2218182724

GS1_hyp_A2_3502n/sn/sn/sn/s9457n/sn/s77331

GS1_chtn_A2_3503n/sn/sn/sn/sn/sn/sn/sn/sPercent35%39%17%11%4%

GS1_mbrn_A2_3504n/sn/sn/sn/sn/sn/sn/sn/sPneuUnkUl/GlTyphPhar

GS1_oxid_A2_3505n/sn/sn/sn/s714n/sn/sn/s1239208.9341.6666666667181.2525

GS1_hyp_A2_3506n/sn/sn/sn/sn/sn/sn/sn/s2n/s128.25n/s150

GS1_hyp_A2_3507n/sn/sn/sn/s2566n/sn/sn/s33832.5

GS1_sulf_A2_3508n/sn/sn/sn/sn/sn/sn/sn/s41828.6

GS1_toler_A2_3509n/sn/sn/sn/s609n/sn/sn/s5131.8235294118

GS1_pept_A2_3510n/sn/sn/sn/sn/sn/sn/sn/s688

GS1_hyp_A2_3511n/sn/s178n/sn/sn/sn/sn/s7204.2727272727

GS1_hyp_A2_3512n/s418n/sn/s36n/sn/sn/s822

GS1_hypm_A2_3513n/sn/sn/sn/sn/sn/sn/sn/s9280.4375

GS1_Hdrx_A2_3514n/sn/sn/sn/sn/sNTn/sn/s10n/s

GS1_hypm_A2_3515n/sn/sn/sn/sn/sNTn/sn/s11527.7

GS1_oxgn_A2_3516n/s127n/sn/s68NTn/sn/s12516


GS1_pilin_A2_3518n/sn/sn/sn/sn/sNTn/sn/s1443.9166666667

GS1_hyp_A2_3519n/sn/sn/sn/sn/sNTn/sn/s151388

GS1_AcpA_A2_3520n/sn/sn/sn/sn/sNTn/sn/s

GS2_pilGly_ A2_3521n/s433n/sNT269NTn/sn/s

GS2_1709_ A2_3522n/sn/sn/sNTNTNTNTn/s

GS2_pilGly_ A2_3523n/sn/sn/sNTNTNTNTn/s



A2 Pooln/s45283n/s18440n/sn/s

PHA759112637429285228012018304810734

713715717720

GS1_rdct_A24_3201n/sn/sn/s43

GS1_hemK_A24_3202n/sn/sn/s57

GS1_mbrn_A24_3204n/s126n/sn/s

GS1_asp_A24_3217n/sn/sn/s43

GS1_lgse_A24_3220NT233n/sNT

GS2_pil4_A24_3221NT101n/sNT

A24 Pooln/s104n/sn/s

PHA2852690728923048

Binding

Peptide ID% Inhibition at 100 μM

FOPA-10274GS1_HAD_A2_35011GS1_hypm_B7_370126GS1_rdct_A24_3201435

FOPA-17674GS1_hyp_A2_35021GS1_mbrn_B7_370222GS1_hemK_A24_3202255

GS1_Secr_II_300129GS1_chtn_A2_350315GS1_mbrn_B7_370318GS1_chnt_A24_320320

GS1_Glyc_II_300287GS1_mbrn_A2_35042GS1_lactm_B7_3704251GS1_mbrn_A24_320410

GS1_hemK_II_300334GS1_oxid_A2_35051GS1_hyp_B7_3705114GS1_hyp_A24_3205180

GS1_Chitn_II_300484GS1_hyp_A2_35061GS1_hyp_B7_370638GS1_hyp_A24_320630

GS1_Epim_II_300599GS1_hyp_A2_35071GS1_asp*_B7_37079GS1_mbrn_A24_320723

GS1_Toler_II_300610GS1_sulf_A2_350824GS1_lactm_B7_3708226GS1_hyp_A24_320818

GS1_Hyp_II_30074GS1_toler_A2_35091GS1_hypm_B7_37094GS1_lgse_A24_320915

GS1_Asp_II_300844GS1_pept_A2_35101GS1_glyc_B7_3710151GS1_hyp_A24_32109

GS1_Hyp_II_300918GS1_hyp_A2_35111GS1_solut_B7_37111862GS1_toler_A24_3211200

GS1_Mbrn_II_301079GS1_hyp_A2_35121GS1_hyp_B7_371274GS1_estr_A24_32125

GS1_Mbrn_II_301140GS1_hypm_A2_35131GS1_hyp_B7_371362GS1_pncl_A24_32138

GS1_Hyp_II_30122GS1_Hdrx_A2_351410GS1_hyp_B7_3714588GS1_hyp_A24_321424

GS1_TolC_II_30133GS1_hypm_A2_35151GS1_chtn_B7_3715>5000GS1_toler_A24_321576

GS1_Hyp_II_30140GS1_oxgn_A2_35161GS1_scrtn_B7_371662GS1_hyp_A24_32164

GS1_FAD_II_30154GS1_hypm_A2_35171GS1_tolC*_B7_37172145GS1_asp_A24_32173

GS1_NADH_II_30160GS1_pilin_A2_351820GS1_pncl_B7_371837GS1_hyp_A24_32183

GS1_Hyp_II_301744GS1_hyp_A2_35191GS1_hyp_B7_3719127GS1_hyp_A24_321933

GS2_pilNBP_II_30180GS1_AcpA_A2_352015GS1_chtn_B7_3720>5000GS1_lgse_A24_322010

GS2_1699_II_301979GS2_pilGly_ A2_35211GS2_pilFib_B7_372111

GS2_1700_II_30200GS2_1709_ A2_35221GS2_pilNBP_B7_37223

GS2_IcID_II_302174GS2_pilGly_ A2_35231GS2_pyruv_B7_37233

GS2_IcID_II_302296GS2_pilGly_ A2_35241GS2_mglA_B7_372446

GS2_1715c_II_3023100GS2_1709_ A2_35251GS2_pilNBP_B7_3725665

GS2_1715c_II_30240

GS2_1715c_II_30250

Calculations

701702703704706707708709710711712713714715716717719720722723725726727

Most Recent Landscaping History on MVNoneNoneNoneNoneNoneNone6-10 yearsNone12 mo.12 mo.12 mo.12 mo.12 mo.12 mo.NoneNoneNoneNone12 mo.12 mo.12 mo.12 mo.12 mo.

Number of Years at Risk> 10> 10> 10> 10> 10 years> 10>10> 103-4>10> 10> 10> 10> 10> 10> 10> 10> 107 - 85 - 67 - 8< 0.57-8

Weeks per Year at RiskUnk++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

Age50-5415-2450-5445-4955-5940-4435-3945-4915-2440-4445-4955-5925-2945-4950-5460+55-5955-5915-2415-2425-2950-5450-54

Form of TularemiaPneuPneuPneuPneuPneuPneuUnkTyphPneuPneuUl/GlPneuPneuUnkPneuPneuPneuPneuTyphUl/GlTyphPharPneu

DiagnosisUnk200019991980’s20042000200120042004UnkUnk20001996Unk197819781978197820052005200520052005

701702703704706707708709710711712713714715716717719720722723725726727

FOPA 102n/sNTn/sn/s11211332583NTn/sn/sNTNTn/sn/sn/sNT32n/sNTn/sNTn/s

FOPA 176n/sNTn/s168293n/sn/sn/sNTn/sn/sNTNTn/sn/sn/sNTn/sNTn/sn/sNTn/s

GS1_Secr_II_3001NTn/sn/sn/sn/sn/sn/sn/sn/sn/sn/sn/sn/sNTn/sn/sn/sn/sn/sn/sn/sn/sn/s

GS1_Glyc_II_300261n/sn/sn/s32n/s32n/s219n/sn/s659n/s218n/sn/s166225n/sn/sn/sn/sn/s

GS1_hemK_II_3003NTn/sn/sn/s30n/s30028233n/sn/s225n/sNT2165516143540n/sn/sn/sn/sn/s

GS1_Chitn_II_300437n/sn/s1768123n/sn/s82673n/s670345n/s198n/sn/s1422103n/sn/sn/sn/s402

GS1_Epim_II_3005n/sn/sn/s179742n/sn/sn/s52n/sn/s145n/sn/s343n/s124373123n/sn/sn/sn/s

GS1_Toler_II_3006NTn/sn/sn/s115n/sNTNT117n/s165124n/sNT182n/s1583n/sn/sn/sn/sNTn/s

GS1_Hyp_II_3007NTn/sn/s207333n/sn/sn/sn/sn/sn/s171n/sNTn/sn/sn/s22n/sn/sn/sn/sn/s

GS1_Asp_II_3008NTn/s38n/s103n/s3732376n/sn/s275n/sNTn/sn/s787n/s160n/sn/sn/sn/s

GS1_Hyp_II_3009NTn/sn/sn/s45n/sn/sn/s88n/sn/s887n/sNTn/sn/sn/s42n/sn/sn/sn/sn/s

GS1_Mbrn_II_301064n/sn/sn/sn/sn/s382n/sn/s22n/s263n/sn/s267n/s136232n/sn/sn/sn/sn/s

GS1_Mbrn_II_3011NTn/sn/sn/s40NT508n/sNTNTNT104n/sNT133n/sn/s20n/sn/sn/sn/sn/s

GS1_Hyp_II_3012NTn/sn/sn/sn/sNTn/sn/sNTNTNTn/sn/sNTn/sn/sn/sn/sn/sn/sn/sn/s1895

GS1_TolC_II_3013NTn/sn/sn/sn/sNTn/sn/sNTNTNTNTn/sNTn/sn/sn/sn/sn/sn/sn/sn/sn/s

GS1_Hyp_II_3014NTNTn/sn/sn/sNTn/sn/sNTNTNTNTn/sNTn/sn/sn/sn/sn/sn/sn/sn/sn/s

GS1_FAD_II_3015NTNTn/sn/s72NT290n/sNTNTNTNTn/sNT1482n/sn/sn/sn/sn/sn/sn/sn/s

GS1_NADH_II_3016NTNTn/sn/sn/sNTn/sn/sNTNTNTNTn/sNT157n/sn/sn/sn/sn/sn/sn/sn/s

GS1_Hyp_II_3017NTNTn/sn/s45NT68n/sNTNTNTNTn/sNTn/sn/sn/sn/sn/sn/sn/sn/sn/s

GS2_pilNBP_II_3018NTNTn/sn/sn/sNTn/sn/sNTNTNTNTn/sNT170n/sn/sn/s75n/sn/sn/sn/s

GS2_1699_II_301963NTn/sn/sn/sn/sn/sn/sn/sn/sn/s37n/sn/sn/sn/sn/sn/sn/sn/sn/sn/s1867

GS2_1700_II_3020NTNTn/sn/sn/sn/sn/sn/s85n/sNT453n/sNT230n/sNTn/sn/sn/sn/sn/sNT

GS2_IcID_II_3021223NTn/s1823n/s38270n/sn/sn/s19056n/s71148n/sNT32n/sn/sn/s25NT

GS2_IcID_II_3022167NTn/sn/s23n/sn/sn/sn/sn/sn/s107n/s26n/sn/sNTn/sn/sn/sn/sn/sNT

GS2_1715c_II_30231056NTn/sn/s98n/sn/sn/s86n/sn/s71n/sn/sn/sn/sNTn/s242n/s25n/sn/s

GS2_1715c_II_3024NTNTn/sn/s30n/sn/sn/s156n/sn/sn/sn/sNTn/sn/sNT28n/sn/s40n/sn/s

GS2_1715c_II_3025NTNTn/sn/s1205113170n/s162n/sn/s565n/sNTn/sn/sNT78n/sn/sn/sn/sn/s

Class II Pool1148250NT1686NT1152963475976NT8031160NT381633553385860565n/s50581867

PHA759723049651126331553242409532374291019331602852438469072801289220183048107343178319822952313

Average238.7142857143n/s381828.6131.823529411888208.9181.25204.272727272722341.6666666667280.4375n/s128.25527.75161356.285714285743.9166666667150n/s32.5251388

1013272727192626171918192510272719272626272424

70151731041113160410171240213

0.700.0370370370.18518518520.62962962960.15789473680.38461538460.15384615380.64705882350.05263157890.16666666670.842105263200.40.37037037040.0370370370.36842105260.44444444440.153846153800.07407407410.04166666670.125

PneuUnkUl/GlTyphPhar

70170471071371671972072251316934210725

GS1_HAD_A2_3501n/s260n/sn/sn/sn/sn/sn/s2218182724

GS1_hyp_A2_3502n/sn/sn/sn/s9457n/sn/s77331

GS1_chtn_A2_3503n/sn/sn/sn/sn/sn/sn/sn/sPercent35%39%17%11%4%

GS1_mbrn_A2_3504n/sn/sn/sn/sn/sn/sn/sn/sPneuUnkUl/GlTyphPhar

GS1_oxid_A2_3505n/sn/sn/sn/s714n/sn/sn/s1239208.9341.6666666667181.2525

GS1_hyp_A2_3506n/sn/sn/sn/sn/sn/sn/sn/s2n/s128.25n/s150

GS1_hyp_A2_3507n/sn/sn/sn/s2566n/sn/sn/s33832.5

GS1_sulf_A2_3508n/sn/sn/sn/sn/sn/sn/sn/s41828.6

GS1_toler_A2_3509n/sn/sn/sn/s609n/sn/sn/s5131.8235294118

GS1_pept_A2_3510n/sn/sn/sn/sn/sn/sn/sn/s688

GS1_hyp_A2_3511n/sn/s178n/sn/sn/sn/sn/s7204.2727272727

GS1_hyp_A2_3512n/s418n/sn/s36n/sn/sn/s822

GS1_hypm_A2_3513n/sn/sn/sn/sn/sn/sn/sn/s9280.4375

GS1_Hdrx_A2_3514n/sn/sn/sn/sn/sNTn/sn/s10n/s


GS1_oxgn_A2_3516n/s127n/sn/s68NTn/sn/s12516


GS1_pilin_A2_3518n/sn/sn/sn/sn/sNTn/sn/s1443.9166666667

GS1_hyp_A2_3519n/sn/sn/sn/sn/sNTn/sn/s151388

GS1_AcpA_A2_3520n/sn/sn/sn/sn/sNTn/sn/s

GS2_pilGly_ A2_3521n/s433n/sNT269NTn/sn/s





A2 Pooln/s45283n/s18440n/sn/s

PHA759112637429285228012018304810734

713715717720

GS1_rdct_A24_3201n/sn/sn/s43

GS1_hemK_A24_3202n/sn/sn/s57

GS1_mbrn_A24_3204n/s126n/sn/s

GS1_asp_A24_3217n/sn/sn/s43

GS1_lgse_A24_3220NT233n/sNT

GS2_pil4_A24_3221NT101n/sNT

A24 Pooln/s104n/sn/s

PHA2852690728923048

Calculations

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1

2

3

4

5

6

7

8

9

10

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12

13

14

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mcmurryjFile AttachmentFT_MS_Tables_1&2_11Dec06.xls

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ARTICLE IN PRESSJVAC 6891 1–13J.A. McMurry et al. / Vaccine xxx (2007) xxx–xxx 7

Table 3Putative epitopes binding to respective HLA

Columns 1 and 2: promiscuous class II peptides at 100 �M and the percent by which each peptide inhibits the binding of a competitor labeled peptide toDRB1*0101. At 100 �M of test peptide, strong (75–100% inhibition), moderate (50–75% inhibition), weak (30–50% inhibition), or non-binding (0–30%inhibition) can be roughly estimated. Peptides that bind are highlighted in grey. Columns 3–8: putative A2, B7, and A24 peptides and the dissociation constant(nM) each exhibits for the corresponding HLA. Weak or no affinity, >500 nM KD; moderate affinity, ≤500 nM K ; high affinity, ≤50 nM K ; very high affinity,≤ 225 hav

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5 nM KD. Peptides that bind are highlighted in grey. A24 peptides 3221–3

roteins was analyzed according to the methods describedbove. From approximately 18,000 possible peptides, thisnalysis yielded 176 unique candidate class II clusters withscore above 15.

The top scoring 50 selected clusters were blasted againsthe protein database at GenBank; 32 (64%) clusters wereiscarded for having at least one 9-mer in which ateast seven of nine amino acids were identical to thosen a human protein. Clusters lacking a significant motif

atch for DRB1*0101 were also discarded. High scor-ng clusters were weighted according to their putativelass I content and resorted. The top ranked 17 candi-ate peptide sequences selected for synthesis. FopA hadreviously been shown to be an immunogenic proteinhus two putative promiscuous epitopes were selected asontrols even though they were not among the highestanked.

.1.4. Selection of class I epitopes for Genome Scan 2We also analyzed all 53 proteins for 9-mer and 10-

er that were putative class I epitopes. After discardingve sequences with 7/9 AA identities to human sequences,e selected the five top scoring 9-mer and 10-mer pep-
U

ides for each of five alleles: A*0201, A*0301, A*2402,*0702, and B*4403. None of the GS2 class I selectedeptides was contained within a class II cluster previouslyhosen.

1a(v

D De yet to be tested.

.2. Binding results

.2.1. Results of class II binding assaysCompetitive in vitro binding assays were performed for

ll 27 putative promiscuous class II epitopes. Though theromiscuous peptides are predicted to bind to several classI HLA molecules, analysis of binding to DRB1*0101 wasiven priority because subsequent studies were planned inice transgenic for DRB1*0101. Results for HLA class IIRB1*0101 binding assays are shown in Table 3. Of the7 putative promiscuous epitopes, 14 (52%) exhibited pos-tive binding to HLA DRB1*0101: 7 peptides bound withigh affinity (75–100% inhibition), 4 peptides with moder-te affinity (50–75% inhibition), and 3 with weak affinity30–50% inhibition).

.2.2. Results of class I binding assaysCompetitive in vitro binding assays were also performed

or A2, B7, and A24; results are shown, respectively. Of the 25utative A2 epitopes, all 25 (100%) exhibited strong bindingo HLA*A2 (≤50 nM KD); of these, 20 exhibited extremelytrong binding (≤2 nM KD). Of the 20 putative A24 epi-opes tested, 4 bound with very high affinity (≤5 nM KD),


1 bound at high affinity (≤50 nM KD), and 5 at moderate 376ffinity (≤500 nM KD). Of the 25 putative B7 epitopes, 19 37776%) exhibited binding to HLA*B7; of these, 3 bound at 378ery high affinity (≤5 nM KD), 3 with high affinity (5–50 nM 379


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D), 8 with moderate (50–500 nM KD), and 6 with low oro binding (≥500 nM KD). Predicted, A3 and B44 epitopesave not yet been tested in binding assays. Taken together,1% of the class I peptides bound as predicted.

.3. ELISpot results

.3.1. Results of class II ELISpotsEven though the Martha’s Vineyard cohort was rela-

ively limited (23 subjects), 24 of 27 EpiMatrix promiscuouspitopes provoked IFN-� secretion by PBMC assessed byLISpot assay (Table 4). The percent of positive responseser subject was 26% on average. Peptide GS2 IcID II 3021as recognized by 50% of the patients. This epitope is from

he intracellular growth locus, subunit D, a known virulenceactor [27]. Two additional epitopes, GS1 hemK II 3003 andS1 Chitin II 3004, were recognized in 48% and 43% of

ubjects, respectively (Table 5).

.3.2. Results of class I ELISpotsFor class I epitopes, positive ELISpot responses were

bserved for 36% (9/25) and 24% (6/25) of the A2 and A24eptides, respectively (Table 6). Two putative B44 epitopesere confirmed to be immunogenic: GS1 phos B44 3402 in

ubjects 710 and 716 and GS1 vitB12 B44 3417 in subject10 (data not shown).

.3.3. Class I and class II ELISpot comparisonIn this study thus far, 3/27 (11%) of the class II puta-

ive promiscuous epitopes induced no significant immuneesponse in ELISpot. It is noteworthy that all three of theseon-antigenic peptides are derived from GS1 ORFs that areutatively secreted but not known to be expressed. In con-rast, 13/25 (76%) putative A2 epitopes and 19/25 (52%) A24eptides induced no significant immune response (Table 7).

.4. Results summary

Fig. 1 illustrates the overall process for identifying candi-ate epitopes for inclusion in a rationally designed tularemiaaccine.

It is encouraging that 88% of the class II peptides selectedor this study were antigenic, though their HLA bindingas not been fully characterized. Likewise it is encouraginghat 91% of the tested class I peptides bound as predicted,lthough their immunogenicity is not yet fully characterized.

. Discussion

.1. Breadth and strength of antigenic recognition

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dx.doi.org/10.1016/j.vaccine.2007.01.039mcmurryjInserted Text, Table 4

mcmurryjCross-Out

mcmurryjInserted Text5

mcmurryjCross-Out

mcmurryjReplacement Text2-5

mcmurryjInserted Text*

mcmurryjCross-Out


mcmurryjCross-Out


mcmurryjCross-Out

mcmurryjReplacement Text2-5*

mcmurryjInserted TextAsterisk (*) indicates positive diagnosis during blood draws in a recent epidemiological survey; thus form and date of tularemia infection unknown.

mcmurryjNoteThis table was submitted as perfectly aligned with Table 5 so that the relationships between subjects characteristics and elispot responses could be seen visually. It does make the text in the table 4 harder to read though, so I understand the rationale for making it vertical on a separate page.

mcmurryjCross-Out

mcmurryjReplacement Textyears since

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Table 5IFN-� ELISpot in response to putative promiscuous class II epitopes

The numbers of PBMC (over background) secreting IFN-� in response to PHA, pool of 27 class II peptides, or to individual class II peptides. For simplicity,statistically non-significant results are denoted by n/s and missing data are omitted; significant results are highlighted in grey. A response in ELISpot wasconsidered positive if two criteria were met: (1) spot-forming cells (SFC) per million PBMC at least 20 over background; (2) spot-forming cells (SFC) permillion PBMC at least two-fold over background. Column headers: human subject ID code. Row labels: promiscuous peptide ID.

Fig. 1. Prediction and triage of candidate epitopes for inclusion in a tularemia vaccine. The number of genes or peptides at each of the levels of selection isshown in parentheses.

dx.doi.org/10.1016/j.vaccine.2007.01.039mcmurryjNoteThe text in Fig 1 renders poorly on screen and in print. A copy of the eps file was sent. Can this be remedied?

mcmurryjNoteWith great effort, these tables were submitted in word format per instructions. They were also provided in excel format. Here in proof stage, they are images and as such, they print and view suboptimally, also making for an extremely large PDF (almost 3MB).

1. Downloaded from GenBank the 1803 ORFs of the Francisella tularensis Schu4 genome

2. Identified sequences for in silico analysis:

(GS1) ORFs that encode putatively secreted proteins (147)

(GS2) published proteins known to be expressed (255)

3. Ranked potential supertype Class I epitopes and promiscuous class II epitopes using EpiMatrix and ClustiMer

4. Identified and discarded human homologues (Blast)

5. Synthesized peptides for top-ranking putative epitopes:

Class I (25 each for A2, A3, A24, B7 and B44 supertypes)

Class II promiscuous (27 each with multiple motifs)

6. Confirmed 91% Class I peptides bind HLA as predicted

Confirmed 88% Class II peptides induce IFN-gamma secretion in PBMC from tularensis-exposed cohort

7. Ranked both ligands and epitopes for inclusion in vaccine

mcmurryjFile AttachmentTuly_MS_Fig1_23Jan07.doc

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Table 6IFN-� ELISpot in response to putative A2 epitopes

The numbers of PBMC (over background) secreting IFN-� in response toPHA, pool of 25 A2 peptides, or to individual A2 peptides. For simplicity,statistically non-significant results are denoted by n/s and missing data areomitted; significant results are highlighted in grey. A response in ELISpotwas considered positive if two criteria were met: (1) spot-forming cells(SFC) per million PBMC at least 20 over background; (2) spot-formingch

T426t427a428a429

Table 7IFN-� ELISpot in response to putative A24 epitopes

The numbers of PBMC (over background) secreting IFN-� in response toPHA, pool of 25 A24 peptides, or to individual A24 peptides. For simplicity,statistically non-significant results are denoted by n/s and missing data areomitted; significant results are highlighted in grey. The A24 peptides notshown here were ELISpot negative in all four subjects tested. A response inELISpot was considered positive if two criteria were met: (1) spot-formingcch

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461

r 462were modest 153 as compared to an average 513 SFC/106 463

NC

ells (SFC) per million PBMC at least two-fold over background. Columneaders: HLA*A2 subject ID.

he epitopes are ranked by percent recognition in Fig. 2;
U

hose with the greatest percentage of subjects respondingre: GS2 IcID II 3021 (50%), GS1 Chitin II 3004 (48%),nd GS1 hemK II 3003 (43%). A lack of response to any

fi(

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eaders: HLA*A24 subject ID code. Row labels: HLA*A24 peptide ID.

iven HLA ligand (class I or class II) could be attributable tonumber of factors: (1) the protein might not be expressed inuman infection, and (2) if expressed, the protein may enterhe MHC presentation pathway in amounts insufficient foresponses to be generated or detected.

The single most immunogenic epitope identified in thistudy was GS1 hyp A2 3507 with 2566 SFC/106 PBMC;owever, on average the class I responses were smaller inagnitude than class II responses. Class II responses, in

ddition to being stronger, were more frequent than classresponses. This may be explained by the restriction of thelass I epitopes to a single HLA supertype as compared tohe multiple overlapping HLA binding motifs within a singleromiscuous class II peptide. From our experience with epi-ope mapping of other pathogens, we expect that the percentf true epitopes among putative epitopes is closer to 75%or any given class I allele and 90% for any given class IIromiscuous peptide. The small number of class I peptidesonfirmed immunogenic thus far is likely to be an artifact ofhe small size of the cohorts: four to eight subjects per A2eptide, and four subjects each per A24 peptide and per B44eptide.

Furthermore, this study was not designed to evaluate theelative contribution of CD4+ or CD8+ T cells: class II pep-ides selected for this study also contained class I MHC motifsnd some of the observed immunogenicity may have been dueo CD8+ T-cell responses. This study does appear to con-rm that both CD4+ and CD8+ T-cell responses are essentialomponents of host defenses against tularemia and that theseesponses are directed toward a broad array of proteins whoseunctions are diverse and even unknown.

It is interesting that the average significant per-peptideesponses of subjects with prior non-pneumonic tularemia


or subjects with prior pneumonic tularemia. This difference 464s not statistically significant by unpaired Student’s t-test 465p < 0.2). Likewise, the most numerous responses were from 466

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ig. 2. Class II putative promiscuous epitopes ranked by percent of testedignificant responses (grey diamonds) for individual and pooled peptides. P

ubjects that had had the pneumonic form. Subject 719 wasne of such subjects; even 27 years after infection, this sub-ect’s response to six individual class II peptides surpassed000 SFC/106 each. Subject 713 responded to 16 of the 19lass II peptides tested (84% as compared to 26% on aver-ge). Subject 713 demonstrated a robust per-peptide positiveesponse averaging 280 SFC/106 and a robust pooled-peptideesponse: 1160 SFC/106. In spite of this subject’s noteworthyreadth of response, the strengths of both the per-peptide andooled-peptide responses approximated the cohort average.iagnosed 5 years prior to the study, subject 713 reported

o have spent over half of each year outdoors on Martha’sineyard for over 10 years suggesting that repeated exposure

o tularensis was possible and might account for the breadthf responses observed.

To date, we identified 39 novel F. tularensis epitopes, ateast 29 of which were derived from proteins not previouslynown to be immunogenic. Some of the confirmed epitopesere derived from the putative protein products of ORFs notreviously known to be expressed. The high rate of success-ul predictions shown herein suggests that the bioinformaticsethods used can be implemented on a broader scale to

elect additional epitopes for this vaccine project, as wells epitopes for projects involving other pathogens. As withraditional antigen discovery, bioinformatics tools cannot yetetermine whether antigenicity, though requisite, will confermmunogenicity and protection.

.2. HLA restriction

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HLA typing was done for each subject (data not shown);owever, the high frequency of responses coupled with themall size of the cohort made it impossible to determinehether the promiscuous epitopes were restricted by certain

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Os with significant ELISpot responses (black bars) vs. the average of those3001, 3013, and 3014 induced no significant responses.

HC. Regardless, other researchers showed one of the criti-al determinants of immunogenicity is the strength of peptideinding to MHC molecules [28], a finding we have confirmedxperimentally in mice.

.3. Correlation between binding and immunogenicity

Every peptide that bound DRB1*0101 (≥30% inhibition)as immunogenic in at least 2 subjects and as many as 11

ubjects (48%) (see Table 3). Because no single HLA alleleas shared by all subjects responding to a given peptide, the

mmune response can be said to be promiscuous. Althoughhis observed promiscuous immunogenicity of the epitopesmplicates, by necessity, their promiscuous HLA binding,nly DRB1*0101 binding assays have yet been performed.

Peptides were considered to be putatively promiscuous-ased EpiMatrix Cluster scores of ≥15, as these scores haveeen indicative in other studies [29]. Putative promiscuouseptides were screened at a single concentration (100 �M)or binding to DRB1*0101. Analysis of binding at severaloncentrations is needed to generate IC50 values (calculatedy non-linear regression analysis using the SigmaPlot analy-is program). Additional class II binding studies are expectedo support the ELISpot data generated in this study; indeed, inur other studies binding to several class II HLA is associatedith promiscuous immunogenicity [29].Of the six A24 peptides confirmed to be immunogenic

hus far, five were confirmed to bind to A24 and one wasot yet tested. All of the putative A2 epitopes bind HLA2 with high affinity. It is perhaps coincidental that within


cohort of only five A2 subjects, the confirmed A2 epi- 527opes were those that bound A2 with extremely high affinity 528

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ARTICLE2 J.A. McMurry et al. /

.4. Summary

The importance of bioinformatics in the evaluation ofhe proteomes of pathogenic organisms is underscored byhe approach outlined herein. Whereas a function has beenssigned to 1080 of the 1603 ORFs in the Schu4 genome,he functions of the remaining 523 ORFs have yet to beharacterized. Among these, 80% have homologues in otherrganisms, and 20% are unique to the Francisella complex.ight out of 39 of the epitopes identified in this study wereerived from “hypothetical proteins”. We also confirmed themmunogenicity of proteins that had previously been studied:clD, 1715c, FopA, pilNBP, FTT 1699, FTT 1700, and Pil4.

In addition, mapping epitopes to under-characterized anti-ens may permit immunologists to evaluate better the breadthf immune response to a pathogen. The immunoinformaticspproach to epitope mapping reveals that no single epi-ope or antigen is overwhelmingly immunodominant [30,31].he breadth of tularensis antigens, coupled with the het-rogeneity of responses to these antigens, suggest that theuman immune response to FT may be more complex andore omnivorous than is generally recognized. We have

bserved this also to be true in studies of human immuneesponse to TB [13,32]. Protective immunity to complexathogens may require recognition of epitopes derived frombroad range of pathogen-associated proteins. This may be

xplain why vaccines focused on a single antigen or a fewntigens do not provide sufficient “breadth” of protectionn humans.

Consequently, a vaccine may need to induce responseso several proteins, or several hundred epitopes in order torovide both sufficient protection and population coverage.his approach would also be concordant with the beliefs ofne of the leading experts in this field, who said, “one aspectf [F. tularensis] vaccine development that has to be con-idered in immunization . . . is the probable heterogeneityf immunogenic epitopes. A large number of T lymphocytelones covering a wide range of specificities may have to beuilt up in order to afford good protection” [4].

Using an immunoinformatics approach, scientists can nowegin to compare the immunomes of pathogens and to mea-ure the breadth and overlap of immunomes that give rise toither favorable or unfavorable consequences. We found, forxample, that 141 of the 152 putative epitopes chosen fromhe Schu4 proteome in this analysis had an identical or closely

atched counterpart in the live vaccine strain. A subset of epi-opes identified here was incorporated into a string-of-beadsNA prime, peptide boost vaccine and was used to immunizeLA transgenic mice. Vaccinated mice exhibited protective

mmunity to intratracheal challenge with a lethal dose of F.ularensis LVS (manuscript in preparation).

Coupling immunoinformatics tools with sensitive
U

mmunoassays offers a powerful approach to identifyinghe immunogenic needles in the proteome haystack. Theuccess of these complementary strategies suggests that theack of protein characterization need not impair our ability

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PRESSxxx (2007) xxx–xxx

o build and test a vaccine comprised of antigens recognizedy T lymphocytes.

cknowledgements

We thank the study subjects for donating their timend specimens, and we thank Donna Enos for coordinatingecruitment and donations. We also thank Bill Martin for per-orming the in silico analysis and to Christine Malboeuf forerforming the ex vivo laboratory work. Both Bill and Chris-ine also provided invaluable assistance in the editing of this

anuscript. This work was supported by two grants from theational Institutes of Health: 1R43AI058326 (PI: A.S. Deroot) and R21AI055657 (PI: S.H. Gregory).One of the contributing authors, Anne S. De Groot, is CEO

nd majority shareholders at EpiVax, Inc., a privately ownedaccine design company located in Providence, RI. Dr. Deroot is also a faculty member at Brown University.Conflict of interest: The authors acknowledge that there

s a potential conflict of interest related to their relationshipith EpiVax and attest to the work contained in this research

eport is free of any bias that might be associated with theommercial goals of the company.

eferences

[1] Dennis DT, Inglesby TV, Henderson DA, et al. Tularemia as a biologicalweapon: medical and public health management. JAMA 2001;285(June(21)):2763–73 [Review].

[2] Larsson P, Oyston PC, Chain P, et al. The complete genome sequenceof Francisella tularensis, the causative agent of tularemia. Nat Genet2005;37(February (2)):153–9.

[3] Elkins KL, Cowley SC, Bosio CM. Innate and adaptive immuneresponses to an intracellular bacterium, Francisella tularensis live vac-cine strain. Microbes Infect 2003;5(February (2)):135–42 [Review].

[4] Tarnvik A. Nature of protective immunity to Francisella tularensis. RevInfect Dis 1989;11(May–June (3)):440–51 [Review].

[5] Fulop M, Mastroeni P, Green M, Titball RW. Role of antibody tolipopolysaccharide in protection against low- and high-virulence strainsof Francisella tularensis. Vaccine 2001;19(August (31)):4465–72.

[6] Yee D, et al. Loss of either CD4+ or CD8+ T cells does not affect themagnitude of protective immunity to an intracellularpathogen, Fran-cisella tularensis strain LVS. J Immunol;157(December (11)):5042–8.

[7] Conlan JW, Sjostedt A, North RJ. CD4+ and CD8+ T-cell-dependentand -independent host defense mechanisms can operate to control andresolve primary and secondary Francisella tularensis LVS infection inmice. Infect Immun 1994;62(December (12)):5603–7.

[8] Duckett NS, Olmos S, Durrant DM, Metzger DW. Intranasalinterleukin-12 treatment for protection against respiratory infectionwith the Francisella tularensis live vaccine strain. Infect Immun 2005;73(April (4)):2306–11.

[9] Rhinehart-Jones TR, Fortier AH, Elkins KL. Transfer of immunityagainst lethal murine Francisella infection by specific antibody dependson host gamma interferon and T cells. Infect Immun 1994;62(August


(8)):3129–37. 63510] Tarnvik A, Ericsson M, Golovliov I, Sandstrom G, Sjostedt A. Orches- 636

tration of the protective immune response to intracellular bacteria: 637Francisella tularensis as a model organism. FEMS Immunol Med 638Microbiol 1996;13(March (3)):221–55. 639

dx.doi.org/10.1016/j.vaccine.2007.01.039mcmurryjCross-Out

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mcmurryjReplacement TextDr. De Groot

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