Patent application title: LYME DISEASE VACCINE
Inventors:
Christopher G. Earnhart (Williamsburg, VA, US)
Richard T. Marconi (Midlothian, VA, US)
IPC8 Class: AA61K3902FI
USPC Class:
4241901
Class name: Antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) amino acid sequence disclosed in whole or in part; or conjugate, complex, or fusion protein or fusion polypeptide including the same disclosed amino acid sequence derived from bacterium (e.g., mycoplasma, anaplasma, etc.)
Publication date: 2011-10-27
Patent application number: 20110262475
Abstract:
Antigenic polypeptides comprising linear immunodominant epitopes of
Borrelia outer surface protein A (OspA) or Borrelia outer surface protein
C (OspC) are useful as vaccines against Lyme disease, and as diagnostics
for detecting Borrelia infections. The OspA and OspC antigenic
polypeptides typically comprise a plurality of peptides representing
epitope containing regions from multiple distinct phyletic groups. The
antigenic polypeptides may also include epitopes from both Borrelia OspA
and Borrelia OspC.Claims:
1. An isolated recombinant or synthetic peptide or polypeptide comprising
at least one linear epitope from Borrelia outer surface protein A (OspA),
said at least one linear epitope having an amino acid sequence selected
from the group consisting of:
TABLE-US-00004
(SEQ ID NO: 1)
STLTITVNSKKTKDLVFTKE;
(SEQ ID NO: 2)
STLTIIVDSKNKTKLVFTKQ
(SEQ ID NO: 3)
STLTISKNRTKTKQLVFTKE
(SEQ ID NO: 4)
STLTISANSKKTKDLVFLTN
(SEQ ID NO: 5)
STLTITVNNKKTKALVFTKQ
(SEQ ID NO: 6)
STLTISVNSKKTTQLVFTKQ
(SEQ ID NO: 7)
STLTISVNSKKTKNIVFTKE
(SEQ ID NO: 8)
STLTISVNSQKTKNLVFTKE
(SEQ ID NO: 9)
NTLTVSADSKKIKDFVFLTD
(SEQ ID NO: 10)
STLTISKNSQKTKQLVFTKE
(SEQ ID NO: 11)
STLTISAKNKKTKDLVFTKQ
(SEQ ID NO: 12)
STLKISKNSKKTKQLVFTKE
(SEQ ID NO: 13)
STLTISAKSKKTKDLVFTKQ
(SEQ ID NO: 14)
STLTISANNKKTKDLVFTKQ
(SEQ ID NO: 15)
KTLTVSADSKKIKDFVFLTD
(SEQ ID NO: 16)
STLTISAKNKKTTDLVFTKQ
(SEQ ID NO: 17)
STLTISKNSRKTKQLVFTKE;
(SEQ ID NO: 18)
STLTISVNSRKTKNLVFTKE;
(SEQ ID NO: 19)
CKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELK
GTS;
(SEQ ID NO: 20)
CKQNVSSLDEKNSVSVDLPGGMTVLVSKEKDKDGKYSLEATVDKLELK
GTS;
(SEQ ID NO: 21)
CKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLKATVDKIELK
GTS;
(SEQ ID NO: 22)
LGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKLITRAD
GTR;
(SEQ ID NO: 23)
LSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEKFNEKGETSEKTIVRAN
GTR;
(SEQ ID NO: 24)
LSKPTFELFKGDGETLVSRKVSSKDKTSTDEMFNEKGELSAKTMTREN
GTK;
(SEQ ID NO: 25)
KSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDSGKAKEVLKGYVL
EG;
(SEQ ID NO: 26)
KSSTEEKFNEKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFT
LEG;
and
(SEQ ID NO: 27)
KTSTDEMFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFT
LEG.
2. The isolated recombinant or synthetic peptide or polypeptide of claim 1, wherein said at least one linear epitope is selected from the group consisting of TABLE-US-00005 STLTITVNSKKTKDLVFTKE; (SEQ ID NO: 1) STLTIIVDSKNKTKLVFTKQ; (SEQ 1D NO: 2) STLTISKNRTKTKQLVFTKE; (SEQ ID NO: 3) STLTISANSKKTKDLVFLTN; (SEQ ID NO: 4) STLTITVNNKKTKALVFTKQ; (SEQ ID NO: 5) STLTISVNSKKTTQLVFTKQ; (SEQ ID NO: 6) STLTISVNSKKTKNIVFTKE; (SEQ ID NO: 7) STLTISVNSQKTKNLVFTKE; (SEQ ID NO: 8) NTLTVSADSKKIKDFVFLTD; (SEQ ID NO: 9) STLTISKNSQKTKQLVFTKE; (SEQ ID NO: 10) STLTISAKNKKTKDLVFTKQ; (SEQ ID NO: 11) STLKISKNSKKTKQLVFTKE; (SEQ ID NO: 12) STLTISAKSKKTKDLVFTKQ; (SEQ ID NO: 13) STLTISANNKKTKDLVFTKQ; (SEQ ID NO: 14) KTLTVSADSKKIKDFVFLTD; (SEQ ID NO: 15) STLTISAKNKKTTDLVFTKQ; (SEQ ID NO: 16) STLTISKNSRKTKQLVFTKE; (SEQ ID NO: 17) and STLTISVNSRKTKNLVFTKE. (SEQ ID NO: 18)
3. The isolated recombinant or synthetic peptide or polypeptide of claim 1, further comprising one or more amino acid sequences that are epitopes of Borrelia outer surface protein C (OspC).
4. The isolated recombinant or synthetic peptide or polypeptide of claim 3, wherein said one or more amino acid sequences that are epitopes of Borrelia OspC are from an OspC loop 5 region or an OspC alpha helix 5 region, or both.
5. The isolated recombinant or synthetic peptide or polypeptide of claim 3, wherein said one or more amino acid sequences that are epitopes of OspC are from OspC types selected from the group consisting of Smar, PLi, H13, PFiM, SL10, PMit, PKi, Pbes, HT22, Pko, PLj7, VS461, DK15, HT25, A,72a, F, E, M, D, U, I, L, H, Szid, PHez, PWa, B, K, N, and C.
6. The isolated recombinant or synthetic peptide or polypeptide of claim 3, wherein said one or more amino acid sequences that are epitopes of Borrelia OspC are selected from the group of polypeptides having sequences as set forth in SEQ ID NOS: 65-73.
7. A method for eliciting an immune response against Borrelia in an individual in need thereof, comprising the step of administering to said individual an isolated recombinant or synthetic peptide or polypeptide comprising at least one linear epitope from Borrelia outer surface protein A (OspA), said at least one linear epitope having an amino acid sequence selected from the group consisting of: TABLE-US-00006 (SEQ ID NO: 1) STLTITVNSKKTKDLVFTKE; (SEQ ID NO: 2) STLTIIVDSKNKTKLVFTKQ; (SEQ ID NO: 3) STLTISKNRTKTKQLVFTKE; (SEQ ID NO: 4) STLTISANSKKTKDLVFLTN; (SEQ ID NO: 5) STLTITVNNKKTKALVFTKQ; (SEQ ID NO: 6) STLTISVNSKKTTQLVFTKQ; (SEQ ID NO: 7) STLTISVNSKKTKNIVFTKE; (SEQ ID NO: 8) STLTISVNSQKTKNLVFTKE; (SEQ ID NO: 9) NTLTVSADSKKIKDFVFLTD; (SEQ ID NO: 10) STLTISKNSQKTKQLVFTKE; (SEQ ID NO: 11) STLTISAKSKKTKDLVFTKQ; (SEQ ID NO: 12) STLKISKNSKKTKQLVFTKE; (SEQ ID NO: 13) STLTISAKSKKTKDLVFTKQ; (SEQ ID NO: 14) STLTISANNKKTKDLVFTKQ; (SEQ ID NO: 15) KTLTVSADSKKIKDFVFLTD; (SEQ ID NO: 16) STLTISAKNKKTTDLVFTKQ; (SEQ ID NO: 17) STLTISKNSRKTKQLVFTKE; (SEQ ID NO: 18) STLTISVNSRKTKNLVFTKE; (SEQ ID NO: 19) CKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELK GTS; (SEQ ID NO: 20) CKQNVSSLDEKNSVSVDLPGGMTVLVSKEKDKDGKYSLEATVDKLELK GTS; (SEQ ID NO: 21) CKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLKATVDKIELK GTS; (SEQ ID NO: 22) LGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRAD GTR; (SEQ ID NO: 23) LSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEKENEKGETSEKTIVRAN GTR; (SEQ ID NO: 24) LSKPTFELFKGDGETLVSRKVSSKDKTSTDEMFNEKGELSAKTMTREN GTK; (SEQ ID NO: 25) KSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYV LEG; (SEQ ID NO: 26) KSSTEEKFNEKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFT LEG; and (SEQ ID NO: 27) KTSTDEMFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFT LEG.
8. The method of claim 7, wherein said at least one linear epitope has an amino acid sequence selected from the group consisting of TABLE-US-00007 STLTITVNSKKTKDLVFTKE; (SEQ ID NO: 1) STLTIIVDSKNKTKLVFTKQ; (SEQ ID NO: 2) STLTISKNRTKTKQLVFTKE; (SEQ ID NO: 3) STLTISANSKKTKDLVFLTN; (SEQ ID NO: 4) STLTITVNNKKTKALVFTKQ; (SEQ ID NO: 5) STLTISVNSKKTTQLVFIKQ; (SEQ ID NO: 6) STLTISVNSKKTKNIVFTKE; (SEQ ID NO: 7) STLTISVNSQKTKNLVFTKE; (SEQ ID NO: 8) NTLTVSADSKKIKDFVFLTD; (SEQ ID NO: 9) STLTISKNSQKTKQLVFTKE; (SEQ ID NO: 10) STLTISAKSKKTKDLVFTKQ; (SEQ ID NO: 11) STLKISKNSKKTKQLVFTKE; (SEQ ID NO: 12) STLTISAKSKKTKDLVFTKQ; (SEQ ID NO: 13) STLTISANNKKTKDLVFIKQ; (SEQ ID NO: 14) KTLTVSADSKKIKDFVFLTD; (SEQ ID NO: 15) STLTISAKNKKTTDLVFTKQ; (SEQ ID NO: 16) STLTISKNSRKTKQLVFTKE; (SEQ ID NO: 17) and STLTISVNSRKTKNLVFTKE. (SEQ ID NO: 18)
9. The method of claim 7, wherein said isolated recombinant or synthetic peptide or polypeptide further comprises one or more epitopes of Borrelia outer surface protein C (OspC).
10. The method of claim 9, wherein said one or more amino acid sequences that are epitopes of Borrelia OspC are selected from the group of polypeptides having sequences as set forth in SEQ ID NOS: 65-73.
11. A method for ascertaining whether an individual has been exposed to or infected with Borrelia, or both, comprising the steps of obtaining a biological sample from said individual, exposing said biological sample to a recombinant or synthetic peptide or polypeptide comprising at least one linear epitope from Borrelia outer surface protein A (OspA), said at least one linear epitope having an amino acid sequence selected from the group consisting of: TABLE-US-00008 (SEQ ID NO: 1) STLTITVNSKKTKDLVFTKE; (SEQ ID NO: 2) STLTIIVDSKNKTKLVFTKQ; (SEQ ID NO: 3) STLTISKNRTKTKQLVFTKE; (SEQ ID NO: 4) STLTISANSKKTKDLVFLTN; (SEQ ID NO: 5) STLTITVNNKKTKALVFTKQ; (SEQ ID NO: 6) STLTISVNSKKTTQLVFTKQ; (SEQ ID NO: 7) STLTISVNSKKTKNIVFTKE; (SEQ ID NO: 8) STLTISVNSQKTKNLVFTKE; (SEQ ID NO: 9) NTLTVSADSKKIKDFVFLTD; (SEQ ID NO: 10) STLTISKNSQKTKQLVFTKE; (SEQ ID NO: 11) STLTISAKNKKTKDLVFTKQ; (SEQ ID NO: 12) STLKISKNSKKTKQLVFTKE; (SEQ ID NO: 13) STLTISAKSKKTKDLVFTKQ; (SEQ ID NO: 14) STLTISANNKKTKDLVFTKQ; (SEQ ID NO: 15) KTLTVSADSKKIKDFVFLTD; (SEQ ID NO: 16) STLTISAKNKKTTDLVFTKQ; (SEQ ID NO: 17) STLTISKNSRKTKQLVFTKE; (SEQ ID NO: 18) STLTISVNSRKTKNLVFTKE; (SEQ 1D NO: 19) CKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELK GTS; (SEQ ID NO: 20) CKQNVSSLDEKNSVSVDLPGGMTVLVSKEKDKDGKYSLEATVDKLELK GTS; (SEQ ID NO: 21) CKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLKATVDKIELK GTS; (SEQ ID NO: 22) LGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRAD GTR; (SEQ ID NO: 23) LSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEKFNEKGETSEKTIVRAN GTR; (SEQ ID NO: 24) LSKPTFELFKGDGETLVSRKVSSKDKTSTDEMFNEKGELSAKTMTREN GTK; (SEQ ID NO: 25) KSSTEEKFNEKGEVSEKIITRADGTRLEYTG1KSDGSGKAKEVLKGYV LEG; (SEQ ID NO: 26) KSSTEEKFNEKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFT LEG; and (SEQ ID NO: 27) KTSTDEMFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFT LEG.
and determining whether antibodies in said biological sample bind to said recombinant or synthetic peptide or polypeptide, wherein detection of antibody binding is indicative of prior exposure to or infection with Borrelia.
12. The method of claim 11, wherein said recombinant or synthetic peptide or polypeptide further comprises one or more epitopes of Borrelia outer surface protein C (OspC).
13. The method of claim 12, wherein said one or more amino acid sequences that are epitopes of Borrelia OspC are selected from the group of polypeptides having sequences as set forth in SEQ ID NOS: 65-73.
14. An antibody to a recombinant or synthetic peptide or polypeptide comprising at least one linear epitope from Borrelia outer surface protein A (OspA), said at least one linear epitope having an amino acid sequence selected from the group consisting of: TABLE-US-00009 (SEQ ID NO: 1) STLTITVNSKKTKDLVFTKE; (SEQ ID NO: 2) STLTIIVDSKNKTKLVFTKQ; (SEQ ID NO: 3) STLTISKNRTKTKQLVFTKE; (SEQ ID NO: 4) STLTISANSKKTKDLVFLTN; (SEQ ID NO: 5) STLTITVNNKKTKALVFTKQ; (SEQ ID NO: 6) STLTISVNSKKITQLVFTKQ; (SEQ ID NO: 7) STLTISVNSKKTKNIVFTKE; (SEQ ID NO: 8) STLTISVNSQKTKNLVFTKE; (SEQ ID NO: 9) NTLTVSADSKKIKDFVFLTD; (SEQ ID NO: 10) STLTISKNSQKTKQLVFTKE; (SEQ ID NO: 11) STLTISAKNKKTKDLVFTKQ; (SEQ ID NO: 12) STLKISKNSKKTKQLVFTKE; (SEQ ID NO: 13) STLTISAKSKKTKDLVFTKQ; (SEQ ID NO: 14) STLTISAKNKKTKDLVFTKQ; (SEQ ID NO: 15) KTLTVSADSKKIKDFVFLTD; (SEQ ID NO: 16) STLTISAKNKKTTDLVFTKQ; (SEQ ID NO: 17) STLTISKNSRKTKQLVFTKE; (SEQ ID NO: 18) STLTISVNSRKTKNLVFTKE; (SEQ ID NO: 19) CKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELK GTS; (SEQ ID NO: 20) CKQNVSSLDEKNSVSVDLPGGMTVLVSKEKDKDGKYSLEATVDKLELK GTS; (SEQ ID NO: 21) CKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLKATVDKIELK GTS; (SEQ ID NO: 22) LGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRAD GTR; (SEQ ID NO: 23) LSQTKFETFREDGKTLVSKKVTLKDKSSTEEKFNEKGETSEKTIVRAN GTR; (SEQ ID NO: 24) LSKPTFELFKGDGETLVSRKVSSKDKTSTDEMFNEKGELSAKTMTREN GTK; (SEQ ID NO: 25) KSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYV LEG; (SEQ ID NO: 26) KSSTEEKFNEKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFT LEG; and (SEQ ID NO: 27) KTSTDEMFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFT LEG.
15. The antibody of claim 19, wherein said antibody is bactericidal for Borrelia spirochetes.
16. An isolated recombinant or synthetic peptide or polypeptide comprising one or more epitopes from Borrelia outer surface protein A (OspA), said one or more epitopes being from an antigenic region selected from the group consisting of: antigenic region 221-240, antigenic region 17-67, antigenic region 94-144, and antigenic region 119-169.
17. The isolated recombinant or synthetic peptide or polypeptide of claim 16, further comprising one or more epitopes of Borrelia outer surface protein C (OspC).
18. An isolated recombinant or synthetic peptide or polypeptide comprising at least one linear epitope from Borrelia outer surface protein C (OspC), said at least one linear epitope having an amino acid sequence selected from the group consisting of: TABLE-US-00010 i) (SEQ ID NO: 65) SETFTNKLKEKHTDLGKEGVTDADAKEAILKTNGTKTKGAEELGKLFE SVEVLSKAAKEMLANSVKELTSSEEFSTKLKDNHAQLGIQGVTDENAK KAILKANAAGKDKGVEELEKLSGSLESLSSEDFTKKLEGEHAQLGIEN VTDENAKKAILITDAAKDKGAAELEKLFKAVENLAAKLKGEHTDLGKE GVTDDNAKKAILKTNNDKTKGADELEKLFESVKNLSKAAKEMLTNSSE KFAGKLKNEHASLGKKDATDDDAKKAILKTHGNTDKGAKELKDLSDSV ESLVSDDFTKKLQSSHAQLGVAGGATTDEEAKKAILRTNAIKDKGADE LEKLFKSVESLAKAAQDALANSVNELTSKKLKEKHTDLGKKDATDVHA KEAILKTNGTKDKGAAELEKLFESVENLAKAAKEMLSNSNKAFTDKLK SSHAELGIANGAATDANAKAAILKTNGTKDKGAQELEKLFESVKNLSK AAQETLNNSSESFTKKLSDNQAELGIENATDDNAKKAILKTHNAKDKG AEELVKLSESVAGLLKAAQAILANSVKELTSPVVAESPKKP; ii) (SEQ ID NO: 66) SEKFTTKLKDSHAELGIQSVQDDNAKKAILKTHGTKDKGAKELEELFK SLESLSKAAQAALTNSVKELTNSDKFTKKLTDSHAQLGAVGGAINDDR AKEAILKTHGTNDKGAKELKELSESVESLAKAAQAALANSSEAFTKKL KDSNAQLGMQNGAATDAHAKAAILKTDATKDKGATELGELFKSVESLS KAAQEASVAFTSKLKSSNAQLGVANGNATDDDAKKAILKTNTPNDKGA KELKELFESVESLAKAAQAALVNSVQELTNSEAFTNRLKGSHAQLGVA AATDDHAKEAILKSNPTKDKGAKELKDLSESVESLAKAAQEALANSVK ELTNSEAFTKKLKDNNAQLGIQNVQDVEAKKAILKTNGDISKSEAFTN KLKEKHAELGVNGGDTTDDNAKAAIFKTHPTKDKGVEDLEKLSESVKS LLKAAQAALSNSAAFTKKLQDGHVDLGKTDVTDDNAKEAILKTNPTKT KGATELEELFKSVEGLVKAAKEA; iii) (SEQ ID NO: 67) SEEFTNKLKSGHADLGKQDATDDHAKAAILKTHATTDKGAKEFKDLFE SVEGLLKAAQVALTNSVKELTSKLKGGHAELGLAAATDENAKKAILKT NGTKDKGAEELEKLFKSVESLAKAAKESLTNSVKELTNTKLRDSHAEL GIQNVQDDNAKRAILKTHGNKDKGAKELKELSESLEKLSKAAQAALAN SVQELTSSEAFTNKLKEKTQELAVAAGAATDIDAKKAILKTNRDKDLG ADERGKLFKSVESLSKAAQEASANSVKELTSSEAFTDKLKNEHASLGK KDATDDDAKKAILKTNVDKTKGADELIKLSGSLESLSKAAQAILANSE AFTKKLQDSNADLGKHNATDADSKEAILKTNGTKTKGAKELEELFKSV ESLSKAAKEALSNSVKELTSSQDFINKLKGGHAELGLVAATDANAKAA ILKTNGDKTKGADEFEKLFKSVEGLLKAAQEALTNSVKELTSSEAFTK KLQDSNADLGKHDATDADAKKAILKTDATKDK; iv) (SEQ ID NO: 68) SETFTNKLKEKHTDLGKEGVTKGAEELGKLFESVEVLSKAAKEMLANS VKELTSSEEFSTKLKDNHAQLGIQGVTKGVEELEKLSGSLESLSSEDF TKKLEGEHAQLGIENVTAAELEKLFKAVENLAKAAKEMAKLKGEHTDL GKEGVTKGADELEKLFESVKNLSKAAKEMLTNSKESEKFAGKLKNEHA SLGKKDATKGAKELKDLSDSVESLVKASDDFTKKLQSSHAQLGVAGGA TTADELEKLFKSVESLAKAAQDALANSVNELTSKKLKEKHTDLGKKDA TAAELEKLFESVENLAKAAKEMLSNSNKAFTDKLKSSHAELGIANGAA TKGAQELEKLFESVKNLSKAAQETLNNSVKESESETKKLSDNQAELGI ENATKGAEELVKLSESVAGLLKAAQAILANSVKELTSPVVAESPKKP; v) (SEQ ID NO: 69) SEKFTTKLKDSHAELGIQSVQDKGAKELEELFKSLESLSKAAQAALTN SVKELTNSDKFTKKLTDSHAQLGAVGGAINDKGAKELKELSESVESLA KAAQAALANSSEAFTKKLKDSNAQLGMQNGAATDKGATELGELFKSVE SLSKAAQEASVAFTSKLKSSNAQLGVANGNATDKGAKELKELFESVES LAKAAQAALVNSVQELTNSEAFTNRLKGSHAQLGVAAATDKGAKELKD LSESVESLAKAAQEALANSVKELTNSEAFTKKLKDNNAQLGIQNVQSE AFTNKLKEKHAELGVNGGDTTDKGVEDLEKLSESVKSLLKAAQAALSN SAAFTKKLQDGHVDLGKTDVTTKGATELEELFKSVEGLVKAAKEA; vi) (SEQ ID NO: 70) SEEFTNKLKSGHADLGKQDATKGAKEEKDLFESVEGLLKAAQVALTNS VKELTSKLKGGHAELGLAAATKGAEELEKLFKSVESLAKAAKESLTNS VKELTNTKLRDSHAELGIQNVQKGAKELKELSESLEKLSKAAQAALAN SVQELTSSEAFTNKLKEKTQELAVAAGAATLGADERGKLFKSVESLSK AAQEASANSVKELTSSEAFTDKLKNEHASLGKKDATKGADELIKLSGS LESLSKAAQAILANSEAFTKKLQDSNADLGKHNATKGAKELEELFKSV ESLSKAAKEALSNSVKELTSSQDFINKLKGGHAELGLVAATKGADEFE KLFKSVEGLLKAAQEALTNSVKELTSSEAFTKKLQDSNADLGKHDAT; vii) (SEQ ID NO: 71) SETFTNKLKEKHTDLGKEGVTKGAEELGKLFESVEVLSKAAKEMLANS VKELTSKGVEELEKLSGSLESLSNKAFTDKLKSSHAELGIANGAATKK LKEKHTDLGKKDATKGADELEKLFESVKNLSKAAKEMLTNSKEIAAEL EKLFKAVENLAKAAKEMAKLKGEHTDLGKEGVTSEEFSTKLKDNHAQL GIQGVTKGAKELKDLSDSVESLVKAAAELEKLFESVENLAKAAKEMLS NSSEKFAGKLKNEHASLGKKDATSEDFTKKLEGEHAQLGIENVTKGAQ ELEKLFESVKNLSKAAQETLNNSVKEADELEKLFKSVESLAKAAQDAL ANSVNELTSSESFTKKLSDNQAELGIENATSDDFTKKLQSSHAQLGVA GGATTKGAEELVKLSESVAGLLKAAQAILANSVKELTSPVVAESPKKP; viii) (SEQ ID NO: 72) SEAFTKKLKDSNAQLGMQNGAATDKGAKELEELFKSLESLSKAAQAAL TNSVKELTNKDKGAKELKELFESVESLAKAAQAALVNSVQELTNSEKF TTKLKDSHAELGIQSVQSDKFTKKLTDSHAQLGAVGGAINDKGAKELK ELSESVESLAKAAQAALANSDKGAKELKDLSESVESLAKAAQEALANS VKELTNSVAFTSKLKSSNAQLGVANGNATSEAFTKKLKDNNAQLGIQN VQTKGATELEELFKSVEGLVKAAKEADKGVEDLEKLSESVKSLLKAAQ AALSNSAAFTKKLQDGHVDLGKTDVTSEAFTNRLKGSHAQLGVAAATD KGATELGELFKSVESLSKAAQEASEAFTNKLKEKHAELGVNGGDTT; and ix) (SEQ ID NO: 73) SEEFTNKLKSGHADLGKQDATKGAKEFKDLFESVEGLLKAAQVALTNS VKELTSKEKGAEELEKLFKSVESLAKAAKESLTNSVKELTNSEAFTDK LKNEHASLGKKDATTKLRDSHAELGIQNVQLGADERGKLFKSVESLSK AAQEASANSVKELTSKEKGAKELEELFKSVESLSKAAKEALSNSVKEL TSSEAFTKKLQDSNADLGKHNATSEAFTKKLQDSNADLGKHDATKGAD EFEKLEKSVEGLLKAAQEALTNSVKELTSELKELSESLEKLSKAAQAA LANSVQELTSSEAFTNKLKEKTQELAVAAGAATKLKGGHAELGLAAAT KGADELIKLSGSLESLSKAAQAILANSQDFINKLKGGHAELGLVAAT.
19. The isolated recombinant or synthetic peptide or polypeptide of claim 18, further comprising one or more epitopes of Borrelia outer surface protein A (OspA).
Description:
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention generally relates to a vaccine and diagnostic for Lyme disease. In particular, the invention provides a Lyme disease vaccine and diagnostic that includes linear Borrelia outer surface protein A (OspA) epitopes and/or Borrelia outer surface protein C (OspC) epitopes, usually from multiple distinct phyletic groups.
[0003] 2. Background of the Invention
[0004] Lyme disease is the most common arthropod-borne disease in North America and Europe, where in some areas up to 3% of the population is infected annually. Lyme disease is caused by the spirochetes Borrelia burgdorferi, B. garinii and B. afzelii. Transmission to mammals occurs through the bite of infected Ixodes ticks. Infection results in a multi-systemic inflammatory disease with early stage symptoms that may include erythema migrans, low-grade fever, arthralgia, myalgia, and headache. Late stage clinical manifestations can be severe and may include in part, arthritis and neurological complications. In addition, Lyme disease has significant socio-economic costs, manifested by reductions in outdoor recreational and social activities due to concerns about tick exposure.
[0005] The antigen used in first generation Lyme disease vaccines (e.g. LYMErix) was Outer surface protein A (OspA). OspA is only expressed by spirochetes in ticks, thus anti-OspA bactericidal activity occurs in the vector. However, a major drawback to the use of full-length OspA was the potential (whether real or perceived) for adverse events secondary to vaccination, such as the development of arthritis caused by immunological cross-reactivity with human proteins (e.g. LFA-1). This was a major factor in the withdrawal from the market of the original OspA-based LYMErix vaccine.
[0006] U.S. Pat. 6,248,562 (Jun. 19, 2001) to Dunn and Luft describes chimeric Borrelia proteins that can be used as immunodiagnostic reagents and vaccine immunogens against Borrelia infection.
[0007] U.S. Pat. Nos. 6,872,550 and 6,486,130 (Mar. 29, 2005, and Nov. 26, 2002, respectively) both to Livey, describe constructs for use a vaccines against Lyme disease.
[0008] U.S. Pat. No. 7,008,625 (Mar. 7, 2006) to Dattwyler et al. discloses chimeric Borrelia proteins that can be used as immunodiagnostic reagents and vaccine immunogens against Borrelia infection
[0009] The publication "Recombinant Chimeric Borrelia Proteins for Diagnosis of Lyme Disease" (Maria J. C. Gomes-Solecki et al. 2000. J. Clin. Microbiol., 38: 2530-2535) also describes recombinant chimeric proteins.
[0010] Despite the above-referenced technologies, to date the prior art has failed to provide an efficacious vaccine for use in the prevention and/or treatment of Lyme disease.
SUMMARY OF THE INVENTION
[0011] In order to address prior art problems with Lyme disease vaccines, the OspA protein from Borrelia burgdorferi has been epitope mapped by assessing the reactivity of sera generated during murine infection with recombinant OspA subfragments. The epitope map demonstrated several linear epitope-containing regions of OspA. While conformational epitopes of OspA have been mapped and described, linear epitopes have not previously been reported for use in a vaccine. The use of one or more of these small, defined epitope-containing sequences in a polypeptide vaccine formulation allows the avoidance of specific regions of OspA that have been implicated in vaccine-mediated adverse events when full-length OspA is used. The inclusion of linear epitopes from a plurality of phyletic groups of Borrelia results in a vaccine that provides broad protection against infection over large geographical areas.
[0012] In addition, in other embodiments of the invention, one or more defined epitope-containing sequences from the Borrelia OspC protein have been used in vaccine formulations. Typically, epitopes or epitope-containing sequences from multiple phyletic groups are utilized.
[0013] Finally, the invention provides vaccine compositions which contain epitopes or epitope regions from both OspA and OspC.
[0014] It is an object of the invention 1. An isolated recombinant or synthetic peptide or polypeptide comprising at least one linear epitope from Borrelia outer surface protein A (OspA), said at least one linear epitope having an amino acid sequence selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26); and SEQ ID NO: 27.
[0015] In one embodiment, the at least one linear epitope is selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; and SEQ ID NO: 18.
[0016] The isolated recombinant or synthetic peptide or polypeptide may further comprise one or more amino acid sequences that are epitopes of Borrelia outer surface protein C (OspC), for example, one or more amino acid sequences that are epitopes of Borrelia OspC from an OspC loop 5 region or an OspC alpha helix 5 region, or both. The epitopes of OspC may be OspC types selected from the group consisting of Smar, PLi, H13, PFiM, SL10, PMit, PKi, Pbes, HT22, Pko, PLj7, VS461, DK15, HT25, A,72a, F, E, M, D, U, I, L, H, Szid, PHez, PWa, B, K, N, and C. In some embodiments, the one or more amino acid sequences that are epitopes of Borrelia OspC are selected from the group of polypeptides having sequences as set forth in SEQ ID NOS: 65-73.
[0017] The invention also provides a method for eliciting an immune response against Borrelia in an individual in need thereof. The method comprises the step of administering to the individual an isolated recombinant or synthetic peptide or polypeptide comprising at least one linear epitope from Borrelia outer surface protein A (OspA). The at least one linear epitope having an amino acid sequence selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26); and SEQ ID NO: 27. In some embodiments, the at least one linear epitope has an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; and SEQ ID NO: 18. According to some embodiments of the method, the isolated recombinant or synthetic peptide or polypeptide may further comprise one or more amino acid sequences that are epitopes of Borrelia outer surface protein C (OspC), for example, one or more amino acid sequences that are epitopes of Borrelia OspC from an OspC loop 5 region or an OspC alpha helix 5 region, or both. The epitopes of OspC may be OspC types selected from the group consisting of Smar, PLi, H13, PFiM, SL10, PMit, PKi, Pbes, HT22, Pko, PLj7, VS461, DK15, HT25, A, 72a, F, E, M, D, U, I, L, H, Szid, PHez, PWa, B, K, N, and C. In some embodiments, the one or more amino acid sequences that are epitopes of Borrelia OspC are selected from the group of polypeptides having sequences as set forth in SEQ ID NOS: 65-73.
[0018] The invention further provides a method for ascertaining whether an individual has been exposed to or infected with Borrelia, or both. The method comprises the steps of 1) obtaining a biological sample from said individual; 2) exposing said biological sample to a recombinant or synthetic peptide or polypeptide comprising at least one linear epitope from Borrelia outer surface protein A (OspA), said at least one linear epitope having an amino acid sequence selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ED NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26); and SEQ ID NO: 27; and 3) determining whether antibodies in the biological sample bind to the recombinant or synthetic peptide or polypeptide, wherein detection of antibody binding is indicative of prior exposure to or infection with Borrelia. In some embodiments of this method, the recombinant or synthetic peptide or polypeptide further comprises one or more epitopes of Borrelia outer surface protein C (OspC), such as those having sequences as set forth in SEQ ID NOS: 65-73.
[0019] The invention further provides antibodies to a recombinant or synthetic peptide or polypeptide comprising at least one linear epitope from Borrelia outer surface protein A (OspA) or outer surface protein C (OspC). The at least one linear epitope having an amino acid sequence selected from the group consisting of: SEQ ID NOS: 1-27 (for OspA) and SEQ ID NOS: 65-73 (for OspC). In some embodiments, the antibody is bactericidal for Borrelia spirochetes.
[0020] The invention further provides an isolated recombinant or synthetic peptide or polypeptide comprising one or more epitopes from Borrelia outer surface protein A (OspA), said one or more epitopes being from an antigenic region selected from the group consisting of: antigenic region 221-240, antigenic region 17-67, antigenic region 94-144, and antigenic region 119-169. The isolated recombinant or synthetic peptide or polypeptide may further comprising one or more epitopes of Borrelia outer surface protein C (OspC).
[0021] The invention also provides an isolated recombinant or synthetic peptide or polypeptide comprising at least one linear epitope from Borrelia outer surface protein C (OspC). The at least one linear epitope may have an amino acid sequence selected from the group consisting of: SEQ ID NO: 65; SEQ ID NO: 66; SEQ ID NO: 67; SEQ ID NO: 68; SEQ ID NO: 69; SEQ ID NO: 70; SEQ ID NO: 71; SEQ ID NO: 72; and SEQ ID NO: 73. The isolated recombinant or synthetic peptide or polypeptide may, in some embodiments, further comprise one or more epitopes of Borrelia outer surface protein A (OspA).
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1. Alignment of the epitope-containing region of OspC protein sequences from all OspC types defined in this study. All sequences within OspC types that differ by more than one amino acid are indicated by a representative sequence. Identity threshold for shading is 80%. Secondary structural alpha helices and loops (corresponding to the B31 structure) are shown below the alignment (Kumaran, D., S. Eswaramoorthy, B. J. Luft, S. Koide, J. J. Dunn, C. L. Lawson, and S. Swaminathan. 2001. Crystal structure of outer surface protein C (OspC) from the Lyme disease spirochete, Borrelia burgdorferi. EMBO J. 20:971-978).
[0023] FIG. 2A-C. General epitope locations for OspC constructs. A, Construct A (SEQ ID NO: 62); B, Construct B (SEQ ID NO: 63), C, Construct C (SEQ ID NO: 64).
[0024] FIG. 3. Schematic of subfragments used to map linear epitopes in OspA. The initial screening of ˜50 amino acid subfragments was followed by targeted screening of overlapping ˜20 amino acid fragments to more accurately define the epitope location. Fragments detected by one or more mouse sera are denoted by a (+).
[0025] FIG. 4. Western blot mapping of OspA demonstrates several linear epitopes. r-OspA subfragments were screened with sera from mice infected for 6 weeks with one of three B. burgdorferi strains. Secondary antibody detection was IgG-specific. While all mice generated an anti-OspA response, as demonstrated by the full length protein (17-273), only a subset recognized linear epitopes on the tested subfragments. Of those fragments recognized, the most frequently and strongly recognized epitope was localized between amino acids 221 and 240.
[0026] FIG. 5. Alignment of OspA sequences from the three B. burgdorferi strains used to infect mice for OspA epitope mapping. B31Mi, SEQ ID NO: 74; B331, SEQ ID NO: 75; LDP74, SEQ ID NO: 76.
[0027] FIG. 6. Structural representation of OspA highlighting a linear epitope-containing region. A ribbon diagram of OspA crystal structure (1FJ1) highlights amino acids 221-240 (gray) that contain a mapped linear epitope. The box surrounds the three loop regions that define the previously described protective Lk-2 conformational epitope. The protein N-terminus is at the bottom of the diagram.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
[0028] The present invention is based on the identification and characterization of linear peptide epitopes from Borrelia OspA protein. Identification of these epitopes has made possible the design and construction of chimeric (fusion) polypeptides that can be used as vaccines against Lyme disease, or as diagnostic tools. The polypeptides, which arc generally isolated and/or purified, comprise at least one copy of an epitope from one of four classes of Borrelia OspA cpitopes or antigenic regions. The four classes are organized based on the amino acid sequences numbering of OspA Borrelia burgdorferi strain B31M1, as follows: [0029] Class I: Antigenic region 221-240; [0030] Class II: Antigenic region 17-67; [0031] Class III: Antigenic region 94-144; and [0032] Class IV: Antigenic region 119-169.
[0033] The prototype Class I sequence is from Borrelia burgdorferi strain B31M1: Class I: STLTITVNSKKTKDLVFTKE (SEQ ID NO: 1), which corresponds to residues 221-240 of OspA protein from B. burgdorferi. Additional corresponding or analogous type specific (i.e. phyletically related) exemplary Class I sequences that may be used in the practice of the invention include the following:
TABLE-US-00001 TABLE 1 Sequence variants at the 221-240 epitope containing region. SEQ Accession ID Species number NO: Sequence B. burgdorferi NP045688.1 1 STLTITVNSKKTKDLVFTKE B. burgdorferi CAB64758.1 2 STLTIIVDSKNKTKLVFTKQ B. burgdorferi CAA56544.1 3 STLTISKNRTKTKQLVFTKE B. burgdorferi CAA01705.1 4 STLTISANSKKTKDLVFLTN B. burgdorferi CAR95557.1 5 STLTITVNNKKTKALVFTKQ B. burgdorferi and ABF29588.1 6 STLTISVNSKKTTQLVFTKQ B. afzelii CAA44092.1 B. burgdorferi and AAB23810.1 7 STLTISVNSKKTKNIVFTKE B. garinii AAT93773.1 B. burgdorferi and CAA82328.1 8 STLTISVNSQKTKNLVFTKE B. garinii CAA56546.1 B. garinii ACD02017.1 9 NTLTVSADSKKIKDFVFLTD B. garinii ABF29554.1 10 STLTISKNSQKTKQLVFTKE B. garinii AA091930.1 11 STLTISAKNKKTKDLVFTKQ B. garinii AAR96307.1 12 STLKISKNSKKTKQLVFTKE B. garinii AA091932.1 13 STLTISAKSKKTKDLVFTKQ B. garinii ACD02015.1 14 STLTISANNKKTKDLVFTKQ B. garinii AAR96306.1 15 KTLTVSADSKKIKDFVFLTD B. garinii AAP94131.1 16 STLTISAKNKKTTDLVFTKQ B. garinii ABF29567.1 17 STLTISKNSRKTKQLVFTKE B. garinii AAP94130.1 18 STLTISVNSRKTKNLVFTKE
[0034] The prototype Class II sequence from Borrelia burgdorferi strain B31M1 is: Class II: CKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLEL KGTS (SEQ ID NO: 19), which corresponds to residues 17-67 of OspA protein from B. burgdorferi.
[0035] The prototype Class III sequence from Borrelia burgdorferi strain B31M1 is: Class III: LGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTR (SEQ ID NO: 22), which corresponds to residues 94-144 of OspA protein from B. burgdorferi.
[0036] The prototype Class IV sequence from Borrelia burgdorferi strain B31M1 is: Class IV: KSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEG (SEQ ID NO: 25), which corresponds to residues 119-169 of OspA protein from B. burgdorferi. Additional exemplary Class II, II and IV type-specific sequences that may be used in the practice of the invention include those depicted in Table 2. The sequences in Table 2 (SEQ ID NOS: 19-27) are exemplary, in that a phylogenetic analysis of Borrelia OspA at the indicated regions would generate additional potential sequences for use in the practice of the invention (i.e. additional sequences from other Borrelia species, strains or types).
TABLE-US-00002 TABLE 2 Example sequence variants for other epitope-containing sequences. Accession SEQ ID Epitope region Species number NO: Sequence Amino acids 17-67 B. burgdorferi NIP045688.1 19 CKQNVSSLDEKNSVSVDLPGEM KVLVSKEKNKDGKYDLIATVDK LELKGTS Amino acids 17-67 B. garinii CAA56545.1 20 CKQNVSSLDEKNSVSVDLPGGM TVLVSKEKDKDGKYSLEATVDK LELKGTS Amino acids 17-67 B. afzelii CAA59724.1 21 CKQNVSSLDEKNSASVDLPGEM KVINSKEKDKDGKYSLKATVD KIELKGTS Amino acids 94-144 B. burgdorferi NP045688.1 22 LGQTTLEVFKEDGKTLVSKKVT SKDKSSTEEKFNEKGEVSEKII TRADGTR Amino acids 94-144 B. garinii CAA56545.1 23 LSQTKFEIFKEDGKTLVSKKVT LKDKSSTEEKFNEKGETSEKTI VRANGTR Amino acids 94-144 B. afzelii CAA59724.1 24 LSKPTFELFKGDGETLVSRKVS SKDKTSTDEMFNEKGELSAKTM TRENGTK Amino acids 119-169 B. burgdorferi NP045688.1 25 KSSTEEKFNEKGEVSEKIITRA DGTRLEYTGIKSDGSGKAKEVL KGYVLEG Amino acids 119-169 B. garinii CAA56545.1 26 KSSTEEKFNEKGETSEKTIVRA NGTRLEYTDIKSDGSGKAKEVL KDFTLEG Amino acids 119-169 B. afzelii CAA59724.1 27 KTSTDEMFNEKGELSAKTMTRE NGTKLEYTEMKSDGTGKAKEVL KNFTLEG
[0037] The use of one or more these linear epitopes in vaccine preparations advantageously avoids exposing the vaccine recipient to regions of OspA that have been implicated in putative adverse vaccine effects. The inclusion of a plurality of these linear sequences provides broad coverage against the development of Lyme disease for individuals over a broad geographical area. Or, when used as a diagnostic, the polypeptides of the invention enable detection of exposure to or infection with Borrelia of most important phyletic types over a broad geographical area. Further, the vaccine and/or diagnostic compositions can be "tuned" if desired, to represent one or more, but not all, geographical regions. For example, a vaccine or diagnostic developed for use in North America might contain only sequences derived from B. burgdorferi, as this is the only species associated with Lyme disease in that geographic area. In addition, in some embodiments of the invention, polypeptides containing one or more linear OspA epitopes or antigenic regions as described herein, may also include one or more OspC epitopes or antigenic regions, as described in detail below.
[0038] In order to facilitate the understanding of the present invention, the following definitions are provided: [0039] Antigen: term used historically to designate an entity that is bound by an antibody, and also to designate the entity that induces the production of the antibody. More current usage limits the meaning of antigen to that entity bound by an antibody, while the word "immunogen" is used for the entity that induces antibody production. Where an entity discussed herein is both immunogenic and antigenic, reference to it as either an immunogen or antigen will typically be made according to its intended utility. The terms "antigen", "antigenic region" "immunogen" and "epitope" may be used interchangeably herein. As used herein, an antigen, immunogen or epitope is generally a portion of a protein (e.g. a peptide or polypeptide). [0040] Epitope or B-cell Epitope: a specific chemical domain on an antigen that is recognized by a B-cell receptor, and which can be bound by secreted antibody. The term as used herein is interchangeable with "antigenic determinant". [0041] Immunodominant epitope: The epitope on a molecule that induces the dominant, or most intense, immune response. The immunodominant epitope would elicit the greatest antibody titer during infection or immunization, as measured by, for example, the fraction of reactivity attributable to a certain antigen or epitope in an enzyme-linked immunosorbant assay as compared with the total responsiveness to an antigen set or entire protein. [0042] Linear epitope: An epitope comprising a single, non-interrupted, contiguous chain of amino acids joined together by peptide bonds to form a peptide or polypeptide. Such an epitope can be described by its primary structure, i.e. the linear sequence of amino acids in the peptide chain. This is in contrast to conformational epitopes, which are comprised of at least some amino acids that are not part of an uninterrupted, linear sequence of amino acids, but which are brought into proximity to other residues in the epitope by secondary, tertiary and/or quaternary interactions of the protein. Residues in conformational epitopes may be located far from other resides in the epitope with respect to primary sequence, but may be spatially located near other residues in the conformational epitope due to protein folding. [0043] Protein: A linear sequence of about 100 or more amino acids covalently joined by peptide bonds. [0044] Polypeptide: A linear sequence of about 55 to about 100 amino acids covalently joined by peptide bonds. [0045] Peptide: A linear sequence of about 55 or fewer amino acids covalently joined by peptide bonds. [0046] Note: The terms "peptide", "polypeptide" and "protein" may be used interchangeably herein. Chimeric or fusion peptide or polypeptide: a recombinant or synthetic peptide or polypeptide whose primary sequence comprises two or more linear amino acid sequences which do not occur together in a single molecule in nature. The two or more sequences may be, for example, a peptide (e.g. an epitope or antigenic region) and a linker sequence, or two or more peptides (which may be the same or different) which are either contiguous or separated by a linker sequences, etc. [0047] Original or native or wild type sequence: The sequence of a peptide, polypeptide, protein or nucleic acid as found in nature. [0048] Recombinant peptide, polypeptide, protein or nucleic acid: peptide, polypeptide, protein or nucleic acid that has been produced and/or manipulated using molecular biology techniques such as cloning, polymerase chain reaction (PCR), etc. [0049] Synthetic peptide, polypeptide, protein or nucleic acid: peptide, polypeptide, protein or nucleic acid that has been produced using chemical synthesis procedures. [0050] Type-specific: associated primarily with a single phyletic group. [0051] Invasive infection: A protein is said to be "associated with invasive infection" if a Borrelia type bearing the protein has been isolated during human infection from locations other than the skin surrounding the initial inoculation by tick bite (e.g. from plasma, cerebrospinal fluid, etc.).
[0052] According to the invention, at least one amino acid sequence from Classes I-IV will be included in a peptide or polypeptide that is administered as a vaccine. In one embodiment of the invention, an antigenic chimeric (or fusion) polypeptide of the invention comprises one or more amino acid sequence from Class I, such as those set forth in, for example, SEQ ID NOS: 1 and 2. Usually, one or more copies of two or more Class I sequences will be included, and each distinct sequence may be present in the polypeptide one or more time, i.e. multiple copies of one or more of the sequences set forth in, for example, SEQ ID NOS: 1 and 2 are included. For example, from about 1 to about 10 of the Class I sequences may be included, and for each separate sequence that is included, that sequence may be present in from about 2 to about 12 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) or more copies. Individual copies of the sequence may be separated by linker sequences (described below), although this need not always be the case, i.e. copies of the same or different antigenic regions may be contiguous in the polypeptide chain.
[0053] In other embodiments, one or more of the amino acid sequences set forth in or represented by the Class II or Class III or Class IV antigenic regions are included in the antigenic polypeptides of the invention. As is the case with the Class I epitopes, one or more of the peptides may be present in the antigenic polypeptide, and, for each distinct sequence that is present, multiple copies(e.g. from about 2 to about 12) may be included. Individual copies of the sequence may be separated by linker sequences, although this need not always be the case.
[0054] In yet other embodiments, mixtures of one or more copies of one or more Class I, Class II, Class III and/or Class IV antigenic regions are present in the antigenic polypeptides of the invention. In other words, the polypeptide includes a mixture antigenic regions from any of the four classes. Each of the sequences that is included may be present in one copy or, more frequently, as multiple copies. In this type of construct, the individual sequences may be present in any order. For example, one segment or section of the polypeptide may include multiple copies of e.g. SEQ ID NO: 1, while another section of the same polypeptide includes multiple copies of e.g. SEQ ID NO: 2, and so on. Alternatively, the sequences of e.g. SEQ ID NO: 1 and e.g. SEQ ID NO: 2 may alternate along the polypeptide chain so that the sequences are, in effect, interspersed amongst each other within the primary sequence of the polypeptide.
[0055] Other antigenic sequences of interest are those from other Borrelia proteins such as OspC. The invention thus provides chimeric polypeptides or proteins that comprise, one or more epitopes from OspC. Exemplary OspC epitopes include but are not limited to linear epitopes from the loop 5 and/or alpha helix 5 regions. The loop 5 region or domain of OspC is the region which includes residues that align with residues 131 to 159 of strain B31 (type A) OspC sequences (together with secondary structural elements such as a portion of alpha helix 3, loop 5, and alpha helix 4, as defined by Kumaran et al. The alpha helix 5 region/domain of OspC is the region which includes residues that align with amino acids 160 to 200 of the strain B31 (OspC type A) sequence and the C-terminal portion of the protein, amino acids 201-210 of the B31 sequence, as well as secondary structural elements including a portion of loop 6, alpha helix 5, and the unstructured C-terminal domain, as defined by Kumaran et al. (2001). According to the present invention, one or more peptides encompassing epitopes located within the primary linear sequence of OspC proteins from residues 131 to 200 (using the strain B31 type A OspC sequence as a reference for residue numbering) may be included in the immunogenic polypeptides of the invention, the polypeptides of the invention. Exemplary OspC regions from which epitopes may be selected are shown in FIG. 1, where the sequences corresponding to residues 131 to 200 of type A OspC from several Borrelia types are listed. One or more epitopes may be selected for inclusion, so that an entire 131-200 (or even 131-210 C-terminal) region encompassing several epitopes may be used. Alternatively, fragments of the 131-200 region may be employed, e.g. any contiguous sequence of from about 10 to about 50 amino acids. One or more of such contiguous sequences may be used in the practice of the invention, with preferable sequences being those corresponding to about amino acids 136 to 150 and about amino acids 168-203 of alpha helix 5.
[0056] In particular, the general epitope locations of suitable OspC-based sequences include Helix 3, Loop5, Loop6, Helix5 and a C-terminus sequence. Three exemplary constructs, denominated constructs A, B and C, are depicted schematically in FIGS. 2A-C (SEQ ID NOS: 62, 63 and 64). Constructs A, B and C differ in that the sequences contained therein are from different phyletic types. For example, as shown in FIG. 2A, Construct A contains sequences from Types A, B, K, I, H, N, C, M and D of B. burgdorferi. In addition, for each of Constructs A, B and C, various versions or variations have been developed. The versions differ, for example, by the inclusion or exclusion of an intervening Helix-4/Loop6 sequence, by the order of the individual epitopes, and by other exclusions and or additions, etc. Exemplary variations of Constructs A, B and C are shown in Table 3.
TABLE-US-00003 TABLE 3 Number of Molecular Theoretical SEQ ID Construct amino acids weight pI NO: Construct A, version 1 569 60657.2 6.35 65 Sequence SETFTNKLKEKHTDLGKEGVTDADAKEAILKTNGTKTKGAEELGKLFESVEVLSKAAKEM <----------------------------Type A------------------------- LANSVKELTSSEEFSTKLKDNHAQLGIQGVTDENAKKAILKANAAGKDKGVEELEKLSGS ---------><-----------------------Type B-------------------- LESLSSEDFTKKLEGEHAQLGIENVTDENAKKAILITDAAKDKGAAELEKLFKAVENLAA ----><---------------------Type K------------------------->< KLKGEHTDLGKEGVTDDNAKKAILKTNNDKTKGADELEKLFESVKNLSKAAKEMLTNSSE --------------------Type I-------------------------------><- KFAGKLKNEHASLGKKDATDDDAKKAILKTHGNTDKGAKELKDLSDSVESLVSDDETKKL --------------------Type H-------------------------><------- QSSHAQLGVAGGATTDEEAKKAILRTNAIKDKGADELEKLEKSVESLAKAAQDALANSVN --------------------Type N---------------------------------- ELTSKKLKEKHTDLGKKDATDVHAKEAILKTNGTKDKGAAELEKLFESVENLAKAAKEML ---><-----------------------Type C-------------------------- SNSNKAFTDKLKSSHAELGIANGAATDANAKAAILKTNGTKDKGAQELEKLFESVKNLSK --><--------------------------Type M------------------------ AAQETLNNSSESETKKLSDNQAELGIENATDDNAKKAILKTHNAKDKGAEELVKLSESVA --------><-------------------------------Type D------------- GLLKAAQAILANSVKELTSPVVAESPKKP ----------------------------> Construct B, version 1 503 53102.4 7.16 66 Sequence SEKFTTKLKDSHAELGIQSVQDDNAKKAILKTHGTKDKGAKELEELFKSLESLSKAAQAA <------------------------------------PWa-------------------- LTNSVKELTNSDKFTKKLTDSHAQLGAVGGAINDDRAKEAILKTHGTNDKGAKELKELSE ---------><------------------------PLi---------------------- SVESLAKAAQAALANSSEAFTKKLKDSNAQLGMQNGAATDAHAKAAILKTDATKDKGATE ---------------><-------------------------PBes-------------- LGELFKSVESLSKAAQEASVAFTSKLKSSNAQLGVANGNATDDDAKKAILKTNTPNDKGA -----------------><---------------------------Pki----------- KELKELFESVESLAKAAQAALVNSVQELTNSEAFTNRLKGSHAQLGVAAATDDHAKEAIL -----------------------------><-----------------PFiM-------- KSNPTKDKGAKELKDLSESVESLAKAAQEALANSVKELTNSEAFTKKLKDNNAQLGIQNV ---------------------------------------><----------Hl3------ QDVEAKKAILKTNGDISKSEAFTNKLKEKHAELGVNGGDTTDDNAKAAIFKTHPTKDKGV -----------------><------------------Smar------------------- EDLEKLSESVKSLLKAAQAALSNSAAFTKKLQDGHVDLGKTDVTDDNAKEAILKTNPTKT ----------------------><-------------------------HT22------- KGATELEELFKSVEGLVKAAKEA ----------------------> Construct C version 1 512 54372.0 8.14 67 Sequence SEEFTNKLKSGHADLGKQDATDDHAKAAILKTHATTDKGAKEFKDLFESVEGLLKAAQVA <-------------------------------------Pko------------------- LTNSVKELTSKLKGGHAELGLAAATDENAKKAILKTNGTKDKGAEELEKLFKSVESLAKA ---------><------------------------------PLj 7-------------- AKESLTNSVKELTNTKLRDSHAELGIQNVQDDNAKRAILKTHGNKDKGAKELKELSESLE -------------><------------------PHez----------------------- KLSKAAQAALANSVQELTSSEAFTNKLKEKTQELAVAAGAATDIDAKKAILKTNRDKDLG ------------------><--------------PMit---------------------- ADERGKLFKSVESLSKAAQEASANSVKELTSSEAFTDKLKNEHASLGKKDATDDDAKKAI ------------------------------><-----------------Szid------- LKTNVDKTKGADELIKLSGSLESLSKAAQAILANSEAFTKKLQDSNADLGKHNATDADSK ---------------------------------><--------------VS461------ EAILKTNGTKTKGAKELEELFKSVESLSKAAKEALSNSVKELTSSQDFINKLKGGHAELG -------------------------------------------><--------------- LVAATDANAKAAILKTNGDKTKGADEFEKLFKSVEGLLKAAQEALTNSVKELTSSEAFTK -------HT25------------------------------------------><----- KLQDSNADLGKHDATDADAKKAILKTDATKDK -----------DK15----------------> Construct A version 2 431 46088.0 5.85 68 Sequence SETFTNKLKEKHTDLGKEGVTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSSEEFST <-------Lp A---------><--------------Hx A--------------><--- KLKDNHAQLGIQGVTKGVEELEKLSGSLESLSSEDFTKKLEGEHAQLGIENVTAAELEKL ---Lp B--------><Hx B------------><--------Lp K--------><--- FKAVENLAKAAKEMAKLKGEHTDLGKEGVTKGADELEKLFESVKNLSKAAKEMLTNSKES --Hx K--------><------Lp I-----><-----------Hx I---------->< EKFAGKLKNEHASLGKKDATKGAKELKDLSDSVESLVKASDDFTKKLQSSHAQLGVAGGA -------Lp H--------><-------Hx H-------><--------Lp N------- TTADELEKLFKSVESLAKAAQDALANSVNELTSKKLKEKHTDLGKKDATAAELEKLFESV -><------------Hx N-------------><-----Lp C------><--------- ENLAKAAKEMLSNSNKAFTDKLKSSHAELGIANGAATKGAQELEKLFESVKNLSKAAQET -Hx C---------><Lp M-------------><----------------Hx M----- LNNSVKESESFTKKLSDNQAELGIENATKGAEELVKLSESVAGLLKAAQAILANSVKELT ------><--------Lp D--------><-----------------Hx D--------- SPVVAESPKKP ----------> Construct B version 2 381 39928.6 5.79 69 Sequence SEKFTTKLKDSHAELGIQSVQDKGAKELEELFKSLESLSKAAQAALTNSVKELTNSDKFT <----Lp PWa------><--------------Hx PWa---------------><---- KKLTDSHAQLGAVGGAINDKGAKELKELSESVESLAKAAQAALANSSEAFTKKLKDSNAQ -----Lp Pli------><------------Hx Pli--------><------Lp PBes LGMQNGAATDKGATELGELFKSVESLSKAAQEASVAFTSKLKSSNAQLGVANGNATDKGA --------><--------Hx PBes-------><--------Lp Pki-------><--- KELKELFESVESLAKAAQAALVNSVQELTNSEAFTNRLKGSHAQLGVAAATDKGAKELKD -------Hx Pki----------------><------Lp PFim------><-------- LSESVESLAKAAQEALANSVKELTNSEAFTKKLKDNNAQLGIQNVQSEAFTNKLKEKHAE ---Hx PFim--------------><-----Lp Hl3--------><---Lp Smar--- LGVNGGDTTDKGVEDLEKLSESVKSLLKAAQAALSNSAAFTKKLQDGHVDLGKTDVTTKG --------><----------Hx Smar---------><------Lp HT22------><-- ATELEELFKSVEGLVKAAKEA ------Hx HT22-------> Construct C version 2 383 40492.5 6.45 70 Sequence SEEFTNKLKSGHADLGKQDATKGAKEFKDLFESVEGLLKAAQVALTNSVKELTSKLKGGH <-----Lp PKo--------><---------Hx PKo----------------><---Lp AELGLAAATKGAEELEKLFKSVESLAKAAKESLTNSVKELTNTKLRDSHAELGIQNVQKG PLj7----><Hx PLj7------------------------><----Lp PHez---><- AKELKELSESLEKLSKAAQAALANSVQELTSSEAFTNKLKEKTQELAVAAGAATLGADER ---------Hx PHez--------------><-------Lp PMit-------><----- GKLFKSVESLSKAAQEASANSVKELTSSEAFTDKLKNEHASLGKKDATKGADELIKLSGS ------Hx PMit-------------><------Lp Szid------><----------- LESLSKAAQAILANSEAFTKKLQDSNADLGKHNATKGAKELEELFKSVESLSKAAKEALS Hx Szid------><------Lp VS461-----><-------------Hx VS461--- NSVKELTSSQDFINKLKGGHAELGLVAATKGADEFEKLFKSVEGLLKAAQEALTNSVKEL -------><------Lp HT25------><------------Hx HT25----------- TSSEAFTKKLQDSNADLGKHDAT -><------Lp DK15------> Construct A version 3 432 46201.2 5.85 71 Sequence SETFTNKLKEKHTDLGKEGVTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSKGVEEL <--------Lp A-------><-------------Hx A--------------><----- EKLSGSLESLSNKAFTDKLKSSHAELGIANGAATKKLKEKHTDLGKKDATKGADELEKLF --Hx B----><---------Lp M---------><-----Lp C------><------- ESVKNLSKAAKEMLTNSKEIAAELEKLFKAVENLAKAAKEMAKLKGEHTDLGKEGVTSEE ----Hx I------------><--------Hx K--------><------Lp I-----> FSTKLKDNHAQLGIQGVTKGAKELKDLSDSVESLVKAAAELEKLFESVENLAKAAKEMLS <------Lp B-------><-------Hx H-------><---------Hx C------- NSSEKFAGKLKNEHASLGKKDATSEDFTKKLEGEHAQLGIENVTKGAQELEKLFESVKNL -><--------Lp H--------><--------Lp K--------><--------Hx M- SKAAQETLNNSVKEADELEKLEKSVESLAKAAQDALANSVNELTSSESETKKLSDNQAEL -------------><------------Hx N-------------><-------Lp D--- GIENATSDDFTKKLQSSHAQLGVAGGATTKGAEELVKLSESVAGLLKAAQAILANSVKEL -----><--------Lp N---------><--------------------Hx D------ TSPVVAESPKKP -----------> Construct B version 3 382 40056.8 5.93 72 Sequence SEAFTKKLKDSNAQLGMQNGAATDKGAKELEELEKSLESLSKAAQAALTNSVKELTNKDK <----------Lp PBes----><-------------Hx PWa-------------><-- GAKELKELFESVESLAKAAQAALVNSVQELTNSEKETTKLKDSHAELGIQSVQSDKFTKK --------Hx PKi-----------------><------Lp PWa-------><------ LTDSHAQLGAVGGAINDKGAKELKELSESVESLAKAAQAALANSDKGAKELKDLSESVES -Lp Pli--------><----------Hx Pli----------><-------------Hx LAKAAQEALANSVKELTNSVAFTSKLKSSNAQLGVANGNATSEAFTKKLKDNNAQLGIQN PFim-------------><------Lp PKi---------><------Lp H13------ VQTKGATELEELFKSVEGLVKAAKEADKGVEDLEKLSESVKSLLKAAQAALSNSAAFTKK -><--------Hx HT22-------><----------Hx Smar--------><------ LQDGHVDLGKTDVTSEAFTNRLKGSHAQLGVAAATDKGATELGELFKSVESLSKAAQEAS Lp HT22------><-----Lp PFim-------><-------Hx PBes-------->< EAFTNKLKEKHAELGVNGGDTT -------Lp Smar-------> Construct C version 3 383 40622.6 6.10 73 Sequence SEEFTNKLKSGHADLGKQDATKGAKEFKDLFESVEGLLKAAQVALTNSVKELTSKEKGAE <------Lp PKo-------><-------------Hx PKo-------------><---- ELEKLFKSVESLAKAAKESLTNSVKELTNSEAFTDKLKNEHASLGKKDATTKLRDSHAEL ----Hx PLj------------------><-----Lp Szid-------><-------Lp GIQNVQLGADERGKLFKSVESLSKAAQEASANSVKELTSKEKGAKELEELFKSVESLSKA PHez-><------------Hx PMit--------><---------------Hx VS461- AKEALSNSVKELTSSEAFTKKLQDSNADLGKHNATSEAFTKKLQDSNADLGKHDATKGAD -------------><------Lp VS461-----><-------Lp DK15-----><--- EFEKLFKSVEGLLKAAQEALTNSVKELTSELKELSESLEKLSKAAQAALANSVQELTSSE -------Hx HT25--------------><-----------Hx PHez---------><- AFTNKLKEKTQELAVAAGAATKLKGGHAELGLAAATKGADELIKLSGSLESLSKAAQAIL -----Lp PMit--------><---Lp PLj7---><---------Hx Szid------- ANSQDFINKLKGGHAELGLVAAT -><-----Lp HT25------->
[0057] In some embodiments, at least two of the OspC epitopes included in the chimeric polypeptide are from different OspC types that are associated with invasive infection. For example, antigenic epitopes representing from about 2 to about 20, and preferably from about 6 to about 10, different OspC types are included in a chimeric protein. When used as a vaccine, such a multivalent (polyvalent) recombinant chimeric protein elicits broad protection against infection with Borrelia spirochetes expressing the OspC types that correspond to those in the chimeric protein, i.e. those Borrelia that are highly infective. While typically at least two of the epitopes are different from one another in primary sequence and originate from different OspC types, it is also possible to include multiple copies of a single type of OspC epitope in a chimera, or to include several sequences that are based on or derived from the original sequence of the same OspC type. While the total number of linear OspC epitopes in a chimera may vary somewhat, in general, the range will be from about 10 to about 20. In one embodiment of the invention, the immunodominant OspC epitopes are selected from OspC types Smar, PLi, H13, PFiM, SL10, PMit, PKi, Pbes, HT22, Pko, PLj7, VS461, DK15, HT25, A,72a, F, E, M, D, U, I, L, H, Szid, PHez, PWa, B, K, N, C. Those of skill in the art will recognize that epitopes from many combinations of OspC types may be used, so long as the resulting chimera produces a suitable immune response and/or is effective as a vaccine in preventing Lyme disease. Examples of suitable combinations of OspC epitopes (which may optionally be combined with OspA epitopes of Classes I-IV) include but are not limited to: 1) E, N, I, C, A, B, K, D; 2) A, B, K, D, E, N, C; 3) I, C, A, B, K, D; and 4) C, A, B, K, D.
[0058] In some embodiments, both the OspC loop 5 and OspC alpha helix 5 regions will be included. For example, an "E, N, I, C, A, B, K, D" construct may contain both the loop 5 and helix 5 regions of each of OspC types E, N, I, C, A, B, K, and D. However, this need not be the case. For example, the loop 5 region of type A and the alpha helix 5 regions of E, N, I, C, B, K, and D may be included; or only the loop 5 region for each OspC type may be included; or only the alpha helix 5 region; or other combinations may be included (e.g. loop 5 region of types E, N, I, and C and the alpha helix 5 region of types A, B, K, and D. Many such combinations will occur to those of skill in the art, and all such variations are intended to be encompassed herein.
[0059] In other embodiments of the invention, one or more of the sequences represented by OspA Classes I-IV are present in an antigenic polypeptide which also includes antigenic sequences of OspC as described above.
[0060] In addition, other peptide sequences may be included in the peptides and polypeptides of the invention. Such sequences include but are not limited to antigenic peptide sequences such as linker sequences which in and of themselves are antigenic.
[0061] Those of skill in the art will recognize that, while in some embodiments of the invention, the amino acid sequences that are chosen for inclusion in the peptides and polypeptides of the invention correspond exactly to the primary amino acid sequence of the original or native sequences of an OspA (or OspC) protein, this need not always be the case. The amino acid sequence of an epitope that is included in the peptides and polypeptides of the invention may be altered somewhat and still be suitable for use in the present invention. For example, certain conservative amino acid substitutions may be made without having a deleterious effect on the ability of the peptides and polypeptides to elicit an immune response. Those of skill in the art will recognize the nature of such conservative substitutions, for example, substitution of a positively charged amino acid for another positively charged amino acid (e.g. K for R or vice versa); substitution of a negatively charged amino acid for another negatively charged amino acid (e.g. D for E or vice versa); substitution of a hydrophobic amino acid for another hydrophobic amino acid (e.g. substitution of A, V, L, I, W, etc. for one another); etc. All such substitutions or alterations of the sequences of the peptides and polypeptides that are disclosed herein are intended to be encompassed by the present invention, so long as the resulting peptides and polypeptides still function to elicit a suitable immune response. In addition, the amino acid sequences that are included in the chimeric proteins of the invention need not encompass a full length native peptide or polypeptide. Those of skill in the art will recognize that truncated versions of amino acid sequences that are known to be or to contain antigenic peptides and/or polypeptides may, for a variety of reasons, be preferable for use in the practice of the invention, so long as the criteria set forth for an epitope is fulfilled by the sequence. Amino acid sequences that are so substituted or otherwise altered may be referred to herein as "based on" or "derived from" the original wild type or native sequence. In general, the OspA or OspC proteins from which the linear epitopes are "derived" or on which the linear epitopes are "based" are the OspA or OspC proteins as they occur in nature. These natural OspA/OspC proteins may alternatively be referred to as native or wildtype proteins.
[0062] Such changes to the primary sequence may be introduced for any of a variety of reasons, for example, to eliminate or introduce a protease cleavage site, to increase or decrease solubility, to promote or discourage intra- or inter-molecular interactions such as folding, ionic interactions, salt bridges, etc, which might otherwise interfere with the presentation and accessibility of the individual epitopes along the length of a peptide or polypeptide. All such changes are intended to be encompassed by the present invention, so long as the resulting amino acid sequence functions to elicit a protective antibody response in a host to whom it is administered. In general, such substituted sequences will be at least about 50% identical to the corresponding sequence in the native protein, preferably about 60 to 70, or even 70 to 80, or 80 to 90% identical to the wild type sequence, and preferably about 95, 96, 97, 98, 99, or even 100% identical to a native OspA (or OspC) sequence. The reference native OspA or OspC sequence may be from any suitable type of Borrelia, e.g. from any Borrelia which is known to infect mammals.
[0063] In some embodiments of the invention, the individual linear epitopes in the chimeric vaccinogen are separated from one another by intervening sequences that are more or less neutral in character, i.e. they do not in and of themselves elicit an immune response to Borrelia. Such sequences may or may not be present between the epitopes of a chimera. If present, they may, for example, serve to separate the epitopes and contribute to the steric isolation of the epitopes from each other. Alternatively, such sequences may be simply artifacts of recombinant processing procedures, e.g. cloning procedures. Such sequences are typically known as linker or spacer peptides, many examples of which are known to those of skill in the art. See, for example, Crasto, C. J. and J. A. Feng. 2000. LINKER: a program to generate linker sequences for fusion proteins. Protein Engineering 13(5): 309-312, which is a reference that describes unstructured linkers. Structured (e.g. helical) sequence linkers may also be designed using, for example, existing sequences that are known to have that secondary structure, or using basic known biochemical principles to design the linkers. In addition, other elements may be present in the chimeric proteins, for example leader sequences or sequences that "tag" the protein to facilitate purification or detection of the protein, examples of which include but are not limited to histidine tags, detection tags (e.g. S-tag, or Flag-tag), other antigenic amino acid sequences such as known T-cell epitope containing sequences and protein stabilizing motifs, etc. In addition, the chimeric proteins may be chemically modified, e.g. by amidation, sulfonylation, lipidation, or other techniques that are known to those of skill in the art.
[0064] The invention further provides nucleic acid sequences that encode the chimeric proteins of the invention. Such nucleic acids include DNA, RNA, and hybrids thereof, and the like. Further, the invention comprehends vectors which contain or house such coding sequences. Examples of suitable vectors include but are not limited to plasmids, cosmids, viral based vectors, expression vectors, etc. In a preferred embodiment, the vector will be a plasmid expression vector.
[0065] The chimeric polypeptides of the invention may be produced by any suitable method, many of which are known to those of skill in the art. For example, they may be chemically synthesized, or produced using recombinant DNA technology (e.g. in bacterial cells, in cell culture (mammalian, yeast or insect cells), in plants or plant cells, or by cell-free prokaryotic or eukaryotic-based expression systems, by other in vitro systems, etc.). In some embodiments, the polypeptides are produced using chemical synthesis methods.
[0066] The present invention also provides compositions for use in eliciting an immune response. The compositions may be utilized as vaccines to prevent or treat Borrelia infection, particularly when manifested as Lyme disease (Lyme borreliosis). By eliciting an immune response, we mean that administration of the antigen causes the synthesis of specific antibodies (at a titer as described above) and/or cellular proliferation, as measured, e.g. by 3H thymidine incorporation, or by other known techniques. By "vaccine" we mean a chimeric or fusion polypeptide that elicits an immune response which results in protection of an organicism against challenge with Borrelia. The protective response either wholly or partially prevents or arrests the development of symptoms related to Borrelia infection (i.e. the symptoms of Lyme disease), in comparison to a non-vaccinated (e.g. adjunct alone) control organisms, in which disease progression is not prevented. The compositions include one or more isolated and substantially purified chimeric peptides or polypeptides as described herein, and a pharmacologically suitable carrier. The chimeric polypeptides or proteins in the composition may be the same or different, i.e. the composition may be a "cocktail" of different chimeras, or a composition containing only a single type of chimera. The preparation of such compositions for use as vaccines is well known to those of skill in the art. Typically, such compositions are prepared either as liquid solutions or suspensions, however solid forms such as tablets, pills, powders and the like are also contemplated. Solid forms suitable for solution in, or suspension in, liquids prior to administration may also be prepared. The preparation may also be emulsified. The active ingredients may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. The vaccine preparations of the present invention may further comprise an adjuvant, suitable examples of which include but are not limited to Seppic, Quil A, Alhydrogel, etc. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like may be added. The composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration. The final amount of chimeric protein in the formulations may vary. However, in general, the amount in the formulations will be from about 0.01-99%, weight/volume.
[0067] The methods involve administering a composition comprising a chimeric recombinant protein in a pharmacologically acceptable carrier to a mammal. The mammal may be a human, but this need not always be the case, as veterinary applications of this technology are also contemplated. The vaccine preparations of the present invention may be administered by any of the many suitable means which are well known to those of skill in the art, including but not limited to by injection, inhalation, orally, intranasally, by ingestion of a food product containing the chimeric protein, etc. In preferred embodiments, the mode of administration is subcutaneous or intramuscular. In addition, the compositions may be administered in conjunction with other treatment modalities such as substances that boost the immune system, various anti-bacterial chemotherapeutic agents, antibiotics, and the like.
[0068] The present invention provides methods to elicit an immune response to Borrelia and to vaccinate against Borrelia infection in mammals. In one embodiment, the mammal is a human However, those of skill in the art will recognize that other mammals exist for which such vaccinations would also be desirable, e.g. the preparations may also be used for veterinary purposes. Examples include but are not limited to companion "pets" such as dogs, cats, etc.; food source, work and recreational animals such as cattle, horses, oxen, sheep, pigs, goats, and the like; or even wild animals that serve as a reservoir of Borrelia (e.g. mice, deer, etc).
[0069] The invention also provides a diagnostic and a method for using the diagnostic to identify individuals who have antibodies to the epitopes contained within the chimeric polypeptides of the invention. A biological sample from an individual (e.g. a human, a deer, or other mammals susceptible to infection by Borrelia spirochetes) suspected of having been exposed to Borrelia, or at risk for being exposed to Borrelia, is contacted with the chimeric proteins of the invention. Using known methodology, the presence or absence of a binding reaction between the chimeric protein and antibodies in the biological sample is detected. A positive result (i.e. binding occurs, thus antibodies are present) indicates that the individual has been exposed to and/or is infected with Borrelia. Further, the diagnostic aspects of the invention are not confined to clinical use or home use, but may also be valuable for use in the laboratory as a research tool, e.g. to identify Borrelia spirochetes isolated from ticks, to investigate the geographical distribution of Borrelia species and strains, etc.
[0070] The present invention also encompasses antibodies to the epitopes and/or to the chimeric polypeptides disclosed herein. Such antibodies may be polyclonal, monoclonal or chimeric, and may be generated in any manner known to those of skill in the art. In a preferred embodiment of the invention, the antibodies are bactericidal (borrelia cidal), i.e. exposure of Borrelia spirochetes to the antibodies causes death of the spirochetes. Such antibodies may be used in a variety of ways, e.g. as detection reagents to diagnose prior exposure to Borrelia, as a reagent in a kit for the investigation of Borrelia, to treat Borrelia infections, etc.
[0071] Alternatively, appropriate antigen fragments or antigenic sequences or epitopes may be identified by their ability, when included in a chimeric protein, to elicit suitable antibody production to the epitope in a host to which the chimeric protein is administered. Those of skill in the art will recognize that definitions of antibody titer may vary. Herein, "titer" is taken to be the inverse dilution of antiserum that will bind one half of the available binding sites on an ELISA well coated with 100 ng of test protein. In general, suitable antibody production is characterized by an antibody titer in the range of from about 100 to about 100,000, and preferably in the range of from about 10,000 to about 10,000,000. Alternatively, and particularly in diagnostic assays, the "titer" should be about three times the background level of binding. For example, to be considered "positive", reactivity in a test should be at least three times greater than reactivity detected in serum from uninfected individuals. Preferably, the antibody response is protective, i.e. prevents or lessens the development of symptoms of disease in a vaccinated host that is later exposed to Borrelia, compared to an unvaccinated host.
[0072] The following Examples are provided to illustrate various embodiments of the invention, but should not be considered as limiting in any way.
EXAMPLES
Example 1
Identification of Linear Immunodominant Epitopes of Borrelia OspA Protein
Materials and Methods
[0073] Recombinant protein production
[0074] Fragments of the outer surface protein A (OspA) gene were amplified by PCR using high fidelity DNA polymerase (Phusion HF, New England Biolabs), and the amplicons purified by agarose gel electrophoresis and gel extraction. Overhangs were generated by treatment with T4 polymerase to allow ligase-independent annealing to the pET-32 Ek/LIC vector. The annealed vector was transformed into Escherichia coli, and the transformants were selected for ampicillin resistance, and were screened for the presence of the OspA gene fragment by PCR. Plasmids were then extracted from the transformants and the sequence confirmed by DNA sequencing (MWG Biotech). The plasmids were then used to transform E. coli BL21 (DE3) cells for protein production.
[0075] To generate recombinant proteins, BL21(DE3) cells were grown at 37° C. to an OD600 of 0.5, then 1 mM IPTG was added to induce protein expression, and the cells were maintained at 37° C. for an additional 3 hours. The pET-32 Ek/LIC vector encodes a 17.1 kDa N-terminal protein tagging sequence which includes a hexahistidine motif that was used to purify the r-proteins by Ni--NTA nickel affinity chromatography, according to the manufacturer's protocol (Qiagen). The purified proteins were quantified by the bicinchoninic acid assay (Pierce).
Generation of Murine Infection Serum
[0076] Borrelia burgdorferi strains B31MI, LDP74, and B331 were grown to late log phase in BSK-H medium (Sigma). The cells were washed with fresh medium, quantified by microscopy, and diluted in BSK-H medium to 104 cells per 0.1 mL of medium. Strain C3H/HeJ mice were infected with 104 cells by subcutaneous injection between the scapulae, with 5 mice infected with each spirochete strain. The mice were bled at week 6 by tail nick, and infection was confirmed by culture of a 2 mm ear punch biopsy. Serum was prepared from the infected mouse blood samples, and will be referred to hereafter as infection serum.
Mapping of Linear B-Cell Epitopes on OspA
[0077] To map linear B-cell epitopes, overlapping recombinant subfragments of OspA were separated by 15% SDS-PAGE and blotted to PVDF. The membranes were blocked with 5% NFDM in PBS-T, and probed with mouse infection sera (1:500 dilution). To assess equality of protein loading, one blot was probed with a mouse monoclonal antibody specific to the hexahistidine motif in the expression tag sequence. The blots were then washed and probed with peroxidase-conjugated goat-anti-mouse IgG antiserum (1:20000; Pierce). The blots were washed, incubated with a chemiluminescent substrate (Supersignal West Pico; Pierce), and exposed to film. When reactive OspA fragments were determined, a series of smaller overlapping subfragments was made to further resolve the location of the linear epitope (FIG. 3).
OspA Sequence Analysis
[0078] The full length ospA gene from the Borrelia strains used to produce infection sera were PCR amplified and cloned into the pET-46 Ek/LIC vector as described above, and the DNA sequences were aligned and compared. In addition, the 204 full length OspA sequences from the Lyme spirochetes B. burgdorferi, B. garinii, and B. afzelii that are available in the NCBI databases were aligned and analyzed for polymorphisms in the epitope containing regions. This was done by multisequence alignment using ClustalX, and generation of neighbor joining phylogenetic trees. These analyses were done on the full length OspA gene, as well as on the epitope-containing subfragments. Once the alignments and trees were complete, representative sequences were extracted that demonstrate extant sequence variants, which may correspond to antigenic variants. Analytical tools included ClustalX, Bioedit, and TreeView.
Results:
Location of OspA Linear Epitopes
[0079] A western blot-based search for linear B-cell epitopes on OspA revealed several amino acid sequences that were reactive with antibodies generated during murine infection (FIG. 4). The reactive subfragments are located at the N-terminus (amino acids 17-67; noted in a single mouse), within a central single layer beta-sheet (amino acids 94-144, and 119-169; seen in multiple mice), and within a membrane-distal loop (amino acids 194-246 and 220-273; highly reactive in multiple mice). High resolution mapping of the beta-sheet epitope containing region failed to further localize the epitope. The most reactive linear epitope was found in the membrane distal loop, and subsequent high resolution mapping localized it to amino acids 221-240. The pattern of reactivity varied somewhat in intensity between mice, but was observed in sera from mice infected with each of the three test Borrelia strains.
OspA Sequence Analysis
[0080] Comparison of sequences among the three Borrelia strains used to infect mice revealed either one or two amino acid differences among them, at amino acid positions 39 (K/N) and 149 (G/E) (FIG. 5). An analysis of available OspA sequences demonstrated several major clades, which split primarily along species lines (B. burgdorferi, B. garinii, and B. afzelii). To better understand the impact of the clades on the development of an epitope-based vaccine, the 221-240 region was analyzed separately, aligned, and a neighbor joining tree created. This also demonstrated the presence of distinct clades of this epitope containing region, again separated primarily by species. There were 2 predominant epitope region sequences within B. burgdorferi, 5 within B. garinii, and 1 within B. afzelii derived OspA sequences. The 221-240 amino acid regions in each of these predominant groups is shown in Table 1. Representative alignments of the other three epitope-containing sequences are shown in Table 2. Using the methods described above, the potential variants of these epitopes can be easily analyzed.
Discussion:
[0081] The mapping of novel linear B-cell epitopes in OspA represents a significant advance in the development of second-generation Lyme disease vaccines. Previous research has primarily focused on known conformational epitopes, primarily the epitope recognized by the LA-2 monoclonal antibody. Linear OspA epitopes have been described, however, this study is novel in its use of serum derived from mice infected with clonal Borrelia populations. The similar epitope recognition pattern during infection with three distinct Borrelia burgdorferi strains further supports the relevance of these epitope containing regions during infection. While OspA is expressed during in vitro culture, it is normally downregulated upon exposure to the mammalian environment or during the tick blood meal during the normal enzootic cycle. Because of this regulation, it is likely that the OspA protein was expressed only for a short time during the infection, which may account for the variability in the intensity of responsiveness between mice. This apparent variability may be enhanced by the IgG-specific screening technique, requiring the B-cells of the infected mice to progress to a more mature immune response and undergo immunoglobulin heavy chain class switching.
[0082] The large degree of intraspecies conservation at the mapped epitope-containing regions is of particular advantage in development of a peptide or chimeric vaccine. For example, since B. burgdorferi is the only species associated with Lyme disease in North America, the occurrence of only a limited number of phylogenetic clades of OspA is particularly advantageous. At the epitope level, the number of major clades is also limited, with only two major antigenically distinct clades representing the large majority of the 221-240 epitope containing region. Borrelia afzelii, which occurs only in Europe and Asia, is limited to a single clade at this region, and B. garinii has approximately 6 clades, several of which are closely related, though it is not yet known if they are antigenically cross-reactive. It is clear that in order for broad protection to be achieved by a peptide vaccine approach, the use of a single OspA epitope sequence is not possible. For that reason, the development of a multi-epitope chimeric vaccine based on OspA epitope variants or on a combination of epitope variants of OspA and OspC is highly desirable. In addition, a significant advantage to the use of defined epitope containing sequences is the ability to avoid segments of the OspA molecule that have been reported to be associated with development of autoimmune responses. While there is disagreement in the literature as to the incidence of these responses caused by anti-OspA antibodies or anti-OspA immune responses, it is advantageous to avoid those portions of OspA that have been implicated (FIG. 6).
Example 2
[0083] When administered to test mammals, this chimeric polypeptide construct comprising at least one 221-240 epitope containing region, and usually two or more 221-240 epitope containing regions from of different phyletic types, is found to elicit a robust immune response, and to provide protection from the development of Lyme disease.
[0084] While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.
Sequence CWU
1
76120PRTBorrelia burgdorferi 1Ser Thr Leu Thr Ile Thr Val Asn Ser Lys Lys
Thr Lys Asp Leu Val1 5 10
15Phe Thr Lys Glu 20220PRTBorrelia burgdorferi 2Ser Thr Leu
Thr Ile Ile Val Asp Ser Lys Asn Lys Thr Lys Leu Val1 5
10 15Phe Thr Lys Gln
20320PRTBorrelia burgdorferi 3Ser Thr Leu Thr Ile Ser Lys Asn Arg Thr Lys
Thr Lys Gln Leu Val1 5 10
15Phe Thr Lys Glu 20420PRTBorrelia burgdorferi 4Ser Thr Leu
Thr Ile Ser Ala Asn Ser Lys Lys Thr Lys Asp Leu Val1 5
10 15Phe Leu Thr Asn
20520PRTBorrelia burgdorferi 5Ser Thr Leu Thr Ile Thr Val Asn Asn Lys Lys
Thr Lys Ala Leu Val1 5 10
15Phe Thr Lys Gln 20620PRTBorrelia burgdorferi and Borrelia
afzelli 6Ser Thr Leu Thr Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val1
5 10 15Phe Thr Lys Gln
20720PRTBorrelia burgdorferi and Borrelia garinii 7Ser Thr Leu
Thr Ile Ser Val Asn Ser Lys Lys Thr Lys Asn Ile Val1 5
10 15Phe Thr Lys Glu
20820PRTBorrelia burgdorferi and Borrelia garinii 8Ser Thr Leu Thr Ile
Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val1 5
10 15Phe Thr Lys Glu 20920PRTBorrelia
garinii 9Asn Thr Leu Thr Val Ser Ala Asp Ser Lys Lys Ile Lys Asp Phe Val1
5 10 15Phe Leu Thr Asp
201020PRTBorrelia garinii 10Ser Thr Leu Thr Ile Ser Lys Asn Ser
Gln Lys Thr Lys Gln Leu Val1 5 10
15Phe Thr Lys Glu 201120PRTBorrelia garinii 11Ser Thr
Leu Thr Ile Ser Ala Lys Asn Lys Lys Thr Lys Asp Leu Val1 5
10 15Phe Thr Lys Gln
201220PRTBorrelia garinii 12Ser Thr Leu Lys Ile Ser Lys Asn Ser Lys Lys
Thr Lys Gln Leu Val1 5 10
15Phe Thr Lys Glu 201320PRTBorrelia garinii 13Ser Thr Leu Thr
Ile Ser Ala Lys Ser Lys Lys Thr Lys Asp Leu Val1 5
10 15Phe Thr Lys Gln
201420PRTBorrelia garinii 14Ser Thr Leu Thr Ile Ser Ala Asn Asn Lys Lys
Thr Lys Asp Leu Val1 5 10
15Phe Thr Lys Gln 201520PRTBorrelia garinii 15Lys Thr Leu Thr
Val Ser Ala Asp Ser Lys Lys Ile Lys Asp Phe Val1 5
10 15Phe Leu Thr Asp
201620PRTBorrelia garinii 16Ser Thr Leu Thr Ile Ser Ala Lys Asn Lys Lys
Thr Thr Asp Leu Val1 5 10
15Phe Thr Lys Gln 201720PRTBorrelia garinii 17Ser Thr Leu Thr
Ile Ser Lys Asn Ser Arg Lys Thr Lys Gln Leu Val1 5
10 15Phe Thr Lys Glu
201820PRTBorrelia garinii 18Ser Thr Leu Thr Ile Ser Val Asn Ser Arg Lys
Thr Lys Asn Leu Val1 5 10
15Phe Thr Lys Glu 201951PRTBorrelia burgdorferi 19Cys Lys Gln
Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val1 5
10 15Asp Leu Pro Gly Glu Met Lys Val Leu
Val Ser Lys Glu Lys Asn Lys 20 25
30Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys
35 40 45Gly Thr Ser
502051PRTBorrelia garinii 20Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys
Asn Ser Val Ser Val1 5 10
15Asp Leu Pro Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys
20 25 30Asp Gly Lys Tyr Ser Leu Glu
Ala Thr Val Asp Lys Leu Glu Leu Lys 35 40
45Gly Thr Ser 502151PRTBorrelia afzelii 21Cys Lys Gln Asn Val
Ser Ser Leu Asp Glu Lys Asn Ser Ala Ser Val1 5
10 15Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser
Lys Glu Lys Asp Lys 20 25
30Asp Gly Lys Tyr Ser Leu Lys Ala Thr Val Asp Lys Ile Glu Leu Lys
35 40 45Gly Thr Ser
502251PRTBorrelia burgdorferi 22Leu Gly Gln Thr Thr Leu Glu Val Phe Lys
Glu Asp Gly Lys Thr Leu1 5 10
15Val Ser Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys
20 25 30Phe Asn Glu Lys Gly Glu
Val Ser Glu Lys Ile Ile Thr Arg Ala Asp 35 40
45Gly Thr Arg 502351PRTBorrelia garinii 23Leu Ser Gln Thr
Lys Phe Glu Ile Phe Lys Glu Asp Gly Lys Thr Leu1 5
10 15Val Ser Lys Lys Val Thr Leu Lys Asp Lys
Ser Ser Thr Glu Glu Lys 20 25
30Phe Asn Glu Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg Ala Asn
35 40 45Gly Thr Arg
502451PRTBorrelia afzelii 24Leu Ser Lys Pro Thr Phe Glu Leu Phe Lys Gly
Asp Gly Glu Thr Leu1 5 10
15Val Ser Arg Lys Val Ser Ser Lys Asp Lys Thr Ser Thr Asp Glu Met
20 25 30Phe Asn Glu Lys Gly Glu Leu
Ser Ala Lys Thr Met Thr Arg Glu Asn 35 40
45Gly Thr Lys 502551PRTBorrelia burgdorferi 25Lys Ser Ser Thr
Glu Glu Lys Phe Asn Glu Lys Gly Glu Val Ser Glu1 5
10 15Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg
Leu Glu Tyr Thr Gly Ile 20 25
30Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys Gly Tyr Val
35 40 45Leu Glu Gly
502651PRTBorrelia garinii 26Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu Lys
Gly Glu Thr Ser Glu1 5 10
15Lys Thr Ile Val Arg Ala Asn Gly Thr Arg Leu Glu Tyr Thr Asp Ile
20 25 30Lys Ser Asp Gly Ser Gly Lys
Ala Lys Glu Val Leu Lys Asp Phe Thr 35 40
45Leu Glu Gly 502751PRTBorrelia afzelii 27Lys Thr Ser Thr Asp
Glu Met Phe Asn Glu Lys Gly Glu Leu Ser Ala1 5
10 15Lys Thr Met Thr Arg Glu Asn Gly Thr Lys Leu
Glu Tyr Thr Glu Met 20 25
30Lys Ser Asp Gly Thr Gly Lys Ala Lys Glu Val Leu Lys Asn Phe Thr
35 40 45Leu Glu Gly
502872PRTBorrelia garinii 28Ser Glu Ala Phe Thr Asn Lys Leu Lys Glu Lys
His Ala Glu Leu Gly1 5 10
15Val Asn Gly Gly Asp Thr Thr Asp Asp Asn Ala Lys Ala Ala Ile Phe
20 25 30Lys Thr His Pro Thr Lys Asp
Lys Gly Val Glu Asp Leu Glu Lys Leu 35 40
45Ser Glu Ser Val Lys Ser Leu Leu Lys Ala Ala Gln Ala Ala Leu
Ser 50 55 60Asn Ser Val Lys Glu Leu
Thr Ser65 702972PRTBorrelia garinii 29Ser Asp Lys Phe
Thr Lys Lys Leu Thr Asp Ser His Ala Gln Leu Gly1 5
10 15Ala Val Gly Gly Ala Ile Asn Asp Asp Arg
Ala Lys Glu Ala Ile Leu 20 25
30Lys Thr His Gly Thr Asn Asp Lys Gly Ala Lys Glu Leu Lys Glu Leu
35 40 45Ser Glu Ser Val Glu Ser Leu Ala
Lys Ala Ala Gln Ala Ala Leu Ala 50 55
60Asn Ser Val Lys Glu Leu Thr Ser65 703070PRTBorrelia
garinii 30Ser Glu Ala Phe Thr Lys Lys Leu Lys Asp Asn Asn Ala Gln Leu
Gly1 5 10 15Ile Gln Asn
Val Gln Asp Val Glu Ala Lys Lys Ala Ile Leu Lys Thr 20
25 30Asn Gly Asp Ile Ser Lys Gly Ala Lys Glu
Leu Lys Glu Leu Phe Glu 35 40
45Ser Val Glu Ser Leu Ala Lys Ala Ala Gln Ala Ala Leu Ala Asn Ser 50
55 60Val Gln Glu Leu Thr Asn65
703170PRTBorrelia garinii 31Ser Glu Ala Phe Thr Asn Arg Leu Lys Gly
Ser His Ala Gln Leu Gly1 5 10
15Val Ala Ala Ala Thr Asp Asp His Ala Lys Glu Ala Ile Leu Lys Ser
20 25 30Asn Pro Thr Lys Asp Lys
Gly Ala Lys Glu Leu Lys Asp Leu Ser Glu 35 40
45Ser Val Glu Ser Leu Ala Lys Ala Ala Gln Glu Ala Leu Ala
Asn Ser 50 55 60Val Lys Glu Leu Thr
Asn65 703270PRTBorrelia garinii 32Ser Glu Ala Phe Thr
Asn Arg Leu Thr Gly Ser His Ala Gln His Gly1 5
10 15Val Ala Ala Ala Thr Asp Asp His Ala Lys Glu
Ala Ile Leu Lys Ser 20 25
30Asn Pro Thr Lys Asp Lys Gly Ala Lys Glu Leu Lys Asp Leu Ser Glu
35 40 45Ser Val Glu Ser Leu Ala Lys Ala
Ala Gln Glu Ala Leu Ala Asn Ser 50 55
60Val Lys Glu Leu Thr Asn65 703372PRTBorrelia garinii
33Ser Glu Ala Phe Thr Asn Lys Leu Lys Glu Lys Thr Gln Glu Leu Ala1
5 10 15Val Ala Ala Gly Ala Ala
Thr Asp Ile Asp Ala Lys Lys Ala Ile Leu 20 25
30Lys Thr Asn Arg Asp Lys Asp Leu Gly Ala Asp Glu Arg
Gly Lys Leu 35 40 45Phe Lys Ser
Val Glu Ser Leu Ser Lys Ala Ala Gln Glu Ala Ser Ala 50
55 60Asn Ser Val Lys Glu Leu Thr Ser65
703472PRTBorrelia garinii 34Ser Val Ala Phe Thr Ser Lys Leu Lys Ser Ser
Asn Ala Gln Leu Gly1 5 10
15Val Ala Asn Gly Asn Ala Thr Asp Asp Asp Ala Lys Lys Ala Ile Leu
20 25 30Lys Thr Asn Thr Pro Asn Asp
Lys Gly Ala Lys Glu Leu Lys Glu Leu 35 40
45Phe Glu Ser Val Glu Ser Leu Ala Lys Ala Ala Gln Ala Ala Leu
Val 50 55 60Asn Ser Val Gln Glu Leu
Thr Asn65 703572PRTBorrelia garinii 35Ser Glu Ala Phe
Thr Lys Lys Leu Lys Asp Ser Asn Ala Gln Leu Gly1 5
10 15Met Gln Asn Gly Ala Ala Thr Asp Ala His
Ala Lys Ala Ala Ile Leu 20 25
30Lys Thr Asp Ala Thr Lys Asp Lys Gly Ala Thr Glu Leu Gly Glu Leu
35 40 45Phe Lys Ser Val Glu Ser Leu Ser
Lys Ala Ala Gln Glu Ala Ser Ala 50 55
60Asn Ser Val Lys Glu Leu Thr Ser65 703670PRTBorrelia
garinii 36Ser Ala Ala Phe Thr Lys Lys Leu Gln Asp Gly His Val Asp Leu
Gly1 5 10 15Lys Thr Asp
Val Thr Asp Asp Asn Ala Lys Glu Ala Ile Leu Lys Thr 20
25 30Asn Pro Thr Lys Thr Lys Gly Ala Thr Glu
Leu Glu Glu Leu Phe Lys 35 40
45Ser Val Glu Gly Leu Val Lys Ala Ala Lys Glu Ala Ser Ala Asn Ser 50
55 60Val Lys Glu Leu Thr Ser65
703770PRTBorrelia afzelii 37Ser Glu Glu Phe Thr Asn Lys Leu Lys Ser
Gly His Ala Asp Leu Gly1 5 10
15Lys Gln Asp Ala Thr Asp Asp His Ala Lys Ala Ala Ile Leu Lys Thr
20 25 30His Ala Thr Thr Asp Lys
Gly Ala Lys Glu Phe Lys Asp Leu Phe Glu 35 40
45Ser Val Glu Gly Leu Leu Lys Ala Ala Gln Val Ala Leu Thr
Asn Ser 50 55 60Val Lys Glu Leu Thr
Ser65 703870PRTBorrelia afzelii 38Ser Glu Glu Phe Thr
Asn Lys Leu Lys Ser Gly His Ala Asp Leu Gly1 5
10 15Lys Gln Asp Ala Thr Asp Glu His Ala Lys Ala
Ala Ile Leu Lys Thr 20 25
30His Ala Thr Thr Asp Lys Gly Ala Lys Glu Phe Lys Asp Leu Phe Glu
35 40 45Ser Val Glu Gly Leu Leu Lys Ser
Ala Gln Val Ala Leu Thr Asn Ser 50 55
60Val Lys Glu Leu Thr Asn65 703970PRTBorrelia afzelii
39Ser Glu Glu Phe Thr Asn Lys Leu Lys Gly Gly His Ala Glu Leu Gly1
5 10 15Leu Ala Ala Ala Thr Asp
Glu Asn Ala Lys Lys Ala Ile Leu Lys Thr 20 25
30Asn Gly Thr Lys Asp Lys Gly Ala Glu Glu Leu Glu Lys
Leu Phe Lys 35 40 45Ser Val Glu
Ser Leu Ala Lys Ala Ala Lys Glu Ser Leu Thr Asn Ser 50
55 60Val Lys Glu Leu Thr Asn65
704070PRTBorrelia afzelii 40Ser Glu Ala Phe Thr Lys Lys Leu Gln Asp Ser
Asn Ala Asp Leu Gly1 5 10
15Lys His Asn Ala Thr Asp Ala Asp Ser Lys Glu Ala Ile Leu Lys Thr
20 25 30Asn Gly Thr Lys Thr Lys Gly
Ala Lys Glu Leu Glu Glu Leu Phe Lys 35 40
45Ser Val Glu Ser Leu Ser Lys Ala Ala Lys Glu Ala Leu Ser Asn
Ser 50 55 60Val Lys Glu Leu Thr
Ser65 704170PRTBorrelia afzelii 41Ser Glu Ala Phe Thr
Lys Lys Leu Gln Asp Ser Asn Ala Asp Leu Gly1 5
10 15Lys His Asp Ala Thr Asp Ala Asp Ala Lys Lys
Ala Ile Leu Lys Thr 20 25
30Asp Ala Thr Lys Asp Lys Gly Ala Lys Glu Leu Glu Glu Leu Phe Lys
35 40 45Ser Val Glu Ser Leu Ser Lys Ala
Ala Lys Glu Ala Leu Ser Asn Ser 50 55
60Val Lys Glu Leu Thr Ser65 704270PRTBorrelia afzelii
42Ser Gln Asp Phe Ile Asn Lys Leu Lys Gly Gly His Ala Glu Leu Gly1
5 10 15Leu Val Ala Ala Thr Asp
Ala Asn Ala Lys Ala Ala Ile Leu Lys Thr 20 25
30Asn Gly Asp Lys Thr Lys Gly Ala Asp Glu Phe Glu Lys
Leu Phe Lys 35 40 45Ser Val Glu
Gly Leu Leu Lys Ala Ala Gln Glu Ala Leu Thr Asn Ser 50
55 60Val Lys Glu Leu Thr Ser65
704370PRTBorrelia afzelii 43Ser Gln Asp Phe Ile Asn Lys Leu Lys Gly Gly
His Ala Glu Leu Gly1 5 10
15Leu Ala Ala Ala Thr Asp Ala Asn Ala Lys Ala Ala Ile Leu Lys Thr
20 25 30Asn Gly Asp Lys Thr Lys Gly
Ala Asp Glu Phe Glu Lys Leu Phe Lys 35 40
45Ser Val Glu Gly Leu Leu Lys Ala Ala Gln Glu Ala Leu Thr Asn
Ser 50 55 60Val Lys Glu Leu Thr
Thr65 704470PRTBorrelia burgdorferi 44Ser Glu Thr Phe
Thr Asn Lys Leu Lys Glu Lys His Thr Asp Leu Gly1 5
10 15Lys Glu Gly Val Thr Asp Ala Asp Ala Lys
Glu Ala Ile Leu Lys Thr 20 25
30Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu Phe Glu
35 40 45Ser Val Glu Val Leu Ser Lys Ala
Ala Lys Glu Met Leu Ala Asn Ser 50 55
60Val Lys Glu Leu Thr Ser65 704572PRTBorrelia
burgdorferi 45Ser Ala Ala Phe Thr Lys Lys Leu Ala Asp Ser Asn Ala Asp Leu
Gly1 5 10 15Val Ala Ala
Gly Asn Ala Thr Asp Asp Asn Ala Lys Arg Ala Ile Leu 20
25 30Lys Thr His Gly His Glu Asp Lys Gly Gly
Lys Glu Leu Lys Glu Leu 35 40
45Ser Glu Ala Val Lys Ser Leu Leu Lys Ala Ala Gln Ala Ala Leu Ala 50
55 60Asn Ser Val Gln Glu Leu Thr Ser65
704670PRTBorrelia burgdorferi 46Ser Glu Asp Phe Thr Asn Lys
Leu Lys Asn Gly Asn Ala Gln Leu Gly1 5 10
15Leu Ala Ala Ala Thr Asp Asp Asn Ala Lys Ala Ala Ile
Leu Lys Thr 20 25 30Asn Gly
Thr Asn Asp Lys Gly Ala Lys Glu Leu Lys Asp Leu Ser Asp 35
40 45Ser Val Glu Ser Leu Val Lys Ala Ala Gln
Val Met Leu Thr Asn Ser 50 55 60Val
Lys Glu Leu Thr Ser65 704770PRTBorrelia burgdorferi
47Ser Thr Glu Phe Thr Asn Lys Leu Lys Ser Glu His Ala Val Leu Gly1
5 10 15Leu Asp Asn Leu Thr Asp
Asp Asn Ala Gln Arg Ala Ile Leu Lys Lys 20 25
30His Ala Asn Lys Asp Lys Gly Ala Ala Glu Leu Glu Lys
Leu Phe Lys 35 40 45Ala Val Glu
Asn Leu Ser Lys Ala Ala Gln Asp Thr Leu Lys Asn Ala 50
55 60Val Lys Glu Leu Thr Ser65
704872PRTBorrelia burgdorferi 48Asn Lys Ala Phe Thr Asp Lys Leu Lys Ser
Ser His Ala Glu Leu Gly1 5 10
15Ile Ala Asn Gly Ala Ala Thr Asp Ala Asn Ala Lys Ala Ala Ile Leu
20 25 30Lys Thr Asn Gly Thr Lys
Asp Lys Gly Ala Gln Glu Leu Glu Lys Leu 35 40
45Phe Glu Ser Val Lys Asn Leu Ser Lys Ala Ala Gln Glu Thr
Leu Asn 50 55 60Asn Ser Val Lys Glu
Leu Thr Ser65 704970PRTBorrelia burgdorferi 49Ser Glu
Ser Phe Thr Lys Lys Leu Ser Asp Asn Gln Ala Glu Leu Gly1 5
10 15Ile Glu Asn Ala Thr Asp Asp Asn
Ala Lys Lys Ala Ile Leu Lys Thr 20 25
30His Asn Ala Lys Asp Lys Gly Ala Glu Glu Leu Val Lys Leu Ser
Glu 35 40 45Ser Val Ala Gly Leu
Leu Lys Ala Ala Gln Ala Ile Leu Ala Asn Ser 50 55
60Val Lys Glu Leu Thr Ser65
705070PRTBorrelia burgdorferi 50Ser Glu Lys Phe Thr Lys Lys Leu Ser Glu
Ser His Ala Asp Ile Gly1 5 10
15Ile Gln Ala Ala Thr Asp Ala Asn Ala Lys Asp Ala Ile Leu Lys Thr
20 25 30Asn Pro Thr Lys Thr Lys
Gly Ala Glu Glu Leu Asp Lys Leu Phe Lys 35 40
45Ala Val Glu Asn Leu Ser Lys Ala Ala Lys Glu Met Leu Ala
Asn Ser 50 55 60Val Lys Asp Leu Gln
Val65 705170PRTBorrelia burgdorferi 51Ser Glu Lys Phe
Thr Lys Lys Leu Ser Glu Ser His Ala Asp Ile Gly1 5
10 15Ile Gln Ala Ala Thr Asp Ala Asn Ala Lys
Asp Ala Ile Leu Lys Thr 20 25
30Asn Pro Thr Lys Thr Lys Gly Ala Glu Glu Leu Asp Lys Leu Phe Lys
35 40 45Ala Val Glu Asn Leu Ser Lys Ala
Ala Lys Glu Met Leu Ala Asn Ser 50 55
60Val Lys Glu Leu Thr Ser65 705270PRTBorrelia
burgdorferi 52Ser Glu Glu Phe Thr Ala Lys Leu Lys Gly Glu His Thr Asp Leu
Gly1 5 10 15Lys Glu Gly
Val Thr Asp Asp Asn Ala Lys Lys Ala Ile Leu Lys Thr 20
25 30Asn Asn Asp Lys Thr Lys Gly Ala Asp Glu
Leu Glu Lys Leu Phe Glu 35 40
45Ser Val Lys Asn Leu Ser Lys Ala Ala Lys Glu Met Leu Thr Asn Ser 50
55 60Val Lys Glu Leu Thr Ser65
705370PRTBorrelia burgdorferi 53Ser Glu Lys Phe Thr Asp Lys Leu Lys
Ser Glu Asn Ala Ala Leu Gly1 5 10
15Lys Gln Asp Ala Ser Asp Asp Asp Ala Lys Lys Ala Ile Leu Lys
Thr 20 25 30His Asn Asp Ile
Thr Lys Gly Ala Lys Glu Leu Lys Glu Leu Ser Glu 35
40 45Ser Val Glu Thr Leu Leu Lys Ala Ala Lys Glu Met
Leu Ala Asn Ser 50 55 60Val Lys Glu
Leu Thr Ser65 705470PRTBorrelia burgdorferi 54Ser Glu
Lys Phe Ala Gly Lys Leu Lys Asn Glu His Ala Ser Leu Gly1 5
10 15Lys Lys Asp Ala Thr Asp Asp Asp
Ala Lys Lys Ala Ile Leu Lys Thr 20 25
30His Gly Asn Thr Asp Lys Gly Ala Lys Glu Leu Lys Asp Leu Ser
Asp 35 40 45Ser Val Glu Ser Leu
Val Lys Ala Ala Lys Glu Met Leu Thr Asn Ser 50 55
60Val Lys Glu Leu Thr Ser65
705570PRTBorrelia burgdorferi 55Ser Glu Ala Phe Thr Asp Lys Leu Lys Asn
Glu His Ala Ser Leu Gly1 5 10
15Lys Lys Asp Ala Thr Asp Asp Asp Ala Lys Lys Ala Ile Leu Lys Thr
20 25 30Asn Val Asp Lys Thr Lys
Gly Ala Asp Glu Leu Ile Lys Leu Ser Gly 35 40
45Ser Leu Glu Ser Leu Ser Lys Ala Ala Gln Ala Ile Leu Ala
Asn Ser 50 55 60Val Lys Glu Leu Thr
Ser65 705670PRTBorrelia garinii 56Ser Glu Lys Phe Thr
Thr Lys Leu Arg Asp Ser His Ala Glu Leu Gly1 5
10 15Ile Gln Asn Val Gln Asp Asp Asn Ala Lys Arg
Ala Ile Leu Lys Thr 20 25
30His Gly Asn Lys Asp Lys Gly Ala Lys Glu Leu Lys Glu Leu Ser Glu
35 40 45Ser Leu Glu Lys Leu Ser Lys Ala
Ala Gln Ala Ala Leu Ala Asn Ser 50 55
60Val Gln Glu Leu Thr Ser65 705770PRTBorrelia garinii
57Ser Glu Lys Phe Thr Thr Lys Leu Lys Asp Ser His Ala Glu Leu Gly1
5 10 15Ile Gln Ser Val Gln Asp
Asp Asn Ala Lys Lys Ala Ile Leu Lys Thr 20 25
30His Gly Thr Lys Asp Lys Gly Ala Lys Glu Leu Glu Glu
Leu Phe Lys 35 40 45Ser Leu Glu
Ser Leu Ser Lys Ala Ala Gln Ala Ala Leu Thr Asn Ser 50
55 60Val Lys Glu Leu Thr Asn65
705871PRTBorrelia burgdorferi 58Ser Glu Glu Phe Ser Thr Lys Leu Lys Asp
Asn His Ala Gln Leu Gly1 5 10
15Ile Gln Gly Val Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu Lys Ala
20 25 30Asn Ala Ala Gly Lys Asp
Lys Gly Val Glu Glu Leu Glu Lys Leu Ser 35 40
45Gly Ser Leu Glu Ser Leu Ser Lys Ala Ala Lys Glu Met Leu
Ala Asn 50 55 60Ser Val Lys Glu Leu
Thr Ser65 705971PRTBorrelia burgdorferi 59Ser Glu Glu
Phe Ser Thr Lys Leu Lys Asp Asn His Ala Gln Leu Gly1 5
10 15Ile Gln Gly Val Thr Asp Glu Asn Ala
Lys Lys Ala Ile Leu Lys Ala 20 25
30Asn Ala Ala Gly Lys Asp Lys Gly Val Glu Glu Leu Glu Lys Leu Ser
35 40 45Gly Ser Leu Glu Ser Leu Ser
Lys Ala Ala Lys Glu Met Leu Ala Asn 50 55
60Ser Val Lys Ser Leu Gln Ser65 706070PRTBorrelia
burgdorferi 60Ser Glu Asp Phe Thr Lys Lys Leu Glu Gly Glu His Ala Gln Leu
Gly1 5 10 15Ile Glu Asn
Val Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu Ile Thr 20
25 30Asp Ala Ala Lys Asp Lys Gly Ala Ala Glu
Leu Glu Lys Leu Phe Lys 35 40
45Ala Val Glu Asn Leu Ala Lys Ala Ala Lys Glu Met Leu Ala Asn Ser 50
55 60Val Lys Glu Leu Thr Ser65
706172PRTBorrelia burgdorferi 61Ser Asp Asp Phe Thr Lys Lys Leu Gln
Ser Ser His Ala Gln Leu Gly1 5 10
15Val Ala Gly Gly Ala Thr Thr Asp Glu Glu Ala Lys Lys Ala Ile
Leu 20 25 30Arg Thr Asn Ala
Ile Lys Asp Lys Gly Ala Asp Glu Leu Glu Lys Leu 35
40 45Phe Lys Ser Val Glu Ser Leu Ala Lys Ala Ala Gln
Asp Ala Leu Ala 50 55 60 Asn Ser Val
Asn Glu Leu Thr Ser65 706280PRTBorrelia burgdorferi
62Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp Leu Gly1
5 10 15Lys Glu Gly Val Thr Asp
Ala Asp Ala Lys Glu Ala Ile Leu Lys Thr 20 25
30Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys
Leu Phe Glu 35 40 45Ser Val Glu
Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala Asn Ser 50
55 60Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser
Pro Lys Lys Pro65 70 75
806380PRTBorrelia garinii 63Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys
His Thr Asp Leu Gly1 5 10
15Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu Lys Thr
20 25 30Asn Gly Thr Lys Thr Lys Gly
Ala Glu Glu Leu Gly Lys Leu Phe Glu 35 40
45Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala Asn
Ser 50 55 60Val Lys Glu Leu Thr Ser
Pro Val Val Ala Glu Ser Pro Lys Lys Pro65 70
75 806480PRTArtificialsynthetic chimeric
polypeptide 64Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp Leu
Gly1 5 10 15Lys Glu Gly
Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu Lys Thr 20
25 30Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu
Leu Gly Lys Leu Phe Glu 35 40
45Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala Asn Ser 50
55 60Val Lys Glu Leu Thr Ser Pro Val Val
Ala Glu Ser Pro Lys Lys Pro65 70 75
8065569PRTArtificialsynthetic chimeric polypeptide 65Ser Glu
Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp Leu Gly1 5
10 15Lys Glu Gly Val Thr Asp Ala Asp
Ala Lys Glu Ala Ile Leu Lys Thr 20 25
30Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu Phe
Glu 35 40 45Ser Val Glu Val Leu
Ser Lys Ala Ala Lys Glu Met Leu Ala Asn Ser 50 55
60Val Lys Glu Leu Thr Ser Ser Glu Glu Phe Ser Thr Lys Leu
Lys Asp65 70 75 80Asn
His Ala Gln Leu Gly Ile Gln Gly Val Thr Asp Glu Asn Ala Lys
85 90 95Lys Ala Ile Leu Lys Ala Asn
Ala Ala Gly Lys Asp Lys Gly Val Glu 100 105
110Glu Leu Glu Lys Leu Ser Gly Ser Leu Glu Ser Leu Ser Ser
Glu Asp 115 120 125Phe Thr Lys Lys
Leu Glu Gly Glu His Ala Gln Leu Gly Ile Glu Asn 130
135 140Val Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu Ile
Thr Asp Ala Ala145 150 155
160Lys Asp Lys Gly Ala Ala Glu Leu Glu Lys Leu Phe Lys Ala Val Glu
165 170 175Asn Leu Ala Ala Lys
Leu Lys Gly Glu His Thr Asp Leu Gly Lys Glu 180
185 190Gly Val Thr Asp Asp Asn Ala Lys Lys Ala Ile Leu
Lys Thr Asn Asn 195 200 205Asp Lys
Thr Lys Gly Ala Asp Glu Leu Glu Lys Leu Phe Glu Ser Val 210
215 220Lys Asn Leu Ser Lys Ala Ala Lys Glu Met Leu
Thr Asn Ser Ser Glu225 230 235
240Lys Phe Ala Gly Lys Leu Lys Asn Glu His Ala Ser Leu Gly Lys Lys
245 250 255Asp Ala Thr Asp
Asp Asp Ala Lys Lys Ala Ile Leu Lys Thr His Gly 260
265 270Asn Thr Asp Lys Gly Ala Lys Glu Leu Lys Asp
Leu Ser Asp Ser Val 275 280 285Glu
Ser Leu Val Ser Asp Asp Phe Thr Lys Lys Leu Gln Ser Ser His 290
295 300Ala Gln Leu Gly Val Ala Gly Gly Ala Thr
Thr Asp Glu Glu Ala Lys305 310 315
320Lys Ala Ile Leu Arg Thr Asn Ala Ile Lys Asp Lys Gly Ala Asp
Glu 325 330 335Leu Glu Lys
Leu Phe Lys Ser Val Glu Ser Leu Ala Lys Ala Ala Gln 340
345 350Asp Ala Leu Ala Asn Ser Val Asn Glu Leu
Thr Ser Lys Lys Leu Lys 355 360
365Glu Lys His Thr Asp Leu Gly Lys Lys Asp Ala Thr Asp Val His Ala 370
375 380Lys Glu Ala Ile Leu Lys Thr Asn
Gly Thr Lys Asp Lys Gly Ala Ala385 390
395 400Glu Leu Glu Lys Leu Phe Glu Ser Val Glu Asn Leu
Ala Lys Ala Ala 405 410
415Lys Glu Met Leu Ser Asn Ser Asn Lys Ala Phe Thr Asp Lys Leu Lys
420 425 430Ser Ser His Ala Glu Leu
Gly Ile Ala Asn Gly Ala Ala Thr Asp Ala 435 440
445Asn Ala Lys Ala Ala Ile Leu Lys Thr Asn Gly Thr Lys Asp
Lys Gly 450 455 460Ala Gln Glu Leu Glu
Lys Leu Phe Glu Ser Val Lys Asn Leu Ser Lys465 470
475 480Ala Ala Gln Glu Thr Leu Asn Asn Ser Ser
Glu Ser Phe Thr Lys Lys 485 490
495Leu Ser Asp Asn Gln Ala Glu Leu Gly Ile Glu Asn Ala Thr Asp Asp
500 505 510Asn Ala Lys Lys Ala
Ile Leu Lys Thr His Asn Ala Lys Asp Lys Gly 515
520 525Ala Glu Glu Leu Val Lys Leu Ser Glu Ser Val Ala
Gly Leu Leu Lys 530 535 540Ala Ala Gln
Ala Ile Leu Ala Asn Ser Val Lys Glu Leu Thr Ser Pro545
550 555 560Val Val Ala Glu Ser Pro Lys
Lys Pro 56566503PRTArtificialsynthetic chimeric
polypeptide 66Ser Glu Lys Phe Thr Thr Lys Leu Lys Asp Ser His Ala Glu Leu
Gly1 5 10 15Ile Gln Ser
Val Gln Asp Asp Asn Ala Lys Lys Ala Ile Leu Lys Thr 20
25 30His Gly Thr Lys Asp Lys Gly Ala Lys Glu
Leu Glu Glu Leu Phe Lys 35 40
45Ser Leu Glu Ser Leu Ser Lys Ala Ala Gln Ala Ala Leu Thr Asn Ser 50
55 60Val Lys Glu Leu Thr Asn Ser Asp Lys
Phe Thr Lys Lys Leu Thr Asp65 70 75
80Ser His Ala Gln Leu Gly Ala Val Gly Gly Ala Ile Asn Asp
Asp Arg 85 90 95Ala Lys
Glu Ala Ile Leu Lys Thr His Gly Thr Asn Asp Lys Gly Ala 100
105 110Lys Glu Leu Lys Glu Leu Ser Glu Ser
Val Glu Ser Leu Ala Lys Ala 115 120
125Ala Gln Ala Ala Leu Ala Asn Ser Ser Glu Ala Phe Thr Lys Lys Leu
130 135 140Lys Asp Ser Asn Ala Gln Leu
Gly Met Gln Asn Gly Ala Ala Thr Asp145 150
155 160Ala His Ala Lys Ala Ala Ile Leu Lys Thr Asp Ala
Thr Lys Asp Lys 165 170
175Gly Ala Thr Glu Leu Gly Glu Leu Phe Lys Ser Val Glu Ser Leu Ser
180 185 190Lys Ala Ala Gln Glu Ala
Ser Val Ala Phe Thr Ser Lys Leu Lys Ser 195 200
205Ser Asn Ala Gln Leu Gly Val Ala Asn Gly Asn Ala Thr Asp
Asp Asp 210 215 220Ala Lys Lys Ala Ile
Leu Lys Thr Asn Thr Pro Asn Asp Lys Gly Ala225 230
235 240Lys Glu Leu Lys Glu Leu Phe Glu Ser Val
Glu Ser Leu Ala Lys Ala 245 250
255Ala Gln Ala Ala Leu Val Asn Ser Val Gln Glu Leu Thr Asn Ser Glu
260 265 270Ala Phe Thr Asn Arg
Leu Lys Gly Ser His Ala Gln Leu Gly Val Ala 275
280 285Ala Ala Thr Asp Asp His Ala Lys Glu Ala Ile Leu
Lys Ser Asn Pro 290 295 300Thr Lys Asp
Lys Gly Ala Lys Glu Leu Lys Asp Leu Ser Glu Ser Val305
310 315 320Glu Ser Leu Ala Lys Ala Ala
Gln Glu Ala Leu Ala Asn Ser Val Lys 325
330 335Glu Leu Thr Asn Ser Glu Ala Phe Thr Lys Lys Leu
Lys Asp Asn Asn 340 345 350Ala
Gln Leu Gly Ile Gln Asn Val Gln Asp Val Glu Ala Lys Lys Ala 355
360 365Ile Leu Lys Thr Asn Gly Asp Ile Ser
Lys Ser Glu Ala Phe Thr Asn 370 375
380Lys Leu Lys Glu Lys His Ala Glu Leu Gly Val Asn Gly Gly Asp Thr385
390 395 400Thr Asp Asp Asn
Ala Lys Ala Ala Ile Phe Lys Thr His Pro Thr Lys 405
410 415Asp Lys Gly Val Glu Asp Leu Glu Lys Leu
Ser Glu Ser Val Lys Ser 420 425
430Leu Leu Lys Ala Ala Gln Ala Ala Leu Ser Asn Ser Ala Ala Phe Thr
435 440 445Lys Lys Leu Gln Asp Gly His
Val Asp Leu Gly Lys Thr Asp Val Thr 450 455
460Asp Asp Asn Ala Lys Glu Ala Ile Leu Lys Thr Asn Pro Thr Lys
Thr465 470 475 480Lys Gly
Ala Thr Glu Leu Glu Glu Leu Phe Lys Ser Val Glu Gly Leu
485 490 495Val Lys Ala Ala Lys Glu Ala
50067512PRTArtificialsynthetic chimeric polypeptide 67Ser Glu Glu
Phe Thr Asn Lys Leu Lys Ser Gly His Ala Asp Leu Gly1 5
10 15Lys Gln Asp Ala Thr Asp Asp His Ala
Lys Ala Ala Ile Leu Lys Thr 20 25
30His Ala Thr Thr Asp Lys Gly Ala Lys Glu Phe Lys Asp Leu Phe Glu
35 40 45Ser Val Glu Gly Leu Leu Lys
Ala Ala Gln Val Ala Leu Thr Asn Ser 50 55
60Val Lys Glu Leu Thr Ser Lys Leu Lys Gly Gly His Ala Glu Leu Gly65
70 75 80Leu Ala Ala Ala
Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu Lys Thr 85
90 95Asn Gly Thr Lys Asp Lys Gly Ala Glu Glu
Leu Glu Lys Leu Phe Lys 100 105
110Ser Val Glu Ser Leu Ala Lys Ala Ala Lys Glu Ser Leu Thr Asn Ser
115 120 125Val Lys Glu Leu Thr Asn Thr
Lys Leu Arg Asp Ser His Ala Glu Leu 130 135
140Gly Ile Gln Asn Val Gln Asp Asp Asn Ala Lys Arg Ala Ile Leu
Lys145 150 155 160Thr His
Gly Asn Lys Asp Lys Gly Ala Lys Glu Leu Lys Glu Leu Ser
165 170 175Glu Ser Leu Glu Lys Leu Ser
Lys Ala Ala Gln Ala Ala Leu Ala Asn 180 185
190Ser Val Gln Glu Leu Thr Ser Ser Glu Ala Phe Thr Asn Lys
Leu Lys 195 200 205Glu Lys Thr Gln
Glu Leu Ala Val Ala Ala Gly Ala Ala Thr Asp Ile 210
215 220Asp Ala Lys Lys Ala Ile Leu Lys Thr Asn Arg Asp
Lys Asp Leu Gly225 230 235
240Ala Asp Glu Arg Gly Lys Leu Phe Lys Ser Val Glu Ser Leu Ser Lys
245 250 255Ala Ala Gln Glu Ala
Ser Ala Asn Ser Val Lys Glu Leu Thr Ser Ser 260
265 270Glu Ala Phe Thr Asp Lys Leu Lys Asn Glu His Ala
Ser Leu Gly Lys 275 280 285Lys Asp
Ala Thr Asp Asp Asp Ala Lys Lys Ala Ile Leu Lys Thr Asn 290
295 300Val Asp Lys Thr Lys Gly Ala Asp Glu Leu Ile
Lys Leu Ser Gly Ser305 310 315
320Leu Glu Ser Leu Ser Lys Ala Ala Gln Ala Ile Leu Ala Asn Ser Glu
325 330 335Ala Phe Thr Lys
Lys Leu Gln Asp Ser Asn Ala Asp Leu Gly Lys His 340
345 350Asn Ala Thr Asp Ala Asp Ser Lys Glu Ala Ile
Leu Lys Thr Asn Gly 355 360 365Thr
Lys Thr Lys Gly Ala Lys Glu Leu Glu Glu Leu Phe Lys Ser Val 370
375 380Glu Ser Leu Ser Lys Ala Ala Lys Glu Ala
Leu Ser Asn Ser Val Lys385 390 395
400Glu Leu Thr Ser Ser Gln Asp Phe Ile Asn Lys Leu Lys Gly Gly
His 405 410 415Ala Glu Leu
Gly Leu Val Ala Ala Thr Asp Ala Asn Ala Lys Ala Ala 420
425 430Ile Leu Lys Thr Asn Gly Asp Lys Thr Lys
Gly Ala Asp Glu Phe Glu 435 440
445Lys Leu Phe Lys Ser Val Glu Gly Leu Leu Lys Ala Ala Gln Glu Ala 450
455 460Leu Thr Asn Ser Val Lys Glu Leu
Thr Ser Ser Glu Ala Phe Thr Lys465 470
475 480Lys Leu Gln Asp Ser Asn Ala Asp Leu Gly Lys His
Asp Ala Thr Asp 485 490
495Ala Asp Ala Lys Lys Ala Ile Leu Lys Thr Asp Ala Thr Lys Asp Lys
500 505
51068431PRTArtificialsynthetic chimeric polypeptide 68Ser Glu Thr Phe Thr
Asn Lys Leu Lys Glu Lys His Thr Asp Leu Gly1 5
10 15Lys Glu Gly Val Thr Lys Gly Ala Glu Glu Leu
Gly Lys Leu Phe Glu 20 25
30Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala Asn Ser
35 40 45Val Lys Glu Leu Thr Ser Ser Glu
Glu Phe Ser Thr Lys Leu Lys Asp 50 55
60Asn His Ala Gln Leu Gly Ile Gln Gly Val Thr Lys Gly Val Glu Glu65
70 75 80Leu Glu Lys Leu Ser
Gly Ser Leu Glu Ser Leu Ser Ser Glu Asp Phe 85
90 95Thr Lys Lys Leu Glu Gly Glu His Ala Gln Leu
Gly Ile Glu Asn Val 100 105
110Thr Ala Ala Glu Leu Glu Lys Leu Phe Lys Ala Val Glu Asn Leu Ala
115 120 125Lys Ala Ala Lys Glu Met Ala
Lys Leu Lys Gly Glu His Thr Asp Leu 130 135
140Gly Lys Glu Gly Val Thr Lys Gly Ala Asp Glu Leu Glu Lys Leu
Phe145 150 155 160Glu Ser
Val Lys Asn Leu Ser Lys Ala Ala Lys Glu Met Leu Thr Asn
165 170 175Ser Lys Glu Ser Glu Lys Phe
Ala Gly Lys Leu Lys Asn Glu His Ala 180 185
190Ser Leu Gly Lys Lys Asp Ala Thr Lys Gly Ala Lys Glu Leu
Lys Asp 195 200 205Leu Ser Asp Ser
Val Glu Ser Leu Val Lys Ala Ser Asp Asp Phe Thr 210
215 220Lys Lys Leu Gln Ser Ser His Ala Gln Leu Gly Val
Ala Gly Gly Ala225 230 235
240Thr Thr Ala Asp Glu Leu Glu Lys Leu Phe Lys Ser Val Glu Ser Leu
245 250 255Ala Lys Ala Ala Gln
Asp Ala Leu Ala Asn Ser Val Asn Glu Leu Thr 260
265 270Ser Lys Lys Leu Lys Glu Lys His Thr Asp Leu Gly
Lys Lys Asp Ala 275 280 285Thr Ala
Ala Glu Leu Glu Lys Leu Phe Glu Ser Val Glu Asn Leu Ala 290
295 300Lys Ala Ala Lys Glu Met Leu Ser Asn Ser Asn
Lys Ala Phe Thr Asp305 310 315
320Lys Leu Lys Ser Ser His Ala Glu Leu Gly Ile Ala Asn Gly Ala Ala
325 330 335Thr Lys Gly Ala
Gln Glu Leu Glu Lys Leu Phe Glu Ser Val Lys Asn 340
345 350Leu Ser Lys Ala Ala Gln Glu Thr Leu Asn Asn
Ser Val Lys Glu Ser 355 360 365Glu
Ser Phe Thr Lys Lys Leu Ser Asp Asn Gln Ala Glu Leu Gly Ile 370
375 380Glu Asn Ala Thr Lys Gly Ala Glu Glu Leu
Val Lys Leu Ser Glu Ser385 390 395
400Val Ala Gly Leu Leu Lys Ala Ala Gln Ala Ile Leu Ala Asn Ser
Val 405 410 415Lys Glu Leu
Thr Ser Pro Val Val Ala Glu Ser Pro Lys Lys Pro 420
425 43069381PRTArtificialsynthetic chimeric
polypeptide 69Ser Glu Lys Phe Thr Thr Lys Leu Lys Asp Ser His Ala Glu Leu
Gly1 5 10 15Ile Gln Ser
Val Gln Asp Lys Gly Ala Lys Glu Leu Glu Glu Leu Phe 20
25 30Lys Ser Leu Glu Ser Leu Ser Lys Ala Ala
Gln Ala Ala Leu Thr Asn 35 40
45Ser Val Lys Glu Leu Thr Asn Ser Asp Lys Phe Thr Lys Lys Leu Thr 50
55 60Asp Ser His Ala Gln Leu Gly Ala Val
Gly Gly Ala Ile Asn Asp Lys65 70 75
80Gly Ala Lys Glu Leu Lys Glu Leu Ser Glu Ser Val Glu Ser
Leu Ala 85 90 95Lys Ala
Ala Gln Ala Ala Leu Ala Asn Ser Ser Glu Ala Phe Thr Lys 100
105 110Lys Leu Lys Asp Ser Asn Ala Gln Leu
Gly Met Gln Asn Gly Ala Ala 115 120
125Thr Asp Lys Gly Ala Thr Glu Leu Gly Glu Leu Phe Lys Ser Val Glu
130 135 140Ser Leu Ser Lys Ala Ala Gln
Glu Ala Ser Val Ala Phe Thr Ser Lys145 150
155 160Leu Lys Ser Ser Asn Ala Gln Leu Gly Val Ala Asn
Gly Asn Ala Thr 165 170
175Asp Lys Gly Ala Lys Glu Leu Lys Glu Leu Phe Glu Ser Val Glu Ser
180 185 190Leu Ala Lys Ala Ala Gln
Ala Ala Leu Val Asn Ser Val Gln Glu Leu 195 200
205Thr Asn Ser Glu Ala Phe Thr Asn Arg Leu Lys Gly Ser His
Ala Gln 210 215 220Leu Gly Val Ala Ala
Ala Thr Asp Lys Gly Ala Lys Glu Leu Lys Asp225 230
235 240Leu Ser Glu Ser Val Glu Ser Leu Ala Lys
Ala Ala Gln Glu Ala Leu 245 250
255Ala Asn Ser Val Lys Glu Leu Thr Asn Ser Glu Ala Phe Thr Lys Lys
260 265 270Leu Lys Asp Asn Asn
Ala Gln Leu Gly Ile Gln Asn Val Gln Ser Glu 275
280 285Ala Phe Thr Asn Lys Leu Lys Glu Lys His Ala Glu
Leu Gly Val Asn 290 295 300Gly Gly Asp
Thr Thr Asp Lys Gly Val Glu Asp Leu Glu Lys Leu Ser305
310 315 320Glu Ser Val Lys Ser Leu Leu
Lys Ala Ala Gln Ala Ala Leu Ser Asn 325
330 335Ser Ala Ala Phe Thr Lys Lys Leu Gln Asp Gly His
Val Asp Leu Gly 340 345 350Lys
Thr Asp Val Thr Thr Lys Gly Ala Thr Glu Leu Glu Glu Leu Phe 355
360 365Lys Ser Val Glu Gly Leu Val Lys Ala
Ala Lys Glu Ala 370 375
38070383PRTArtificialsynthetic chimeric polypeptide 70Ser Glu Glu Phe Thr
Asn Lys Leu Lys Ser Gly His Ala Asp Leu Gly1 5
10 15Lys Gln Asp Ala Thr Lys Gly Ala Lys Glu Phe
Lys Asp Leu Phe Glu 20 25
30Ser Val Glu Gly Leu Leu Lys Ala Ala Gln Val Ala Leu Thr Asn Ser
35 40 45Val Lys Glu Leu Thr Ser Lys Leu
Lys Gly Gly His Ala Glu Leu Gly 50 55
60Leu Ala Ala Ala Thr Lys Gly Ala Glu Glu Leu Glu Lys Leu Phe Lys65
70 75 80Ser Val Glu Ser Leu
Ala Lys Ala Ala Lys Glu Ser Leu Thr Asn Ser 85
90 95Val Lys Glu Leu Thr Asn Thr Lys Leu Arg Asp
Ser His Ala Glu Leu 100 105
110Gly Ile Gln Asn Val Gln Lys Gly Ala Lys Glu Leu Lys Glu Leu Ser
115 120 125Glu Ser Leu Glu Lys Leu Ser
Lys Ala Ala Gln Ala Ala Leu Ala Asn 130 135
140Ser Val Gln Glu Leu Thr Ser Ser Glu Ala Phe Thr Asn Lys Leu
Lys145 150 155 160Glu Lys
Thr Gln Glu Leu Ala Val Ala Ala Gly Ala Ala Thr Leu Gly
165 170 175Ala Asp Glu Arg Gly Lys Leu
Phe Lys Ser Val Glu Ser Leu Ser Lys 180 185
190Ala Ala Gln Glu Ala Ser Ala Asn Ser Val Lys Glu Leu Thr
Ser Ser 195 200 205Glu Ala Phe Thr
Asp Lys Leu Lys Asn Glu His Ala Ser Leu Gly Lys 210
215 220Lys Asp Ala Thr Lys Gly Ala Asp Glu Leu Ile Lys
Leu Ser Gly Ser225 230 235
240Leu Glu Ser Leu Ser Lys Ala Ala Gln Ala Ile Leu Ala Asn Ser Glu
245 250 255Ala Phe Thr Lys Lys
Leu Gln Asp Ser Asn Ala Asp Leu Gly Lys His 260
265 270Asn Ala Thr Lys Gly Ala Lys Glu Leu Glu Glu Leu
Phe Lys Ser Val 275 280 285Glu Ser
Leu Ser Lys Ala Ala Lys Glu Ala Leu Ser Asn Ser Val Lys 290
295 300Glu Leu Thr Ser Ser Gln Asp Phe Ile Asn Lys
Leu Lys Gly Gly His305 310 315
320Ala Glu Leu Gly Leu Val Ala Ala Thr Lys Gly Ala Asp Glu Phe Glu
325 330 335Lys Leu Phe Lys
Ser Val Glu Gly Leu Leu Lys Ala Ala Gln Glu Ala 340
345 350Leu Thr Asn Ser Val Lys Glu Leu Thr Ser Ser
Glu Ala Phe Thr Lys 355 360 365Lys
Leu Gln Asp Ser Asn Ala Asp Leu Gly Lys His Asp Ala Thr 370
375 38071432PRTArtificialsynthetic chimeric
polypeptide 71Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp Leu
Gly1 5 10 15Lys Glu Gly
Val Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu Phe Glu 20
25 30Ser Val Glu Val Leu Ser Lys Ala Ala Lys
Glu Met Leu Ala Asn Ser 35 40
45Val Lys Glu Leu Thr Ser Lys Gly Val Glu Glu Leu Glu Lys Leu Ser 50
55 60Gly Ser Leu Glu Ser Leu Ser Asn Lys
Ala Phe Thr Asp Lys Leu Lys65 70 75
80Ser Ser His Ala Glu Leu Gly Ile Ala Asn Gly Ala Ala Thr
Lys Lys 85 90 95Leu Lys
Glu Lys His Thr Asp Leu Gly Lys Lys Asp Ala Thr Lys Gly 100
105 110Ala Asp Glu Leu Glu Lys Leu Phe Glu
Ser Val Lys Asn Leu Ser Lys 115 120
125Ala Ala Lys Glu Met Leu Thr Asn Ser Lys Glu Ile Ala Ala Glu Leu
130 135 140Glu Lys Leu Phe Lys Ala Val
Glu Asn Leu Ala Lys Ala Ala Lys Glu145 150
155 160Met Ala Lys Leu Lys Gly Glu His Thr Asp Leu Gly
Lys Glu Gly Val 165 170
175Thr Ser Glu Glu Phe Ser Thr Lys Leu Lys Asp Asn His Ala Gln Leu
180 185 190Gly Ile Gln Gly Val Thr
Lys Gly Ala Lys Glu Leu Lys Asp Leu Ser 195 200
205Asp Ser Val Glu Ser Leu Val Lys Ala Ala Ala Glu Leu Glu
Lys Leu 210 215 220Phe Glu Ser Val Glu
Asn Leu Ala Lys Ala Ala Lys Glu Met Leu Ser225 230
235 240Asn Ser Ser Glu Lys Phe Ala Gly Lys Leu
Lys Asn Glu His Ala Ser 245 250
255Leu Gly Lys Lys Asp Ala Thr Ser Glu Asp Phe Thr Lys Lys Leu Glu
260 265 270Gly Glu His Ala Gln
Leu Gly Ile Glu Asn Val Thr Lys Gly Ala Gln 275
280 285Glu Leu Glu Lys Leu Phe Glu Ser Val Lys Asn Leu
Ser Lys Ala Ala 290 295 300Gln Glu Thr
Leu Asn Asn Ser Val Lys Glu Ala Asp Glu Leu Glu Lys305
310 315 320Leu Phe Lys Ser Val Glu Ser
Leu Ala Lys Ala Ala Gln Asp Ala Leu 325
330 335Ala Asn Ser Val Asn Glu Leu Thr Ser Ser Glu Ser
Phe Thr Lys Lys 340 345 350Leu
Ser Asp Asn Gln Ala Glu Leu Gly Ile Glu Asn Ala Thr Ser Asp 355
360 365Asp Phe Thr Lys Lys Leu Gln Ser Ser
His Ala Gln Leu Gly Val Ala 370 375
380Gly Gly Ala Thr Thr Lys Gly Ala Glu Glu Leu Val Lys Leu Ser Glu385
390 395 400Ser Val Ala Gly
Leu Leu Lys Ala Ala Gln Ala Ile Leu Ala Asn Ser 405
410 415Val Lys Glu Leu Thr Ser Pro Val Val Ala
Glu Ser Pro Lys Lys Pro 420 425
43072382PRTArtificialsynthetic chimeric polypeptide 72Ser Glu Ala Phe
Thr Lys Lys Leu Lys Asp Ser Asn Ala Gln Leu Gly1 5
10 15Met Gln Asn Gly Ala Ala Thr Asp Lys Gly
Ala Lys Glu Leu Glu Glu 20 25
30Leu Phe Lys Ser Leu Glu Ser Leu Ser Lys Ala Ala Gln Ala Ala Leu
35 40 45Thr Asn Ser Val Lys Glu Leu Thr
Asn Lys Asp Lys Gly Ala Lys Glu 50 55
60Leu Lys Glu Leu Phe Glu Ser Val Glu Ser Leu Ala Lys Ala Ala Gln65
70 75 80Ala Ala Leu Val Asn
Ser Val Gln Glu Leu Thr Asn Ser Glu Lys Phe 85
90 95Thr Thr Lys Leu Lys Asp Ser His Ala Glu Leu
Gly Ile Gln Ser Val 100 105
110Gln Ser Asp Lys Phe Thr Lys Lys Leu Thr Asp Ser His Ala Gln Leu
115 120 125Gly Ala Val Gly Gly Ala Ile
Asn Asp Lys Gly Ala Lys Glu Leu Lys 130 135
140Glu Leu Ser Glu Ser Val Glu Ser Leu Ala Lys Ala Ala Gln Ala
Ala145 150 155 160Leu Ala
Asn Ser Asp Lys Gly Ala Lys Glu Leu Lys Asp Leu Ser Glu
165 170 175Ser Val Glu Ser Leu Ala Lys
Ala Ala Gln Glu Ala Leu Ala Asn Ser 180 185
190Val Lys Glu Leu Thr Asn Ser Val Ala Phe Thr Ser Lys Leu
Lys Ser 195 200 205Ser Asn Ala Gln
Leu Gly Val Ala Asn Gly Asn Ala Thr Ser Glu Ala 210
215 220Phe Thr Lys Lys Leu Lys Asp Asn Asn Ala Gln Leu
Gly Ile Gln Asn225 230 235
240Val Gln Thr Lys Gly Ala Thr Glu Leu Glu Glu Leu Phe Lys Ser Val
245 250 255Glu Gly Leu Val Lys
Ala Ala Lys Glu Ala Asp Lys Gly Val Glu Asp 260
265 270Leu Glu Lys Leu Ser Glu Ser Val Lys Ser Leu Leu
Lys Ala Ala Gln 275 280 285Ala Ala
Leu Ser Asn Ser Ala Ala Phe Thr Lys Lys Leu Gln Asp Gly 290
295 300His Val Asp Leu Gly Lys Thr Asp Val Thr Ser
Glu Ala Phe Thr Asn305 310 315
320Arg Leu Lys Gly Ser His Ala Gln Leu Gly Val Ala Ala Ala Thr Asp
325 330 335Lys Gly Ala Thr
Glu Leu Gly Glu Leu Phe Lys Ser Val Glu Ser Leu 340
345 350Ser Lys Ala Ala Gln Glu Ala Ser Glu Ala Phe
Thr Asn Lys Leu Lys 355 360 365Glu
Lys His Ala Glu Leu Gly Val Asn Gly Gly Asp Thr Thr 370
375 38073383PRTArtificialsynthetic chimeric polypeptide
73Ser Glu Glu Phe Thr Asn Lys Leu Lys Ser Gly His Ala Asp Leu Gly1
5 10 15Lys Gln Asp Ala Thr Lys
Gly Ala Lys Glu Phe Lys Asp Leu Phe Glu 20 25
30Ser Val Glu Gly Leu Leu Lys Ala Ala Gln Val Ala Leu
Thr Asn Ser 35 40 45Val Lys Glu
Leu Thr Ser Lys Glu Lys Gly Ala Glu Glu Leu Glu Lys 50
55 60Leu Phe Lys Ser Val Glu Ser Leu Ala Lys Ala Ala
Lys Glu Ser Leu65 70 75
80Thr Asn Ser Val Lys Glu Leu Thr Asn Ser Glu Ala Phe Thr Asp Lys
85 90 95Leu Lys Asn Glu His Ala
Ser Leu Gly Lys Lys Asp Ala Thr Thr Lys 100
105 110Leu Arg Asp Ser His Ala Glu Leu Gly Ile Gln Asn
Val Gln Leu Gly 115 120 125Ala Asp
Glu Arg Gly Lys Leu Phe Lys Ser Val Glu Ser Leu Ser Lys 130
135 140Ala Ala Gln Glu Ala Ser Ala Asn Ser Val Lys
Glu Leu Thr Ser Lys145 150 155
160Glu Lys Gly Ala Lys Glu Leu Glu Glu Leu Phe Lys Ser Val Glu Ser
165 170 175Leu Ser Lys Ala
Ala Lys Glu Ala Leu Ser Asn Ser Val Lys Glu Leu 180
185 190Thr Ser Ser Glu Ala Phe Thr Lys Lys Leu Gln
Asp Ser Asn Ala Asp 195 200 205Leu
Gly Lys His Asn Ala Thr Ser Glu Ala Phe Thr Lys Lys Leu Gln 210
215 220Asp Ser Asn Ala Asp Leu Gly Lys His Asp
Ala Thr Lys Gly Ala Asp225 230 235
240Glu Phe Glu Lys Leu Phe Lys Ser Val Glu Gly Leu Leu Lys Ala
Ala 245 250 255Gln Glu Ala
Leu Thr Asn Ser Val Lys Glu Leu Thr Ser Glu Leu Lys 260
265 270Glu Leu Ser Glu Ser Leu Glu Lys Leu Ser
Lys Ala Ala Gln Ala Ala 275 280
285Leu Ala Asn Ser Val Gln Glu Leu Thr Ser Ser Glu Ala Phe Thr Asn 290
295 300Lys Leu Lys Glu Lys Thr Gln Glu
Leu Ala Val Ala Ala Gly Ala Ala305 310
315 320Thr Lys Leu Lys Gly Gly His Ala Glu Leu Gly Leu
Ala Ala Ala Thr 325 330
335Lys Gly Ala Asp Glu Leu Ile Lys Leu Ser Gly Ser Leu Glu Ser Leu
340 345 350Ser Lys Ala Ala Gln Ala
Ile Leu Ala Asn Ser Gln Asp Phe Ile Asn 355 360
365Lys Leu Lys Gly Gly His Ala Glu Leu Gly Leu Val Ala Ala
Thr 370 375 38074257PRTBorrelia
burgdorferi 74Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser
Val1 5 10 15Asp Leu Pro
Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 20
25 30Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val
Asp Lys Leu Glu Leu Lys 35 40
45Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 50
55 60Ala Asp Lys Ser Lys Val Lys Leu Thr
Ile Ser Asp Asp Leu Gly Gln65 70 75
80Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val
Ser Lys 85 90 95Lys Val
Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 100
105 110Lys Gly Glu Val Ser Glu Lys Ile Ile
Thr Arg Ala Asp Gly Thr Arg 115 120
125Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu
130 135 140Val Leu Lys Gly Tyr Val Leu
Glu Gly Thr Leu Thr Ala Glu Lys Thr145 150
155 160Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser
Lys Asn Ile Ser 165 170
175Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala
180 185 190Ala Thr Lys Lys Thr Ala
Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 195 200
205Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr
Lys Glu 210 215 220Asn Thr Ile Thr Val
Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu225 230
235 240Gly Ser Ala Val Glu Ile Thr Lys Leu Asp
Glu Ile Lys Asn Ala Leu 245 250
255Lys75257PRTBorrelia burgdorferi 75Cys Lys Gln Asn Val Ser Ser Leu
Asp Glu Lys Asn Ser Val Ser Val1 5 10
15Asp Leu Pro Gly Glu Met Asn Val Leu Val Ser Lys Glu Lys
Asn Lys 20 25 30Asp Gly Lys
Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 35
40 45Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val
Leu Glu Gly Val Lys 50 55 60Ala Asp
Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln65
70 75 80Thr Thr Leu Glu Val Phe Lys
Glu Asp Gly Lys Thr Leu Val Ser Lys 85 90
95Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys
Phe Asn Glu 100 105 110Lys Gly
Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 115
120 125Leu Glu Tyr Thr Glu Ile Lys Ser Asp Gly
Ser Gly Lys Ala Lys Glu 130 135 140Val
Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr145
150 155 160Thr Leu Val Val Lys Glu
Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 165
170 175Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr
Asp Ser Ser Ala 180 185 190Ala
Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 195
200 205Ile Thr Val Asn Ser Lys Lys Thr Lys
Asp Leu Val Phe Thr Lys Glu 210 215
220Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu225
230 235 240Gly Ser Ala Val
Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 245
250 255Lys76257PRTBorrelia burgdorferi 76Cys Lys
Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val1 5
10 15Asp Leu Pro Gly Glu Met Lys Val
Leu Val Ser Lys Glu Lys Asn Lys 20 25
30Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu
Lys 35 40 45Gly Thr Ser Asp Lys
Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 50 55
60Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu
Gly Gln65 70 75 80Thr
Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys
85 90 95Lys Val Thr Ser Lys Asp Lys
Ser Ser Thr Glu Glu Lys Phe Asn Glu 100 105
110Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly
Thr Arg 115 120 125Leu Glu Tyr Thr
Glu Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 130
135 140Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr
Ala Glu Lys Thr145 150 155
160Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser
165 170 175Lys Ser Gly Glu Val
Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 180
185 190Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr
Ser Thr Leu Thr 195 200 205Ile Thr
Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 210
215 220Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn
Gly Thr Lys Leu Glu225 230 235
240Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu
245 250 255Lys
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