fiologi™ ‡—rf—re wedi™ine

hefense eg—inst fiologi™ ‡—rf—re with ƒuper—ntigen „oxins

‚—ymond u—empfer €hhD qil— er—d €hhD ‚evit—l vevy fe —nd h—li— rillm—n fe

hep—rtment of wole™ul—r †irologyD re˜rew niversityEr—d—ss—h wedi™—l ƒ™hoolD terus—lemD ssr—el

uey wordsX super—ntigenD „ ™ell —™tiv—tionD toxi™ sho™kD in™—p—™it—tionD —nt—gonist peptide

pro™essing ˜y —ntigenEpresenting ™ells ‘R±V“F „he „g‚ inter—™ts with

e˜str—™t

super—ntigens vi— the outer f—™e of its †˜ dom—inD — region not

f—™kgroundX ƒuper—ntigens produ™ed ˜y ƒt—phylo™o™™us —ureus

involved in ordin—ry —ntigen re™ognition ‘W“F fyp—ssing the —nd ƒtrepto™o™™us pyogenes —re —mong the most leth—l of toxinsF

restri™ted present—tion of ™onvention—l —ntigensD super—ntigens „oxins in this f—mily trigger —n ex™essive ™ellul—r immune response

le—ding to toxi™ sho™kF ™—n —™tiv—te up to SH7 of „ ™ells to divide —nd produ™e ™ytokinesF

y˜je™tivesX „o design —n —nt—gonist th—t is effe™tive in vivo

„husD super—ntigens —™tiv—te the ™ellul—r immune response —t le—st

—g—inst — ˜ro—d spe™trum of super—ntigen toxinsF

SDHHHEfold more strongly th—n do ordin—ry —ntigensF „oxi™ sho™k

wethodsX ƒhort peptide —nt—gonists were sele™ted for their —˜ility

results from — sudden —nd m—ssive indu™tion of „ helper I ™ellE to inhi˜it super—ntigenEindu™ed expression of hum—n genes for

derived ™ytokines th—t in™lude interleukinEPD interferonEg —nd tumor ™ytokines th—t medi—te sho™kF „he —˜ility of these peptides to prote™t

mi™e —g—inst leth—l toxin ™h—llenge w—s ex—minedF ne™rosis f—™tor ‘IH“F he—th from ™—pill—ry le—k syndrome results

‚esultsX ent—gonist peptide prote™ted mi™e —g—inst leth—l ™h—lE

within PR±RV hoursD ˜ut even —t ™on™entr—tions sever—l logs ˜elow

lenge with st—phylo™o™™—l enterotoxin f —nd toxi™ sho™k syndrome

leth—l ones these toxins ™—n eli™it severe —nd prolonged in™—p—™it—E

toxinEID super—ntigens th—t sh—re only T7 over—ll —mino —™id

tion in hum—nsD in™luding n—use—D vomiting —nd di—rrhe— ‘PDII“F homologyF woreoverD it res™ued mi™e undergoing toxi™ sho™kF

ƒt—phylo™o™™—l food poisoning is ™—used ˜y low ™on™entr—tions of ent—gonist peptides show homology to — ˜Estr—ndGhingeG—Ehelix dom—in

th—t is stru™tur—lly ™onserved —mong super—ntigensD yet remote from super—ntigen toxinsF

known ˜inding sites for the m—jor histo™omp—ti˜ility ™l—ss ss mole™ule

„he f—mily of —˜out two dozen pyrogeni™ exotoxin super—ntigens

—nd „ ™ell re™eptor th—t fun™tion in toxi™ „ ™ell hyper—™tiv—tionF

se™reted ˜y ƒt—phylo™o™™us —ureus —nd ƒtrepto™o™™us pyogenes in™ludes

gon™lusionsX „he leth—l effe™t of super—ntigens ™—n ˜e ˜lo™ked

—t le—st IH st—phylo™o™™—l enterotoxins —mong whi™h ƒif is most with — peptide —nt—gonist th—t inhi˜its their —™tion —t the top of the

prominentD —s well —s toxi™ sho™k syndrome toxinEID —nd streptoE toxi™ity ™—s™—de ˜efore —™tiv—tion of „ ™ells o™™ursF ƒuper—ntigeni™

toxin —nt—gonists m—y serve not only —s ™ounterme—sures to ˜iologi™ ™o™™—l pyrogeni™ exotoxinsD —mong them ƒ€ieF „o ™ompound the

w—rf—re ˜ut m—y ˜e useful in the tre—tment of st—phylo™o™™—l —nd

pro˜lem of prote™tionD the —mino —™id sequen™es of super—ntigens

strepto™o™™—l toxi™ sho™kD —s well —s in some ™—ses of septi™ sho™kF

—re highly divergentF „husD ƒie —nd ƒif —re PU7 homologous ‘IP“D

swet PHHPYRXSPH±SPQ where—s „ƒƒ„EI sh—res only T7 sequen™e homology with ƒif ‘IQ“F

ƒif w—s we—ponized ˜y the nited ƒt—tes ˜efore IWTWF roweverD

™urrent s™en—rios of ˜iologi™ w—rf—re —nd ˜ioterror —re more likely to

fiologi™ toxins m—y soon repl—™e tr—dition—l ™hemi™—l we—ponsD ent—il the use of super—ntigen toxins in n—tur—l mixturesD re—dily

given the e—se —nd low ™ost of toxin produ™tion ™oupled with their o˜t—ined ˜y ™ulturing the ˜—™teri— F ell the st—phylo™o™™—l enteroE

high leth—lityF „husD the development of prote™tive ™ounterme—E toxins —re potent emeti™ —gents —nd most of the ˜—™teri—l

sures h—s ˜e™ome —n urgent missionF st is —lso the ˜est me—ns to super—ntigens indu™e toxi™ sho™k syndromeD though with individu—l

deter the use of toxin we—ponsD ˜e it in the milit—ry —ren— or ˜y ™h—r—™teristi™s ‘II“F „his ™omplexity dem—nds the development of

terroristsF ˜ro—dEspe™trum ™ounterme—suresF

„he m—in mi™ro˜i—l thre—ts ± —nthr—xD ™holer—D pl—gueD tul—remi— €revious efforts to develop —ntidotes —g—inst toxi™ sho™k

—nd  fever ± ™—n ˜e ™om˜—ted to — v—rying degree with —nti˜ioti™sY ™on™entr—ted on ˜lo™king downstre—m phenomen— in the toxi™ity

—nthr—x spores —re sensitive to surf—™t—nt n—noemulsions ‘I“F ™—s™—deD m—inly ˜y inhi˜iting the —™tion of tumor ne™rosis f—™tor

‡here—s —nti˜odies —g—inst ˜otulinum toxins —re —v—il—˜le —nd with mono™lon—l —nti˜odies or solu˜le re™eptorsF roweverD the

v—™™ines —re under developmentD there is ™urrently no effe™tive extremely high levels of ™ytokines produ™ed in response to

defenseD whether —s —ntidote or v—™™ineD —g—inst the ˜—™teri—l super—ntigens render this —ppro—™h diffi™ult —nd ineffe™tiveF

exotoxins of the super—ntigen f—milyD whi™h r—te with ˜otulinum ‡e h—ve explored the possi˜ility of ˜lo™king super—ntigen —™tion

toxin —mong the most leth—l of toxins ‘P“F fy ™omp—risonD other —t the top of the toxi™ity ™—s™—deD ˜efore —™tiv—tion of „ ™ells o™™ursF

™—ndid—te toxinsD su™h —s ri™in or the „EP my™otoxinsD require f—r ‡e des™ri˜e —nt—gonist peptides th—t inhi˜it the indu™tion of

gre—ter —mounts to —™hieve — simil—r degree of leth—lityD m—king hum—n „hI ™ytokine gene expression ˜y — v—riety of super—ntigensF

them less suit—˜le —s we—ponsF „hough de—dlyD tons of ri™in would

˜e needed for —n effe™tive openE—ir —tt—™k ‘P“F

„g‚ a „ ™ell re™eptor

ƒuper—ntigens —re st—˜le proteins th—t ˜ind dire™tly to most

†˜ a v—ri—˜le portion of the ˜ ™h—in of the „ ™ell re™eptor

m—jor histo™omp—ti˜ility ™l—ss ss mole™ules ‘Q“ —nd stimul—te

ƒif a st—phylo™o™™—l enterotoxin f

virtu—lly —ll „ ™ells ˜e—ring p—rti™ul—r dom—ins in the v—ri—˜le ƒie a st—phylo™o™™—l enterotoxin e

portion of the ˜ ™h—in of the „ ™ell re™eptorD without need for „ƒƒ„EI a toxi™ sho™k syndrome toxinEI

SPH ‚F u—empfer et —lF swe F †ol R F tuly PHHP t

fiologi™ ‡—rf—re wedi™ine

„hese —nt—gonist peptides —re ™—p—˜le of prote™ting mi™e from the ˜e expe™ted to exhi˜it th—t propertyD yet we ™onje™tured th—t should

leth—l effe™ts of super—ntigen toxins —s widely different —s ƒif —nd it ™ompete with ƒif for —n essenti—l siteD it might prevent

„ƒƒ„EI —nd ™—n res™ue —nim—ls —lre—dy deep into toxi™ sho™kF ™ooper—tive inter—™tionsF ‡e thus —ss—yed e—™h peptide for its

—˜ility to inhi˜it ƒifEmedi—ted indu™tion of hum—n svEPD spxg —nd

wethods tumor ne™rosis f—™torE˜ m‚xe expression ‘PH“F xo peptide derived

„oxin —nt—gonist peptides from ƒif dom—ins th—t inter—™t with the „g‚ —ndGor wrg ™l—ss ss

€eptides were synthesized using fluoronylEmethoxy™—r˜onyl ™hemE mole™ule w—s inhi˜itoryD ˜ut strong —nt—gonist —™tivity w—s

istryF „hey were ™le—ved —nd the side ™h—in deprote™ted with IP whose sequen™eD „yrEesnEvysE exhi˜ited ˜y dode™—peptide p

triflouro—™eti™ —™idF „riflouro—™eti™ —™idEpeptide s—lts were solu˜le vysEvysEel—E„hrE†—lEqlnEqluEveuEespD is — v—ri—nt of ƒif dom—in

in ™ulture mediumF ISHEITIF p hEel— w—s linked to xE —nd gEtermini using the IP inhi˜ited not only the —™tion of ƒif ˜ut —lso th—t of

s—me pro™edureF righ pressure liquid ™hrom—togr—phy showed th—t the st—phylo™o™™—l —nd strepto™o™™—l super—ntigens ƒieD „ƒƒ„EID

peptides were b WS7 pureF —nd ƒ€ie ‘PH“F p IRD — deriv—tive of pIPD ™—rries two extr— —mino

—™ids from ƒif @†—lEqlnA pre™eding the pIP sequen™e ‘PI“F IRVEIRW

p €rote™tion of mi™e —g—inst toxi™ sho™k IPe —nd pIRe ™—rry hEel— residues —˜utted to the xE —nd gE

termini to render them more prote—seEresist—ntF qroups of IH fem—le fevfG™ mi™e @r—rl—nD terus—lemD ssr—elAD —ged

IH±IP weeksD were ™h—llenged ˜y intr—peritone—l inje™tion of IH mg

of ƒif @ƒigm—D ƒeA mixed with PH mg of hEg—l—™tos—mineD or of S €rote™tion —nd res™ue of mi™e from leth—l sho™k ‘pigure I“

mg „ƒƒ„EI @ƒigm—A mixed with RH mg of hEg—l—™tos—mineF nless „he hEg—l—™tos—mineEsensitized mouseD —n —™™epted —nim—l model

otherwise shownD —n —mount of PS mg of —nt—gonist peptide w—s for studying leth—lity of the super—ntigensD w—s used to investig—te

inje™ted intr—peritone—lly QH minutes ˜efore the toxinF ƒurviv—l w—s the prote™tive —™tivity of p IPe ‘PH“ —nd pIRe ‘PI“F wi™e exposed to

monitoredF ƒ€ƒƒ for ‡indows WFH softw—re w—s used for —n—lysis of —nt—gonist peptide —lone rem—ined vi—˜le —nd showed no dete™t—˜le

v—ri—n™e ˜etween groupsF ell experiments involving the use of mi™e side effe™tsF yf the mi™e ™h—llenged with ƒifD only QH7 were still

were in —™™ord—n™e with proto™ols —pproved ˜y the enim—l g—re —nd —live PR hours —fter toxin exposure —nd PH7 —t l—ter timesF roweverD

se gommittee of the re˜rew niversityEr—d—ss—h wedi™—l ƒ™hoolF —ll of the ƒifE™h—llenged mi™e were prote™ted ˜y p IRe given QH min

˜efore toxin @ € a HFHHHQA ‘pigure Ie“F ƒurviving —nim—ls showed no

signs of distress —nd rem—ined indistinguish—˜le from norm—l ƒtru™tures of super—ntigens

wole™ul—r modeling using ‚—swol PFT softw—re w—s ˜—sed on

—tomi™ ™oordin—tes derived ˜y ˆEr—y diffr—™tion for ƒif @€rotein

h—t— f—nk ™ode IƒifA ‘IR±IV“ —nd „ƒƒ„EI @I„ƒƒA ‘IW“F hom—ins

with p—rti—l homology to —nt—gonist peptide p IP —re ƒif ISHEITI

@„hrEesnEvysEvysEvysE†—lE„hrEel—EqlnEqluEveuEespA —nd „ƒƒ„EI IIWE

@€heEespEvysEvysEqlnEveuEel—EsleEƒerE„hrEveuEespAF IQH

‚esults

ƒuper—ntigen toxin —nt—gonist

sn —n —ttempt to o˜t—in —nt—gonists of ƒifD short peptides were

synthesized th—t ™onsist of —mino —™id sequen™es from ƒif

dom—ins known to ˜e essenti—l for ˜inding to the „g‚D to the

wrg ™l—ss ss mole™uleD or ˜oth ‘PH“F sn —dditionD peptides were

synthesized with p—rti—l homology to ƒif residues ISHEITID —

dom—in th—t is ™onserved —mong super—ntigens not yet known to ˜e

involved in ˜inding to either „g‚ or wrg ™l—ss ss mole™ule ‘IR±IV“F

‡hen present in up to PHHEfold higher mol—r —mounts th—n ƒifD

none of these peptides h—d signifi™—nt ƒif —gonist —™tivityD shown

˜y the l—™k of —˜ility to indu™e expression of m‚xe en™oding the

„hI ™ytokinesD svEP —nd spxgD in hum—n peripher—l ˜lood monoE

nu™le—r ™ells ‘PH“F

„he powerful —˜ility of super—ntigens to —™tiv—te „ ™ells involves

their tight ˜inding to the „g‚ —nd wrg ™l—ss ss mole™uleD st—˜ilized

˜y inter—™tions —t multiple sites ‘IR±IV“F e short peptide would not

pigure IF ent—gonist peptide prote™ts —nd res™ues mi™e from ƒifEindu™ed „hI a „ helper I

leth—l sho™k —nd prote™ts mi™e from „ƒƒ„EIEindu™ed leth—l sho™kF qroups of IH wrg a m—jor histo™omp—ti˜ility ™omplex

mi™e were ™h—llenged with ƒif ‘e“ or „ƒƒ„EI ‘f“D —lone or in the presen™e of sv a interleukin

—nt—gonist peptideF spx a interferon

swe F †ol R F tuly PHHP hefense eg—inst fiologi™ ‡—rf—re with „oxins SPI t

fiologi™ ‡—rf—re wedi™ine

™ontrols in ˜eh—viorY they survived for —s long —s they were

monitoredD P weeksF €—rti—l prote™tion w—s o˜t—ined when —dminE

istr—tion of the —nt—gonist peptide w—s del—yed to Q hr @ € a HFHSAD S

hr @ € a HFHVA or even U hr —fter leth—l ™h—llengeF e progressively

de™re—sing prote™tive effe™t of p IRe w—s seen ˜etween PH —nd RH hrD

yielding UH7D TH7 —nd SH7 surviv—lD respe™tivelyF „husD p IRe is not

only fully prote™tive when given ˜efore ƒif ™h—llenge ˜ut is —lso

—˜le to res™ue mi™e —fter they were exposed to toxinF €rote™tion or

res™ue w—s o˜served when the —nt—gonist peptide w—s in only — PHE

fold mol—r ex™ess over ƒifD showing th—t the peptide is — potent

super—ntigen —nt—gonist in vivoF

iven though „ƒƒ„EI exhi˜its — mere T7 over—ll sequen™e

homology with ƒifD p IPeD whi™h prote™ts mi™e from ƒif ‘PH“D w—s

—lso prote™tive —g—inst this toxinF ‚el—tive to ƒifD „ƒƒ„EI killed

mi™e f—r more slowlyD with h—lfEm—xim—l mort—lity o™™urring ˜y RH hr

‘pigure If“F sn the ™ontrol groupD —ll mi™e died upon ™h—llenge with

„ƒƒ„EIF p IPe did not prote™t when —dministered just ˜efore „ƒƒ„EI

˜ut —fforded signifi™—nt prote™tion @up to UH7A when —lso inje™ted

—fter toxin ™h—llengeF „he prote™tive effe™t ˜e™—me progressively

more pronoun™ed with repe—ted —dministr—tion —t Q —nd IV hrF

ƒurviv—l of mi™e from „ƒƒ„EIEmedi—ted toxi™ sho™k thus depends on

sust—ined presen™e of the peptideD suggesting th—t —lthough its

h—lfElife is limitedD it is long enough to prote™t mi™e —g—inst de—thF

p IPe is — ˜ro—dEspe™trum super—ntigen —nt—gonist in vivoF

wi™e prote™ted from leth—l toxin ™h—llenge ˜y —nt—gonist peptide pigure PF ƒtru™tures of super—ntigen toxins ƒif —nd „ƒƒ„EIF „he ƒif dom—in

h—ving homology to —nt—gonist peptide p IP —nd the ™orresponding dom—in in r—pidly ˜e™—me resist—nt to su˜sequent toxin ™h—llengesF ‡ithin P

„ƒƒ„EI —re ™olored redF finding sites of „g‚ †˜ ™h—in —nd wrg ™l—ss ss weeksD prote™tive —nti˜odies —g—inst super—ntigens were found in

mole™ule —re indi™—tedF the serum of mi™e th—t were shielded from leth—l sho™k ˜y the

—nt—gonist peptideF „his immunity is ™rossEprote™tive —g—inst

™onserv—tionF sn p—rti™ul—rD the ˜Estr—ndGhingeG—Ehelix dom—in different super—ntigen toxins ‘PHDPI“F epp—rentlyD ˜y ˜lo™king the

™orresponding to residues ISHEITI in ƒif is found in „ƒƒ„EI —˜ility of the toxin to indu™e — ™ellul—r immune response le—ding to

residues IIWEIQHF „his dom—in in „ƒƒ„EI sh—res only QQ7 sequen™e toxi™ sho™kD the peptide —nt—gonist —llows the super—ntigen to

simil—rity with the ™orresponding ƒif dom—inD yet the dom—in indu™e — vigorous humor—l immune response dire™ted —g—inst itselfF

folding is ™onservedF en —nt—gonist peptide th—t sh—res — higher ‰etD —nti˜odies —g—inst —nt—gonist peptide ™ould not ˜e dete™tedF

homology with ƒif w—s nevertheless ™—p—˜le of prote™ting —g—inst sndeedD the sm—ll size —nd rel—tively r—pid ™le—r—n™e of — short

leth—l sho™k indu™ed ˜y „ƒƒ„EIF peptide @IP±IR —mino —™idsA ™onstitute ther—peuti™ —dv—nt—gesF

his™ussion „he —nt—gonists t—rget — stru™tur—lly ™onserved dom—in ‘pigure P“

helivery of live ˜—™teri— is diffi™ultD whi™h limits their we—pons €eptides deriving from the ƒif dom—in exhi˜it super—ntigen ISHEITI

potenti—lF sn ™ontr—stD ™ert—in mi™ro˜i—l toxins —re ex™eption—lly —nt—gonistD where—s peptides from regions known to ˜e essenti—l

st—˜leD f—™ilit—ting their dissemin—tionF „he super—ntigen toxins for the inter—™tion of ƒif with the „g‚ —ndGor ™l—ss ss wrg

rem—in fully —™tive even —fter prolonged ˜oilingF „his he—t mole™ule l—™k su™h —™tivity ‘PH“F sndeedD the IPE—mino —™id ISHEITI

resist—n™e is one re—son for their potent —˜ility to indu™e food dom—in in ƒif is well removed from regions th—t p—rti™ip—te in

poisoningY indeedD most ™—ses of food poisoning with high —nd ˜inding of „g‚ —ndGor ™l—ss ss wrgF woreoverD this ƒif dom—in lies

prolonged mor˜idity —re ™—used ˜y ƒifF outside the region suffi™ient for mitogeni™ —™tivityD the xEtermin—l

nlike ™hemi™—l we—ponsD ˜iologi™ toxins —re not vol—tile —nd IQV —mino —™ids ‘PPDPQ“F „husD the —˜ility of p IRe —nd pIPe ‘PH“ to

tend to ˜e more toxi™ per weight th—n m—ny ™hemi™—l —gentsF —™t —s ƒif —nt—gonists is surprisingF „he ƒif dom—in forms — ISHEITI

fe™—use they —re produ™ed ˜y ™ulturing mi™roorg—nisms ™ommonly ™entr—l turn st—rting within — ˜Estr—nd —nd ™onne™ting itD vi— —nother

found in n—tureD whi™h in the ™—se of st—phylo™o™™—l super—ntigen short ˜Estr—ndD to —n —EhelixF „his dom—in is ™onserved —mong

toxins is re—dily —™™omplished ˜y personnel with ordin—ry skills —nd pyrogeni™ toxinsD with IHGIP —mino —™id identities for ƒieD ƒigID

equipmentD they —re —lso more e—sily —v—il—˜leD enh—n™ing their ƒigP —nd ƒ€ieD WGIP for ƒiiD —nd RGIP for „ƒƒ„EID the most

thre—t potenti—lF por this re—sonD toxins m—y repl—™e ™hemi™—l remotely rel—ted mem˜er of the st—phylo™o™™—l super—ntigen f—mily

we—pons in the futureF ‘PR“F xotwithst—nding the gre—t diversity in over—ll sequen™es of

„he pro˜—˜ility th—t — p—rti™ul—r toxin m—y ˜e sele™ted for ƒif —nd „ƒƒ„EID refle™ted —lso ˜y distin™t ˜inding sites for the „g‚

˜iologi™ w—rf—re in™re—ses when —n —ntidote or v—™™ine is l—™kingF †˜ ™h—in in these toxins ‘IIDIT“D their stru™tures show sp—ti—l

SPP ‚F u—empfer et —lF swe F †ol R F tuly PHHP t

fiologi™ ‡—rf—re wedi™ine

SF pr—ser th righE—ffinity ˜inding of st—phylo™o™™—l enterotoxins e —nd f to yn™e ™ounterme—sures —re developedD its —ttr—™tiveness —s —

x—ture IWVWYQQWXPPI±QF rveEh‚F potenti—l ˜iologi™ we—pon is diminishedF „he —nt—gonist peptides

TF ghoi ‰D rerm—n eD hiqiusto hD ‡—de „D w—rr—™k €D u—ppler tF ‚esidues of

des™ri˜ed here prote™t or res™ue mi™e from leth—l sho™k —t — mol—r

the v—ri—˜le region of the „E™ellEre™eptor ˜et—E™h—in th—t inter—™t with ƒF

ex™ess of —s low —s PHEfold over the toxinD implying th—t they ˜ind

—ureus toxin super—ntigensF x—ture IWWHYQRTXRUI±QF

tightly to — ™ellul—r t—rget th—t is ™riti™—l for super—ntigen —™tionF UF u—ppler tD uotzin fD rerron vD et —lF †˜Espe™ifi™ stimul—tion of hum—n „

„hese —nt—gonist peptides show homology to — super—ntigen ™ells ˜y st—phylo™o™™—l toxinsF ƒ™ien™e IWVWYPRRXVII±IRF

VF t—new—y geD ‰—gi tD u—tz wiD tones fD †regop ƒD fuxser ƒF „ ™ell dom—in ™onsisting of — ˜Estr—ndGhingeG˜Estr—ndG—Ehelix motif th—t is

responses to wls —nd to ˜—™teri—l proteins th—t mimi™ its ˜eh—viorF

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