CN101374858A - Polyvalent chimeric OSPC vaccinogen and diagnostic antigen - Google Patents

Abstract

A chimeric polyvalent recombinant protein for use as a vaccine and diagnostic for Lyme disease is provided. The chimeric protein comprises epitopes of the loop 5 region and/or the alpha helix 5 region of outer surface protein C (OspC) types. The OspC types may be associated with mammalian Borrelia infections.

Description

Polyvalent chimeric OSPC vaccinogen and diagnostic antigen

Background of invention

Invention field

Generally speaking, the present invention relates to vaccine and the diagnosis of Lai Mushi disease.Specifically, the invention provides chimeric polyvalent recombinant protein, ring 5th district that it contains the outer surface protein relevant with mammalian infections (OspC) C type and/or the immunodominant epitope of α spiral 5 districts/structural domain.

Background of invention

Lyme disease (Lyme disease) is the modal arthropod-borne disease in North America and Europe.It is caused by spirochete Borrelia burgdoyferi (Borrelia burgdorferi), Borrelia garinii (B.garinii) and Erichsen burgdorferi (B.afzelii).Mammiferous propagate through that infected hard tick belongs to tick bite generation [Burgdorfer etc., 1982, Benach etc., 1983].Lai Mushi disease has quite high sickness rate, in the certain areas of US and European, has every year nearly 3% population to be infected [Fahrer etc., 1991].Infection has caused the inflammatory diseases of multisystem, and its early symptom may comprise migratory erythema, low fever, arthrodynia, myalgia and headache [Steere etc., 1977a].Clinical manifestation in late period can be serious, and part may comprise sacroiliitis [Steere etc., 1977a; Eiffert etc., 1998; Steere etc., 2004], carditis [Asch etc., 1994; Nagi etc., 1996; Barthold etc., 1991] and nervosa complication [Nachman and Pontrelli, 2003; Coyle and Schutzer 2002].In addition, Lyme disease has significant society-Financial cost, shows as owing to worrying that contact tick reduces out-of-home entertainment and social activity.

Pharmacoeconomics research shows, ImuLyme is existed to clear and definite demand, particularly [Meltzer etc., 1999 in annual onset risk surpasses 1% crowd; Shadick etc., 2001].But, also do not have at present business-like vaccine to use.First mankind's ImuLyme is the LYMErix (GlaxoSmithKline) based on OspA; But its usage period is short, cause selling decline, in 2002, automatically withdrawn from market.The decline of selling can be traced back to the real or sensorial misgivings to possible side effect, and these side effects are included in the receptor of the HLA-DR4 positive contingent chronic inflammatory arthritis [Kalish etc., 1993] in theory.Although developing the new vaccinogen based on OspA to alleviate this potential complication [Koide etc., 2005; Willett etc., 2004], but still exist about the problem of the viability of the vaccine based on OspA.Misgivings are the frequencies that keep the needed reinforcement of long-term protection.OspA expresses in the middle intestines of tick, after tick feed, is lowered fast, does not express [Gilmore etc., 2001 in Mammals; Schwan etc., 1995].The mechanism of action of the vaccine based on OspA is the spirochete in target tick and prevents their propagation [de Silva etc., 1999].Because propagation occurs in 48 hours of tick feed, therefore effectively protection depends on the height circulation titre of anti-OspA antibody, and this just needs frequent reinforcement.The intrinsic problem that vaccine based on OspA relates to can be avoided by using at the antigen that infects early stage high level expression and cause bactericidal properties antibody.

In ImuLyme exploitation, OspC has received suitable concern.It is the lipoprotein [Fuchs etc., 1992] that the surface of 22kDa exposes, ubiquitous 26kb cyclic plasmid coding [Marconi etc., 1993 in the chorista at Borrelia burgdoyferi (B.burgdorferisensu lato) mixture; Sadziene etc., 1993].Its expression is induced while being taken food by tick, and at mammalian infections, is maintained in early days [Schwan, 2004], and between period of infection inheritance stability [Hodzic etc., 2000; Stevenson etc., 1994].Anti-OspC antibody has been proved to be has provide protection to infecting, but only for strain [Gilmore etc., 1996 of expressing in sequence with the closely-related OspC of vaccinogen; Bockenstedt etc., 1997; Gilmore and Mbow, 1999; Mathiesen etc., 1998; Scheiblhofer etc., 2003; Jobe etc., 2003; Rousselle etc., 1998; Wallich etc., 2001; Mbow etc., 1999; Probert etc., 1997; Brown etc., 2005; Probert and LeFebvre 1994].The sequential analysis of OspC has been depicted to about 21 OspC germline groups or type, by letter designation, distinguished (from A to U) [Seinost etc., 1999; Wang etc., 1999].Although the variation of sequence is generally less than 2% in group, between different OspC types, it may be up to 22%[Wang etc., 1999; Theisen etc., 1995; Brisson and Dykhuizen, 2004].The change of epi-position between this type is most possible explains that to use single OspC type to carry out the protection domain that immunization can bear limited.

The United States Patent (USP) 6 of Dunn and Luff, 248, describe the chimeric burgdorferi albumen being comprised of at least two polypeptide 562 (June 19 calendar year 2001), these polypeptide are from the accordingly and/or corresponding albumen of same and/or different burgdorferi kind.Mix the chimeric polyeptides of chimeric protein from any burgdorferi albumen of any burgdorferi bacterial strain, comprise OspA, OspB, OspC, OspD, p12, p39, p41, p66 and p93.Chimeric protein can be used as immune diagnostic reagent, and as the vaccine immunogens for borrelia infection.But there is no ring 5 and α 5 epi-positions in reference and OspC albumen.

The United States Patent (USP) 6,872,550 and 6,486,130 of Livey (being respectively on March 29th, 2005 and on November 26th, 2002) has been described the construct of the vaccine of the Lyme disease that contains OspC antigen as antagonism.But, in these patents, do not mention ring 5 and α 5 epi-positions.

The United States Patent (USP) 7,008,625 of Dattwyler etc. (on March 7th, 2006) discloses the antigenic polypeptide of in single albumen multiple different burgdorferi bacterial strains and/or albumen.Chimeric burgdorferi albumen consists of the polypeptide fragment of outer surface protein OspA and outer surface protein OspC.These albumen can be effectively for lime borreliosis and for immune diagnostic reagent.But, do not mention ring 5 and the feature of α 5 epi-positions.

Publication " for diagnosing the chimeric burgdorferi albumen of restructuring (RecombinantChimeric Borrelia Proteins for Diagnosis of Lyme Disease) of Lyme disease " (Maria J.C.Gomes-Solecki etc., 2000.J.Clin.Microbiol., 38:2530-2535) relate to two above-described patents.Author's engineering has been manufactured restructuring mosaic, a part of antigenic protein OspA, the OspB that each contains Borrelia burgdoyferi key, OspC, flagellin (Fla or p41) and albumen p93.This piece of paper relates to diagnosis, but described in the application aspect immunogen in finishing paragraph.The hereditary variability that author has mentioned by the important epi-position of further research can produce better mosaic, but does not mention ring 5 and α 5 epi-positions of OspC.

Position up till now, prior art fails to provide the vaccine that extensive protection is provided for multiple OspC type, for preventing and/or treating Lyme disease.

Invention summary

The invention provides as the vaccine of Lyme disease and the chimeric polyvalent recombinant protein of diagnosis.

The new protective epitope of the present invention's part based on to from several different OspC germline groups (type) discovery and character, these epi-positions each for example, infect relevant with Mammals (mankind) Lyme disease.The evaluation of these epi-positions makes to build the chimeric protein contain from a plurality of epi-positions of different OspC infection types becomes possibility.Therefore,, when when the vaccine, chimeric recombinant protein causes for the protection widely of expressing these OspC types a plurality of burgdorferi bacterial strains relevant to Mammals Lyme disease.In addition, chimeric protein can be used as diagnostic tool and has the individuality for the antibody of epi-position to differentiate, thereby and determines whether individuality has been exposed to or has been subject to the infection of lyme bacteria body.In certain embodiments of the invention, epi-position is B cell epitope and/or immunodominant epitope.

The object of this invention is to provide and contain from the two chimeric recombinant protein of ring 5th district of two or more outer surface protein C (OspC) type or the epi-position in α spiral 5th district or its.In one embodiment, OspC type is selected from 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 one embodiment, chimeric recombinant protein contains the epi-position from OspC type A, B, K and D.In another embodiment, chimeric recombinant protein contains the epi-position from OspC type E, N, I, C, A, B, K and D.In another embodiment, chimeric recombinant protein has the one-level aminoacid sequence showing in SEQID NO:75 or SEQ ID NO:249.In certain embodiments, OspC type is relevant with aggressive borrelia infection.

The present invention also provides and in the individuality of needs, has caused the method for the immunne response of burgdorferi.The method has comprised the step of using chimeric recombinant protein to individuality, this chimeric recombinant protein contain from ring 5th district of two or more outer surface protein C (OspC) type or the epi-position in α spiral 5th district or its two.In one embodiment of the invention, OspC type is selected from 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 one embodiment of the invention, chimeric recombinant protein contains the epi-position from OspC type A, B, K and D.In another embodiment, chimeric recombinant protein contains the epi-position from OspC type E, N, I, C, A, B, K and D.In another embodiment, chimeric recombinant protein has the one-level aminoacid sequence showing in SEQ ID NO:75 or SEQ IDNO:249.In certain embodiments, OspC type is relevant with aggressive borrelia infection.

The present invention also provides definite individuality whether to be exposed to or to be subject to the method for the infection of burgdorferi.The method comprising the steps of 1) from individuality, obtain biological sample; 2) biological sample is exposed to at least one restructuring chimeric protein, wherein at least one chimeric protein contains from ring 5th district of two or more outer surface protein C (OspC) type or the epi-position in α spiral 5th district or the two; And 3) determine whether antibody is combined with at least one chimeric protein in described biological sample, wherein the detection of antibodies is exposed to or is subject to the infection of burgdorferi before having indicated.In one embodiment of the invention, OspC type is selected from 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 one embodiment of the invention, chimeric recombinant protein contains the epi-position from OspC type A, B, K and D.In another embodiment of the invention, chimeric recombinant protein contains the epi-position from OspC type E, N, I, C, A, B, K and D.In another embodiment of the invention, chimeric recombinant protein has the one-level aminoacid sequence showing in SEQ ID NO:75 or SEQ ID NO:249.In certain embodiments, OspC type is relevant with aggressive borrelia infection.

The present invention also provides for ring 5th district or the epi-position in α spiral 5th district or the antibody of the chimeric recombinant protein of the two that contain from two or more outer surface protein C (OspC) type.In one embodiment of the invention, OspC type is selected from 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 one embodiment, chimeric recombinant protein contains the epi-position from OspC type A, B, K and D.In another embodiment, chimeric recombinant protein contains the epi-position from OspC type E, N, I, C, A, B, K and D.In another embodiment, chimeric recombinant protein has the one-level aminoacid sequence showing in SEQ ID NO:75 or SEQ ID NO:249.In certain embodiments, OspC type is relevant with aggressive borrelia infection.Antibody can be polyclonal or monoclonal.In one embodiment, antibody has germicidal action for Borrelia spirochete.

The present invention also provides the immunogenicity mixture (cocktail) of chimeric recombinant protein.Each chimeric recombinant protein in mixture contains from ring 5th district of two or more outer surface protein C (OspC) type or the epi-position in α spiral 5th district or the two.

Accompanying drawing summary

Fig. 1, from the evolutionary relationship of the OspC sequence of the human patients of Maryland: OspC type identification.OspC gene is by pcr amplification, order-checking, and then constructing system is schemed.Analysis has comprised that the database sequence of 22 ospC types describes (having pointed out registration number).For the typonym (capitalization) of each germline group appointment is marked in each branch by capitalization.The value of bootstrapping (1000 tests) is displayed on each node for the differentiation key of group.

Fig. 2, shown that antibody is mainly OspC type specific to the reaction of OspC in course of infection.Produced the detection from Borrelia burgd albumen (indicating in the drawings) of several OspC types, separated by SDS-PAGE, the serum that the S albumen of the HRP coupling of use indication or the mouse infecting from the clone and separate thing by known OspC type are collected carries out immunoblotting and screening.

The location of the immunodominant epitope of Fig. 3, A type OspC.The category-A type OspC of brachymemma is as producing and express in intestinal bacteria with S-Tag fusion rotein.Figure A has shown the schematic diagram of brachymemma OspC.Numbering has reflected the residue numbering of Borrelia burgdoyferi B31 MI OspC.In figure A, (+) on the right or (-) represent the albumen and the ability that infects antibodies of each brachymemma.Left-hand digit represents to comprise the amino-acid residue that each blocks.In figure B and C, to use the immunoblotting of the S protein screening recombinant protein of HRP coupling to express and loading with confirmation, or use the MI by Borrelia burgdoyferi B31, the serum (α-B31 MI infects serum) of the mouse that a kind of category-A type OspC generation strain is infected screens.For reference, the arrow in figure b and c has been indicated with α-B31 MI and has been infected the migration position that serum does not have immunoreactive recombinant chou.The right at each immunoblotting has shown molecular weight marker.

Fig. 4, with tabulated form, shown the comparative analysis of the ring 5 of level between type and in type or the fragment of α 5 epi-positions.

In Fig. 5, a plurality of animals of having confirmed to infect with different A type OspC producing bacterial strain, encircle the reaction of 5 antibody.With infecting serum screening 1) total length A type OspC or 2) immunoblotting of fragment (comprising ring 5) that contains 130-150 amino acids.For generation of the specific mouse (m) that infects the bacterial strain of serum and be collected serum, be indicated at the top of each figure.Time point while collecting serum in course of infection is also marked.Every kind is all carried out immunoblotting with the albumen of equivalent, and it is also all identical to be exposed to the time quantum of film.

Fig. 6, ELISA: identify and comprise the serum sample that A type OspC target is determined antibody.Restructuring A type total length OspC, restructuring A type ring 5 and bovine serum albumin are used to the hole of coated elisa plate.Use the serum screening hole from mankind's lime body patient.Three parts of all analyses repeat, and provide mean value and standard deviation.All methods are all described in text.The antibody being confirmed as for Borrelia burgdoyferi B31 MI from No. 15 patients' serum is that IgG is negative, as negative control.

Fig. 7 A and B, by PepSpot, analyze, identify and comprise the specific residue that A type OspC encircles 5 epi-positions.The overlapping peptide of crossing over ring 5 structural domains produces and spotting on nitrocellulose filter.Then use from the serum of the mouse being infected by the clone population of A type OspC producing bacterial strain (B31 MI) or the peptide of using serum (the indicating) screening from mankind's Lyme disease patient to be fixed.(A) encircle the immunoblotting result of 5 structural domains; (B) peptide sequence.

Fig. 8, confirmed to encircle 5 and be that surface exposes and there is germicidal action for the antibody of ring 5.IFA and sterilization are analyzed and are used the antiserum(antisera) producing for A type ring 5 to carry out.(A) result has confirmed the sero-fast specificity of anti-ring 5.The full cell pyrolysis liquid of Borrelia burgdoyferi B31 MI, B.parkeri and restructuring A type ring 5 fragments are carried out separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and use anti-A type ring 5 antiserum(antisera)s (1:1000) to screen.The molecular weight of protein labeling is on the right of figure.

The generation of Fig. 9 A and B, the chimeric OspC test of tetravalence construct.A, the schema that produces the former construct of tetravalence ABKD chimeric is presented in figure A.In the type specific OspC of the former middle use of ABKD chimeric epi-position, by different strip shades, represented.Ring 5 epi-positions of A type OspC and α spiral 5 epi-positions of B, K and D type increase and gel-purified in the 1st takes turns PCR.Then in the PCR of subsequent rounds, these initial amplicons are merged, to produce chimeric construct body completely.Because the end (joint sequence) of amplicon is complementary, after sex change, they can be annealed, to can extend after PCR overlappingly.Last amplicon is annealed in pET46Ek/LIC carrier.B, in figure B, has shown the final protein sequence of the former construct of ABKD chimeric, has marked region and joint sequence that moiety contains epi-position.

Figure 10, show anti-ABKD antiserum(antisera) and ABKD chimeric is former and the immunoreactive Western trace of the OspC of total length.Immunogenicity is carried out immunoblotting by, A former to ABKD chimeric, B, K and D type total length detection from Borrelia burgd albumen (by indicating) and rBBN39 (negative control) and is assessed.Trace screens with anti-His label mAb, with the volume containing the sample that confirms substantially to equate, or with representational anti-ABKD antiserum(antisera) screen (below indicate).Molecular weight is presented at the right.Observed the strong IgG reaction to A, B and K (but not to D).

The ELISA titration of the serum reactivity of the mouse of Figure 11 A and B, the former immunity of use ABKD chimeric.A, to the mouse with the former immunity of ABKD chimeric (n=12) or with the serum of the mouse (n=3) of the false immunity of PBS/ adjuvant, carry out titration, to measure reactivity former with ABKD chimeric or A, B, K and D type rOspC albumen.Figure A has shown that all serum is to the immunoreactive titration of the former construct of ABKD chimeric (every mouse is used the solid line with distinct symbols).In the mouse of false immunity, do not observe Ab reaction (dotted line).B, has also completed the titration (not showing curve) for the specific reaction of every kind of OspC type, and the titre of measuring at 1/2 maximum OD405 place is presented at (1 point of each mouse, sea line is average titer) in figure B.Control mice does not have titre, not mapping.

The sero-fast Immunoglobulin Isotype distribution situation of Figure 12, anti-ABKD.ELISA hole is coated with (100ng/ hole) with the former construct of ABKD chimeric, and detects (1:10000 by anti-ABKD antiserum(antisera) double; N=12).In conjunction with biotinylated isotype specificity second antibody (mouse isotype parting kit for Ig; Zymed Laboratories) and the streptavidin of HRP coupling detect.Coloured product that the conversion of the ABTS substrate mediating by measurement HRP produces carries out quantitatively reactivity.

The schematic diagram of Figure 13 A-D, structure ABKD vaccine varient.ABKDppa (figure A) has 5 ' overhang by use to build to add the amino acid whose reverse primer of the C-terminal original construct that increases.ABKDgg (not shown) builds in the same way, but use is OCDH5ggLIC primer.ABKDD (figure B), ADBK (figure C) and ADBKD (figure D) have added by use composition sequence to be carried out the primer of the afterbody of coding joint sequence to pcr amplification and prepare.The PCR product obtaining, by gel-purified, couples together by overlapping annealing and extension.Final product is cloned in pET-46Ek/LIC carrier.The region letter representation that contains OspC type specific epi-position, numeral for joint sequence (referring to the encoding amino acid sequence inserting).Arrow represents primer, the afterbody that 5 ' outstanding LIC afterbody or joint sequence are labeled in each primer arrow.

The SDS-PAGE gel of the coomassie brilliant blue staining of Figure 14, the former test construct of chimeric.Vaccinogen recombinant protein is expressed in intestinal bacteria, by nickel chromatogram, carries out affinity purification, and quantitative by BCA method.By the albumen of 2 μ g purifying at 15% SDS-PAGE gel (Criterion; Biorad) on, carry out electrophoresis, and dye with Xylene Brilliant Cyanine G G-250.Do not notice the albumen of pollution, recombinant protein degraded seldom or is not degraded.

The assessment of Figure 15 A and B, mouse vaccine serum identification total length detection from Borrelia burgd.In figure A, A, B, K and D type detection from Borrelia burgd electrophoresis form trace (type is indicated at top on PVDF; 500ng/ road), and with the representative serum of the immune mouse of every kind of variation construct (being labeled in the left side) of use by oneself of 1:2500 dilution detect.Secondary detection is used goat anti-mouse IgG (1:40000) and the chemoluminescence of peroxidase coupling to carry out.Molecular weight is labeled in the right.Figure B is the result of reactive quantitative ELISA titration of mouse vaccine serum and total length detection from Borrelia burgd.The serum producing for every kind of vaccine constructs (being labeled in bottom) carries out titration for fixing total length restructuring A, B, K and D type OspC.Also comprised the ABKD construct (ABKD with PBS dialysis ^*) titre of [17].Bar represents the average titer for the OspC of A (black), B (grey), K (hollow) and D (oblique line) type.Titre from individual mouse represents by hollow triangle.What list below is the numerical value of average titer, and for using PBS (ABKD ^*) or the titre of the corresponding titre indication of the ABKD construct of Arg/Glu damping fluid (ABKD) dialysis.

The reaction of the special isotype of Figure 16 A-C, epi-position to three kinds of vaccine constructs.A, B, K and D type OspC are fixed on elisa plate, use the immune serum double of the mouse of use by oneself ABKD, ABKDD or the immunity of ADBKD construct to detect.In conjunction with Ig isotype with the streptavidin of the special second antibody of biotinylated isotype and peroxidase coupling, detect.

The splenocyte of the mouse of Figure 17, immunity is to being used the immunogenic external IFN-gamma reaction stimulating again.With being collected without erythrocytic splenocyte of every kind of 6 kinds of vaccine constructs 3 immune mouse, merge, with three parts of parallel (every mL10 in 24 orifice plates that stimulate again of initial immunizing antigen ⁷individual cell, antigen is 10 or 5 μ g/mL).In three control wells, use 10mg/mLBSA or do not use albumen.Hatch (37 ℃, 5%CO2), after 96 hours, collect acellular supernatant liquor, by ELISA, measure the concentration of IFN-γ.In all cases, BSA and without the IFN-γ concentration in albumen hole lower than the detectability of analyzing.

Figure 18 A and B, the reaction of assessment antibody to the ABBCD vaccinogen of using in freund's adjuvant or alum.Figure A is for the ABKD vaccine (solid bars) of emulsification in freund's adjuvant or is adsorbed in the result of the quantitative ELISA titration of IgG in the mice serum that the ABKD vaccine (oblique line bar) of alum produces.Serum carries out titration for fixing ABKD vaccinogen or A, B, K and D type total length detection from Borrelia burgd.Figure B has shown the isotype distribution situation of the serum of being combined with fixing ABKD vaccinogen.In conjunction with Ig isotype with the streptavidin of the special second antibody of biotinylated isotype and peroxidase coupling, detect.

The distribution of Figure 19, the conforming paired comparison of OspC protein sequence.Use PAM40 score matrix that the OspC protein sequence from 280 burgdorferi strain isolateds has been carried out to Clustal comparison, and calculated paired percentage consistence.Histogram interval is 1%, and percentage consistence is not lower than 50%.

The species of the OspC type of Figure 20, appointment, geography and biology separate data.

The sharing system evolutionary tree of Figure 21 A-C, representative OspC protein sequence.The OspC sequence that comprises amino acid 20-200 (A), 20-130 (B) and 131-300 (C) is bootstrapped (n=1000), and computed range produces contiguous combination tree, and consistent with stric consistency tree.The Vmp33 sequence of Hermes burgdorferi (B.hermsii) is used as outgroup.Mark represents that species are that the name of Borrelia burgdoyferi (Bb), Borrelia garinii (Bg) or Erichsen burgdorferi (Ba), strain isolated is, the quantity (in bracket) of the OspC sequence consistent with other bacterial strain of the OspC type (runic) of appointment and this unique sequence representative.From drawing support, be presented on the node of all differentiation OspC types.

The ospC sequence of Figure 22, PLj7 type OspC (Erichsen burgdorferi) and Pki type (Borrelia garinii; Solid black lines), F type (Borrelia burgdoyferi; Grey solid line) and M type (Borrelia burgdoyferi; The startup scanning (bootscan) of the comparison of OspC black dotted lines) is analyzed.Starting scanning window is 40 bases, 10 bases of every step.By comparing from the stric consistency that draws duplicate with 100.Figure has been simplified, and has only shown that the percentage ratio of the tree (permutedtrees) of those schedules surpasses 50% peak, and level is 70% to be considered to represent possible restructuring, and they are indicated.

Figure 23, from the comparison in the region of containing epi-position of the OspC protein sequence of all OspC types of definition in this research.In OspC type, there is all sequences that surpasses an amino acid difference to be pointed out by representational sequence.The consistence threshold value of shade is 80%.Secondary structure α spiral and ring (corresponding to B31 structure) are displayed on the below (Kumaran etc., 2001) of aligned sequences.

Figure 24, for the ClustalX comparison of the parent OspC sequence of the former structure of ABKD chimeric be included in the physical location in the region of containing epi-position of vaccinogen.In the Clustal of parental array X comparison, the position in the region of containing epi-position of A type (encircling 5th district, light grey frame) and B, K and D type (α spiral 5th district, Dark grey frame) OspC is highlighted.

The chimeric of the present invention of Figure 25 A-J, example is former.The title of construct shows ring 5 and α spiral 5 epi-positions of the OspC type specific that construct is integrated, and order.Runic X represents the position of optional joint sequence.

Figure 26, from albumen and the DNA registration number of the OspC of several burgdorferi bacterial strains.

The specific descriptions of embodiment of the present invention

The present invention is evaluation and the sign of epi-position based on infecting new, protectiveness, the type specific of relevant OspC type with mankind's Lyme disease.This new epi-position is arranged in half two structural domains (region) of C-terminal of OspC.These two structural domains were not all accredited as the immunogenicity with height in the past.First structural domain (be called as in this article " α spiral 5 districts/structural domain " or “α 5 districts/structural domain " or " spiral 5 districts/structural domain ") between 160 and 200 residues, and the secondary structure element (Kumaran etc., 2001) that contains the part, α spiral 5 and the structureless C-terminal structural domain that comprise ring 6.Second structural domain (be referred to herein as “Huan 5 districts/structural domain ") between 131 and 159 residues, and contain comprise α spiral 3 a part, encircle 5 and the secondary structure element (Kumaran etc., 2001) of spiral α.Each contains the epi-position that at least one can be used for practice of the present invention these regions.From the epi-position of these two structural domains or from antigenic peptide or polypeptide in these two regions, other epi-position combination separately or preferably and in chimeric immunogen, can be in practice of the present invention.In certain embodiments of the invention, epi-position is immunodominant epitope.Conventionally epi-position is B cell epitope, although also do not get rid of t cell epitope.

The discovery of new epi-position and the mapping to the epi-position of different OspC types, make the polyvalent chimeric albumen that builds the immunodominant epitope from a plurality of OspC types that contains a plurality of linearities, type specific become possibility.When as vaccine; polyvalent recombinant chimeric protein causes the extensive provide protection for the spirochetal infection of Borrelia; corresponding in the OspC type that wherein this Borrelia spirochete is expressed and chimeric protein, i.e. tool, has those burgdorferis of hyperinfection.In addition, chimeric protein can be used as diagnostic tool and has the antibody for the epi-position comprising in chimeric protein to differentiate individuality, thereby determines whether this individuality has been exposed to and/or has been subject to the infection of lyme bacteria body.

For the ease of understanding the present invention, provide definition below:

Antigen: in history for censuring by the term of the entity of antibodies, be also used in reference to the entity that induction antibody produces.Recent usage is limited to the meaning of antigen by the entity of antibodies, and the entity of " immunogen " word for inducing antibody to produce.When the entity of discussing has immunogenicity and antigenicity concurrently, in general will be referred to as immunogen or antigen according to the application of its expection herein.Term " antigen ", " immunogen " and " epi-position " in this article can Alternates.

B cell epitope: on antigen by B-cell receptor identification, and the specific chemical structural domain of antibodies that can be secreted.This term can with " antigenicity determinant " Alternate.

Immunodominant epitope: the epi-position of inducing dominant or the strongest immunne response on molecule.

Linear epitope: comprise the epi-position that is joined together to form single, the continual continuous amino acid chain of peptide or polypeptide by peptide bond.Such epi-position can be passed through its primary structure, and in chain, amino acid whose linear order is described.

Conformational epitope: at least some amino acid that this epi-position comprises is not a part for continual linear aminoacid sequence, but the secondary of these amino acid by albumen, three grades and/or level Four interact be brought to described epi-position in other residue approach.From primary structure, these residues may be far apart from other residue position in epi-position, but due to albumen folding may with conformational epitope in other residue on locus, approach.

Encircle 5 districts/structural domain: OspC comprised with Figure 23 in the region of 131 to 159 residues of the A type OspC sequence that shows residue arranged together.The OspC secondary structure element of the B31 bacterial strain comprising in this region be α spiral 3 a part, encircle 5 and α spiral 4, as defined in (2001) such as Kumaran.

α spiral 5 districts/structural domain: OspC comprised with Figure 23 in 160 of B31 bacterial strain (the A type OspC) sequence that the shows regions to the C-terminal part (the 201-210 amino acids of B31 sequence) of the albumen that does not have in 200 amino acids residue arranged together and Figure 23 to show.The OspC secondary structure element of the B31 bacterial strain comprising in this region is a part, α spiral 5 and the structureless C-terminal structural domain of ring 6, as defining (2001) in institutes such as Kumaran.

Albumen: about 100 or more amino acid are by the covalently bound linear order of peptide bond.

Polypeptide: about 20 are passed through the covalently bound linear order of peptide bond to about 100 amino acid.

Peptide: about 20 or amino acid are still less by the covalently bound linear order of peptide bond.

Term " albumen ", " polypeptide " and " peptide " in this article can Alternates.

Chimeric protein: its primary sequence contains the recombinant protein that can simultaneously not appear at a plurality of peptides, polypeptide and/or protein sequence in the single molecule in nature.

The valency of chimeric protein (for example " multivalence ") refers to the quantity of the polypeptide with OspC type specific epi-position that chimeric comprises in former.For example divalence mosaic can consist of A type 5 spiral α and Type B α spiral 5, or consists of A type α spiral 5 and A type ring 5.At each, may there are a plurality of different epi-positions in the region of polypeptide epitope.

Original or natural or wild-type sequence: with the sequence of the same peptide, polypeptide, albumen or the nucleic acid found in nature.

Recombinant peptide, polypeptide, albumen or nucleic acid: the peptide, polypeptide, albumen or the nucleic acid that use Protocols in Molecular Biology to produce and/or operated such as clone, polymerase chain reaction (PCR) etc.

Type specific: main relevant with single germline group.

Invasive infection: if the place (such as blood plasma, cerebrospinal fluid etc.) of biting outside the surrounding skin of initial inoculation from tick in human infection's process has been separated to the burgdorferi with OspC type, OspC albumen be known as " relevant with invasive infection " so.

Therefore, the invention provides and contain a plurality of restructuring chimeric proteins from ring 5 and/or the linear epitopes in α spiral 5th district, in these epi-positions at least two from the different OspC types relevant from invasive infection.Preferably, represented about 2 to about 20, preferably from about 6 antigenic epitopes to about 10 different OspC types, be comprised in single chimeric protein.Although conventionally have at least two epi-positions to differ from one another and derive from different OspC types in primary structure, also may in mosaic, comprise a plurality of copies of single type epi-position, comprise based on or from several sequences of the original series of same OspC type.Although the sum of mosaic neutral line epi-position can be slightly different, in general scope is from about 10 to 20.In one embodiment of the invention, immunodominant epitope is selected from two or more 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, C type OspC.In one embodiment, chimeric protein is tetravalence, contains from A, B, K and D type epi-position.In another embodiment, chimeric protein is octavalence, contains the epi-position from E, N, I, C, A, B, K and D type OspC.But those skilled in the art will recognize that, also can use the epi-position from other OspC type combination, as long as the mosaic obtaining produces suitable immunne response and/or effectively prevents Lyme disease as vaccine.Other appropriately combined example includes but 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.

In certain embodiments, ring 5 and α spiral 5th district will be comprised simultaneously.For example, " E, N, I, C, A, B, K, D " construct can contain ring 5 and spiral 5th district of each E, N, I, C, A, B, K and D type OspC simultaneously.But situation must not be like this.For example, can comprise ring 5th district of A type and α spiral 5th district of E, N, I, C, B, K and D; Or ring 5th district that can only comprise every kind of OspC type; Or only comprise α spiral 5th district; Or can comprise other combination (for example ring 5th district of E, N, I and C type and α spiral 5th district of A, B, K and D type).For those skilled in the art, can produce many such combinations, all such variations are all intended to comprise in the present invention.

In addition, in mosaic, the linear precedence of epi-position can change.In general, order Wei“Huan 5th district from amino to C-terminal, α spiral 5 district ，Huan 5th district, α spiral 5th district ... " etc.For example, the in the situation that of E, N, I, C, A, B, K, D construct, preferably order is, along chimeric length direction, and " E Xing Huan 5th district, E type α spiral 5th district; NXing Huan 5th district, N-type α spiral 5th district; I Xing Huan 5th district, I type α spiral 5th district; " etc., different OspC types and/or different structural domains are selected to be separated by neutral joint sequence.But this order can change, this for example depends on and is selected and is included in the element in mosaic.Can use any OspC type and the order of structural domain, as long as the mosaic obtaining produces applicable immunne response and/or effectively prevents Lyme disease, maybe can be effective to diagnosis as vaccine.The example of exemplary mosaic sequence provides in Figure 25 A-J.Form from the crucial albumen of the OspC of several burgdorferi bacterial strains and DNA registration number with form is presented in Figure 26.

The aminoacid sequence comprising in chimeric protein can comprise α spiral 5th district and/or encircle 5th district, or its antigenicity fragment.For " antigenicity fragment ", we refer to the fragment of the one-level OspC sequence that contains at least one linear epitope being identified in infection.Such epi-position, when expressing in the OspC protein protomer of restructuring, has retained to be similar in conjunction with expressing the ability in conjunction with infection induced antibody in the mode of the wild-type protein of cell surface.Single antigenicity fragment can comprise more than one different epi-position.Those skilled in the art will recognize that, some is inaccurate slightly to the affinity of epi-position or avidity to measure antibody, and in immunne response process, due to for example Affinity maturation/somatic hypermutation, affinity/avidity can change significantly.But, in general, affinity/avidity that antibody is combined with chimeric protein is with natural complete α 5 or encircle 5 affinities that show and compare, in at least about 50% scope, preferably approximately 60%, more preferably about 70%, be more preferably about 80%, about 90-100% or even higher most preferably.In general, the antigenicity sequence comprising in chimera protein will contain about 20 to about 100 amino acid, preferably from about 30 to about 70 amino acid, chimeric protein itself will contain altogether from about 160 to about 800 amino acid, preferably from about 240 to about 560 amino acid.In addition, antigenicity sequence can be called as " epi-position " in this article, and no matter whether they have comprised complete " natural " epi-position, as long as they have antibodies characteristic described herein.

Or suitable antigen fragment or antigenicity sequence or epi-position be in being included in chimeric protein time, can in using the host of chimeric protein, cause by them the ability that produces suitable antibody for epi-position and identify.Those skilled in the art will recognize that the definition of antibody titers can change.Here, " titre " is taken as in the ELISA hole coated by 100ng test proteins the sero-fast dilution inverse in conjunction with the available binding site of half.In general, the antibody titers that is characterized as that produces suitable antibody arrives in about 100,000 scope about 100, preferably about 10,000, arrives in about 10,000000 scope.Or particularly in diagnositc analysis, " titre " should be about 3 times of the background level of combination.For example, be considered to " positive ", the reactivity in test should be at least high 3 times than the reactivity detecting in the individual serum not infecting.Preferred antibody reaction is protectiveness, compares with nonvaccinated host, prevents or reduced the development of disease symptoms in the inoculation host who is exposed to afterwards burgdorferi.

The aminoacid sequence of an exemplary chimeric protein of the present invention is presented in Fig. 9 B.In this illustrative embodiment, mosaic contains from ring 5 region amino acid sequences of A type OspC with from α spiral 5 region sequences of B, K and D type OspC.In this case, A type OspC sequence is from bacterial strain LDP56, and Nucleotide registration number is EF053513, and albumen registration number is ABK41054; The sequence of Type B OspC is from bacterial strain LDP73, and nucleic acid registration number is EF053525, and albumen registration number is ABK41066; K type OspC is from bacterial strain LDP89, and Nucleotide registration number is EF053523, and albumen registration number is ABK41064; D type OspC is from bacterial strain LDP116, and Nucleotide registration number is EF053527, and albumen registration number is ABK41068.Those skilled in the art will recognize that, from the OspC of many burgdorferi bacterial strains, are known maybe can finding, and can be in practice of the present invention.

Those skilled in the art will recognize that, although in certain embodiments of the invention, selected be included in aminoacid sequence in chimeric protein of the present invention directly corresponding to the one-level aminoacid sequence of the original or native sequences of OspC albumen, but this situation not necessarily.The aminoacid sequence that is included in the epi-position in chimeric protein of the present invention can slightly change and still be suitable in the present invention.For example, can carry out some conserved amino acid replacement and the ability of epi-position initiation immunne response is not had to injurious effects.Those skilled in the art will recognize that the essence of this conservative replacement, for example, use another positively charged amino acid of positively charged aminoacid replacement; With another electronegative amino acid of electronegative aminoacid replacement; With another hydrophobic amino acid of hydrophobic aminoacid replacement; Etc..All this replacement or changes that are included in the sequence of the epi-position in chimeric protein of the present invention are all intended to be included in the invention, as long as the epi-position producing still can cause suitable immunne response.In addition the aminoacid sequence being included in chimeric protein of the present invention, must not forgiven the natural epi-position of total length or the structural domain that contains epi-position.Those skilled in the art will recognize that, the version of the brachymemma of the known aminoacid sequence that is or contains epi-position can be preferred in the present invention due to a variety of causes, as long as sequence has met the standard for epi-position setting.The aminoacid sequence that is substituted like this or otherwise changes can be called as in this article " based on " or " coming from " original wild-type or native sequences.In general, linear epitope institute " from " or linear epitope institute " based on " OspC albumen be the OspC albumen occurring in nature.These natural OspC albumen also can be called as natural or wild-type protein.

Can import for a variety of reasons such change to primary sequence, for example, in order to eliminate or introduce proteolytic enzyme cutting site, in order to increase or reduce solvability, in order to promote or to hinder in molecule or such as folding, ionic interaction, salt bridge etc. of molecular interaction, otherwise they may disturb single epi-position presenting and accessibility along mosaic major diameter.All such changes are all intended to forgive within the scope of the present invention, as long as the aminoacid sequence obtaining can act on the protection antibody reaction that causes the OspC type being derived from for epi-position.In general, such replacement sequence has the sequence with corresponding native protein at least about 50% consistence, preferably there is about 60-70,70 to 80 or 80 to 90% consistence even, the consistence of preferably approximately 95 to about 100% with wild-type sequence.

In certain embodiments of the invention, the single linear epitope during chimeric is former is separated by intervening sequence each other, and these intervening sequences are being more or less neutral in nature, and they do not participate in or itself does not cause the immunne response for burgdorferi.Such sequence can exist between chimeric epi-position, also can not exist.If existed, they can, for example, for separating epi-position, and contribute to epi-position spatial separation each other.Or such sequence can be only the artificial product that restructuring work program is for example cloned program.Such sequence is commonly referred to as joint or spacer peptide, and its many examples are known by those skilled in the art.Referring to for example Crasto, C.J. and J, A.Feng.2000.LINKER:a program togenerate linker sequences for fusion proteins. (LINKER: produce the program for the joint sequence of fusion rotein) Protein Engineering13 (5): 309-312, it is the reference of having described non-structure joint.Structure (for example spiral) sequence joint also can use for example existing known sequence with secondary structure to design, or carrys out designed joint by the basic known organism principles of chemistry.In addition, other element also can appear in chimeric protein, leader sequence or " tag " so that the purifying of albumen or the sequence of detection to albumen for example, that its example includes but not limited to is histidine-tagged, detection label (for example S-tag or Flag-tag), other antigenicity aminoacid sequence, such as the sequence that contains known t cell epitope and protein stabilized motif etc.In addition, chimeric protein can be by chemically modified, for example, by amidation, sulfonylation, esterified or known other technology of those skilled in the art.

The present invention also provides the nucleotide sequence of the chimeric protein of the present invention of encoding.Such nucleic acid comprises DNA, RNA and hybrid thereof etc.In addition, the present invention has comprised the carrier that contains or include these encoding sequences in.The example of applicable carrier includes but not limited to plasmid, clay, the carrier based on viral, expression vector etc.In preferred embodiments, carrier is plasmid expression vector.

Chimeric protein of the present invention can be produced by any appropriate means, and many in them are known by those skilled in the art.For example, albumen can be by chemosynthesis, or produce (for example, in bacterial cell by recombinant DNA technology, in cell culture (Mammals, yeast or insect cell), in plant or vegetable cell, or by acellular protokaryon or the expression system based on eucaryon, by other vitro system etc.).The present invention also provides for causing the composition of immunne response, and it can be used as vaccine with prevention or treatment borrelia infection, particularly when turning out to be Lyme disease (lime borreliosis).For causing immunne response, we refer to that using of antigen caused synthetic (with the above-mentioned titre) of specific antibody, and/or the propagation of cell, as measured by mixing of for example 3H thymus pyrimidine.For " vaccine "; we refer to the chimeric protein that causes immunne response; for example, compare with the organism that contrasts that is not vaccinated (only using adjuvant); it produces the provide protection of attacking for burgdorferi, prevents wholly or in part or contained the development of the symptom relevant with borrelia infection (being Lyme disease symptom).Composition comprises the restructuring chimeric protein of one or more purifying substantially described herein and suitable pharmaceutical carrier.Multiple chimeric protein in composition can be identical or different, and composition can be different chimeric " mixture ", or composition only contains the mosaic of single type.The preparation of this composition as vaccine is known for those skilled in the art.In general, such composition is prepared to liquor or suspension, but also can consider solid form such as tablet, pill, powder etc.Also can prepare and be suitable for before administration, dissolving or being suspended in the solid form in liquid.Preparation also can be emulsified.Activeconstituents can with mixed with excipients pharmaceutically useful and can be compatible with activeconstituents.Applicable vehicle is such as water, salt solution, dextrose, glycerine, ethanol etc. or its combination.In addition, composition can contain a small amount of auxiliary substance, such as wetting agent or emulsifying agent, pH buffer reagent etc.Vaccine preparation of the present invention can also contain adjuvant, and applicable example includes but not limited to Seppic, Quil A, Alhydrogel etc.If need oral form to use composition, can add various thickening materials, seasonings, thinner, emulsifying agent, dispersing auxiliary or tackiness agent etc.Composition of the present invention can contain any such supplementary component so that the composition that is suitable for form of medication to be provided.In preparation, the final content of chimeric protein can change.But in general, the amount in preparation is about 0.01-99% weight/volume.

Method comprises the composition that contains chimeric recombinant protein to administration in pharmaceutically useful carrier.Vaccine preparation of the present invention can be by any the using in the applicable method of the known many kinds of those skilled in the art, include but not limited to by injection, suction, oral, nose, the food that contains chimeric protein by digestion etc.In preferred embodiments, mode of administration is subcutaneous or intramuscular.In addition, composition can be used with together with other form of therapy, such as strengthening immune material, chemotherapeutics etc.

The invention provides in Mammals and to cause for the immunne response of burgdorferi and for borrelia infection, to carry out the method for immunization.In one embodiment, Mammals is the mankind.But those skilled in the art will recognize that, may also there are the needs to this immunization in other Mammals, and for example, preparation also can be for animal doctor's object.Example includes but not limited to accompany " pet " such as dog, cat etc.; Food source, work and amusement animal such as ox, horse, bull, sheep, pig, goat etc.; Or or even for example, as the main wildlife (mouse, deer) of burgdorferi storage.The present invention also provides diagnostic reagent and has differentiated that with diagnostic reagent individuality has the method for the antibody of the epi-position comprising in chimeric protein of the present invention.For example, from the biological sample that has been exposed to burgdorferi or has been in the individuality (mankind of burgdorferi spirochete susceptible, deer or other Mammals) being exposed in burgdorferi risk under a cloud, contact with chimeric protein of the present invention.Use existing method, detect in chimeric protein and biological sample whether have association reaction between antibody.Positive findings (in conjunction with having occurred, so antibody exists) shows that individuality has been exposed to and/or has infected burgdorferi.In this connection, the object according to analyzing, can build specificity for the mosaic of any subclass of paid close attention to OspC type, and all possible OspC type can comprise, maybe can be not included in diagnosis mosaic.

In addition, diagnosis of the present invention aspect is not limited to clinical use and family is used, and for being also valuable as research tool in laboratory, for example, identifies the Borrelia spirochete being separated to from tick, the regional distribution of research OspC type etc.

The antibody for epi-position disclosed herein and/or chimera protein has also been contained in the present invention.Such antibody can be polyclonal, monoclonal or chimeric, and can produce in the known any mode of those skilled in the art.In a preferred embodiment of the invention, antibody is sterilization (killing burgdorferi), is about to Borrelia spirochete and is exposed to antibody and causes spirochetal death.Such antibody can be used in many ways, for example, as the previously exposure in burgdorferi of detection reagent diagnosis, as the reagent of the test kit of research burgdorferi, treatment borrelia infection etc.

Provide the following examples to set forth various embodiments of the present invention, but they should not be considered to be construed as limiting by any way.

Embodiment

Embodiment 1, in aggressive mankind Lyme disease strain isolated, prove OspC type diversity and identify to limit OspC antibody response specific before the epi-position of sign not

Brief introduction

Lyme disease propagates into the mankind by being belonged to biting of tick by the hard tick of Borrelia burgdoyferi, Borrelia garinii or Erichsen borrelia infection.Outer surface protein C (OspC) is considered to participate in the important virulence factor of communication process, and may participate in the foundation of early infection in Mammals (Grimm etc., 2004; Parl etc., 2004; Schwan etc., 1995).OspC is variable, approximately 22kDa, surperficial lipoprotein (Fuchs etc., 1992 that expose, plasmid-encoded; Marconi etc., 1993; Sadziene etc., 1993).Crystalline structure (Eicken etc., 2001 of three OspC albumen have been determined; Kumaran etc., 2001).Albumen is mainly spiral helicine, has 5 α spirals, by variable ring, connects.Ring has formed ligand binding domains (Eicken etc., 2001 by inference; Kumaran etc., 2001).Evidence shows, OspC may by as with sialisterium in the adhesin of unidentified receptors bind help the transfer (Pal etc., 2004) of spirochete intestines from tick.In several species of typhinia group, identified the ortholog of OspC, this has improved possibility (Marconi etc., 1993 that OspC associated protein is carried out said function in other burgdorferi species; Margolis etc. 1994).The expression of OspC is subject to the regulation and control of environment, be subject to the induction of tick feed, and OspC is dominant antigen (Alverson etc., 2003 in Mammals early infection; Schwan etc., 1998; Stevenson etc., 1995).Be transcribed into the regulation and control (Hubner etc., 2001) that small part is subject to RpoN/S regulated and control network.Should be noted that, about between propagation periods and the report of the fine detail of the occasional nature that during early infection, OspC expresses have conflict (Ohnishi etc., 2001; Schwan etc., 1995).

OspC shows obvious heredity and antigen diversity (Theisen etc., 1995; Theisen etc., 1993).21 OspC germline groups (being called below OspC type) (Seinost etc., 1999 have been described; Wang etc., 1999).OspC letter designation for type (from A to U) is distinguished.For the hundreds of in database, an analysis that OspC aminoacid sequence carries out shows, the divergence between OspC type can be up to 30%, and is generally less than 6% in one type.The existence associated (Seinost etc., 1999) between A, B, I and K type OspC and the mankind's invasive infection of Seinost etc. hypothesis.Lagal etc. have also reported by the definite specificity OspC varient of single-strand conformation polymorphism analysis relevant with aggressive human infection (Lagal etc., 2003).But Alghaferi and colleague's thereof nearest research has proposed query (Alghaferi etc., 2005) for the intensity of this association.The type of OspC or sequence have represented a research field important, that display great creativity to function and the interactional impact of host-cause of disease.Application for OspC in ImuLyme exploitation is studied (Bockenstedt etc., 1997; Gilmore etc., 2003; Gilmore etc., 1999a; Probert etc., 1994; Theisen etc., 1993; Wilske etc., 1996).But, the variation of OspC and make these work very complicated to the limited understanding of OspC antigenicity structure.OspC has protectiveness power, but only for same bacterial strain (Bockenstedt etc., 1997; Gilmore etc., 1999; Gilmore etc., 1999b; Probert and LeFebvre, 1994; Wilske etc., 1996).This shows that the epi-position of protectiveness is positioned at the albumen region of sequence alterable height.

Which floor the object of this research has.First, by definite aggressive reclaiming from the patient crowd of Maryland State restriction and the OspC type of Non-Invasive strain isolated, seek the further assessment of inferring cognation between OspC type and invasive infection.The second, in trial, understand better in the reaginic antibody for OspC, seek to determine whether this reaction is type specific.Finally, be tested and appraised the epi-position that causes antibody response in mouse infection process, seek determining of OspC antigenicity structure.The data that present herein show that the quantity of the OspC type relevant with invasive infection surpasses former supposition (Seinost etc., 1999).In addition, we have identified two epi-positions that do not characterize in the past, and confirmed antibody response for OspC shown be type specific.These analyses provide important information, strengthen the understanding of our effect in Lyme disease pathogenesis to OspC, and will be convenient to the structure of the vaccine based on OspC.

Experimental arrangement

Bacterium strain isolated, infects cultivation and the generation of serum.The Lyme disease strain isolated reclaiming from Maryland State mankind patient is used in these analyses (table 1).Patient has supplied Informed Consent Form in research prerequisite, and this research is ratified by examination board of John's Thelma Hopkins medical research institute (JohnHopkins Medicine Institutional Review Board).Spirochete is cultivated in BSK-H perfect medium (Sigma) at 33 ℃, monitors, by centrifugal collection by dark-field microscope.For some strain isolated, by bed board under the liquid level of former description (Sung etc., 2000), produced clone population.In order to determine the ospC type of individual bacterium colony, ospC gene has been carried out to pcr amplification, order-checking, and carried out comparative sequential analysis (as described below).In order to produce the antiserum(antisera) for the clone population of the OspC albumen of a series of expression known types, by 10 ³individual spirochete cleans in phosphate buffered saline buffer (PBS), then with pin be inoculated in C3H-HeJ mouse (subcutaneous, between omoplate; Jackson Labs).The infection of mouse is according to former description (Zhang etc., 2005), by 2 or 4 weeks after inoculation, ear puncture biological tissue carried out to PCR in real time with the primer that target is determined flaB gene and confirms.In the time of 0,2,4 and 8 week, by cutting truncation bar, from every mouse, collect blood, results infect serum.Antiserum(antisera) that other uses in these are analyzed and infect that serum is former described (McDowell etc., 2002).

Table 1, bacterium strain isolated, source-information and OspC type

Borrelia burgdoyferi strain isolated	Source	OspC type
			B31?MI	Tick	A
5A4	Clone from B31 MI	A
			LDP56	Human blood	A
LDP61	Human blood	A
			LDP60	Human blood	A
LDP80	Human blood	A
			LDP76	Human blood	A

LDS106	Human skin	A
			LDP73	Human blood	B
LDS79	Human skin	H
			LDS101	Human skin	H
LDP84	Human blood	C
			LDP63	Human blood	N
LDC83	Human cerebrospinal fluid	N
			LDP120	Human blood	N
LDP74	Human blood	K
			LDS81	Human skin	K
LDS88	Human skin	K
			LDP89	Human blood	K
LDP116	Human blood	D

DNA separation, OspC somatotype and area of computer aided structural analysis.In order to determine OspC type, according to former description, from the total DNA of each strains separation (Marconi etc., 2003), and carry out PCR (table 2) with OspC20 (+) LIC as template together with OspC210 (-) LIC primer.PCR is used exo+ polymerase amplification (Expand High Fidelity polymerase (Roche)) to carry out, and uses cycling condition below: 94 ℃ of initial sex change 2 minutes; 94 ℃ 15 seconds, 50 ℃ 30 seconds, 60 ℃ are carried out 10 circulations in 60 seconds; 94 ℃ 15 seconds, 50 ℃ 30 seconds, 60 ℃ 60 seconds, last 20 circulation each additionally add 5 seconds; Finally at 68 ℃, extend 7 minutes.Use QiaQuick PCR purification kit (Qiagen) to reclaim amplicon, with T4 archaeal dna polymerase, process to produce strand overhang, be annealed in pET-32 Ek/LIC carrier (Novagen), and be transformed in intestinal bacteria NovaBlue (DE3) cell (Novagen).The method of these programs is according to the description of manufacturers.According to amicillin resistance (50 μ g/ml) screening bacterium colony, by PCR, screen ospC Insert Fragment.By the colony lift of selecting (Fisher) in LB meat soup, at 37 ℃, vibration (300rpm) is cultivated, and uses QiaFilter Midi plasmid separating kit (Qiagen) separation quality grain.OspC Insert Fragment is by charge service order-checking (MWG Biotech).The sequence of mensuration is translated and used ClustalX (35) and default parameter to compare.In order to determine the type of OspC, produced in abutting connection with tree, and calculated the value of bootstrapping (1000 tests).There is figure and use N-J Plotter to show in the biosystem obtaining.In database, other available OspC sequence is also comprised in analysis.The CN3D software that uses NCBI molecular simulation database file 1GGQ, IflM and 1G5Z (4,15) and provide on ncbi.nlm.nih.gov/Stnicture/CN3D/cn3d.shtml site has produced the structural models of OspC.

Table 2, the chain reaction primer of polymerase using in this research

^alIC afterbody sequence is by underscore.

The generation of recombinant protein.In order to produce the OspC of total length and brachymemma, A type ospC sequence (Fraser etc., 1997) the design primer based on Borrelia burgdoyferi B31MI.Primer has the annealing of permission and is connected to the afterbody sequence in pET-32Ek/LIC carrier (Novagen), and this carrier is clone (LIC) and the expression vector that does not rely on ligase enzyme.All LIC programs the same with former description (Hovis etc., 2004).In order to confirm the sequence of all constructs, use QiaFilter Midi plasmid purification test kit (Qiagen) from intestinal bacteria NovaBlue (DE3) cell purifying recombinant plasmid, and to Insert Fragment check order (MWG Biotech).

SDS-PAGE and immunoblotting assay.According to former description (Roberts etc., 2002), albumen is carried out to separation by SDS-PAGE in 12.5% standard precast gel (Biorad), and immunoblotting (Millipore) to pvdf membrane.Use S-albumen horseradish peroxidase (HRP) conjugate (Novagen) to confirm the expression of recombinant protein, this conjugate detects the N-end S-label that all recombinant proteins used all carry in this research and merges.The S-albumen 1:10 of HRP coupling, 000 dilution is used.For immunoblotting assay, the serum of collecting from infected mouse is used with 1:1000 dilution.The goat anti-mouse IgG of HRP coupling is used as second antibody (Pierce), 1:10, and 000 dilution is used.General western blotting method the same with former description (Metts etc., 2003).

Result

The ospC phenotypic analysis of the strain isolated reclaiming from mankind's Lyme disease patient of the Maryland State.The strain isolated reclaiming from analyzed each mankind's Lyme disease patient from the Maryland State ospC that successfully increased.The sequence of each amplicon is determined, and has carried out comparative sequential analysis to determine the type (Fig. 1) of OspC.Identify the representative of several different OspC types, comprised A (n=6), B (n=1), C (n=1), D (n=1), H (n=2), K (n=4) and N (n=3).Reported in the past only have A, B, I relevant with the invasive infection in the mankind with K type OspC (Seinost etc., 1999).In this research, aggressive strain isolated is restricted to the strain isolated reclaiming from blood, organ or cerebrospinal fluid, and the strain isolated (Seinost etc., 1999) that Non-Invasive strain isolated reclaims, still do not find in other position of health from skin.But, confirmed that some strain isolated of expressing C, D and N-type OspC reclaims from blood (LDP84, LDP63, LDPl 16 and LDPl 20) or cerebrospinal fluid (LDC83) here, be therefore invasive.This observations shows that the dependency between specific OspC type and invasive infection may not be strict, and the intensity of cognation need to be reappraised.

Type specific for the antibody response of OspC in mouse infection process is analyzed.In order to determine whether the antibody response causing for OspC is type specific in course of infection, A, B, C, D, H, K and N-type detection from Borrelia burgd albumen have been produced as test antigen.Recombinant protein is by immunoblotting, and the serum that the mouse of using the Borrelia burgdoyferi strain isolated (as determined) from A, B or D OspC type to infect is above collected screens (Fig. 2).The expression of recombinant protein in intestinal bacteria (E.coli) and the equal carrying capacity of albumen, immunoblotting of S-protein screening of identifying the HRP coupling of the S-label in the fusion of N-end by use confirms.When using the anti-Borrelia burgdoyferi B31 MI antiserum(antisera) (A type OspC) of collecting when infecting 2 weeks to screen, only have the A of use type albumen just strong reactivity can be detected.For the strong and early stage IgG reaction of OspC, (Theisen etc. 1995 with the report of morning conforms to; Wilske etc., 1993).At the serum that infects collection in the 8th week, also mainly react with A type OspC, still observed the weak cross-immunoreactivity with other OspC type.In the mouse infecting with LDP116 and LDP73 (being respectively D and Type B OspC strain isolated), to the antibody response of OspC, be also type specific.Can reach a conclusion, have the type specific of certain degree in the antibody response for OspC, in this specificity prompting body, immunodominant epitope is arranged in the type specific structural domain of albumen.

In mouse infection process, cause the location of the OspC linear epitope of antibody response.In order to identify the linear epitope of the A type OspC that causes antibody response in course of infection, produced several detection from Borrelia burgd fragments, and with α-Borrelia burgdoyferi B31MI, infected serum (the 8th week) and screen (Fig. 3).B31MI is the bacterial strain that produces A type OspC.The expression of recombinant protein confirms by the immunoblotting of the S-albumen with HRP coupling.In order to locate the linear epitope of OspC, with the immunoblotting that infects serum screening OspC fragment.Two structural domains that contain one or more epi-positions have been located, one between 168 and 203 residues of half α spiral 5 of the C-of albumen end, another is at spiral 3 and encircle between 136 and 150 residues of 5 and (be called below α 5 and ring 5 epi-positions).These epi-positions were not characterized in the past in the literature.

The computer simulation of ospC sequential analysis and OspC structure.In order to determine α 5 and the locus of ring 5 epi-positions on OspC albumen, assessment is analyzed (Eicken etc., 2001 by X-radiocrystallography; Kumaran etc., 2001) definite coordinate, and the A type OspC of monomer and disome form is produced to band and space-filling model (data do not show).The I of monomeric form and E type OspC albumen also simulated.These analyses show on A, the E of monomer and disome form and I type OspC albumen, and encircling 5 epi-positions is all that surface exposes.In initial X-radiocrystallography is analyzed, the part of N-and C-end or be not a part for recombinant protein, or can not simulated.Under any circumstance, the structure of being determined shows that the position of N-and C-end is very close to each other, and approaches cytolemma.

For the sequence variations in assessment ring 5 and α 5 epi-positions in level in type, 227 OspC sequences are compared.These analyses show, encircle 5 and α 5 epi-positions in level between type, be all alterable height, but in type, have quite high conservative property.Fig. 4 provides ring 5 and the α 5 structural domain sequences of (with the form of form) every kind of OspC type, and has indicated the frequency of each particular sequence detecting in the OspC sequence of analyzing.As the evidence of the conservative property of horizontal pressed on ring 5 in type, to 57 A type ring 5 epitope sequences relatively to demonstrate 53 sequences that show with outside identical, only have the difference of 1 or two residue.For α 5 epi-positions, also observed similar result.In 43 A type OspC sequences, there are 42 between 168 and 203 residues, to be identical.Notice that analyzed α 5 epitope sequences are less, this is because the sequence that can obtain in database is in many cases incomplete, has lacked the variable quantity of C-end.

Proof is not only for individual mouse for the antibody response of ring 5 epi-positions.In view of conservative property and relatively short length thereof in the type of ring 5, encircling 5 epi-positions may be extraordinary candidate, can be used for developing the chimeric vaccinogen based on OspC ring 5.In order to confirm that antibody response for ring 5 epi-positions occurs conventionally in course of infection and not to be only for individual mouse, use the serum from other several the mouse that infected by A type OspC producing bacterial strain B31MI, LDP56 and 5A4, the immunoblotting of the ring 5 that examination contains 130-150 bit slice section.In all cases, the antibody (Fig. 5) of identifying this epi-position detected.Although in the infection serum of the mouse 2 infecting from LDP56, a little less than the reaction for ring 5, it is antigenic that the exposure of long period clearly shows ring 5 in this animal.This has confirmed that the immunne response for these epi-positions is not only for individual animals, and provides further support for its possible application in vaccine development.

Discuss

OspC is clearly defined as the nosogenetic important factor of Lyme disease (Grimm etc., 2004; Pal etc., 2004; Schwan etc., 1995).Have strong evidence show it lyme disease spirochete therefrom intestines in the transfer of sialisterium, brought into play vital role (Pal etc., 2004).In addition, it is expressed by selectivity in early days in infection, is advantage immunizing antigen (Fingerle etc., 1995; Schwan etc., 1998; Wilske etc., 1993), and by others, to be assumed to be in the transmission capacity of Lyme disease strain isolated be crucial determinant (Seinost etc., 1999).The object of this research is the potential cognation between check OspC type and invasive infection, determine whether the antibody response for OspC is type specific, and by the linear epitope of presenting in course of infection being positioned to the antigenicity structure that further defines OspC.

21 different OspC types (Seinost etc., 1999) have been depicted in the sequential analysis of OspC, have inferred and in them, have only had 4 kinds of (A, B, I and K type) relevant with the invasive infection in the mankind (Seinost etc., 1999).But nearest research has proposed query (Alghaferi etc., 2005) to the cognation of this supposition.In order further to illustrate this problem, determined from the aggressive of Maryland State mankind patient recovery and the OspC type of Non-Invasive Lyme disease strain isolated.In order to complete this task, total length ospC gene is by pcr amplification, order-checking, and carried out comparative sequential analysis.These analyses show that OspC type relevant with aggressive human infection in this patient crowd also comprises C, D and N-type.Although proposed I type OspC producing bacterial strain, be the main Types (Seinost etc., 1999) relevant with aggressive human infection, in the patient crowd of the Maryland State, do not identify the aggressive strain isolated that carries I type ospC.Similarly, Alghaferi etc. do not detect I type OspC producing bacterial strain (Alghaferi etc., 2005) yet.Integrate, these two researchs have identified 18 kinds of aggressive strain isolateds in area, large Baltimore, are classified as follows: A, n=5; B, n=2; C, n=1; D, n=1; H, n=1; K, n=3 and N, n=7.Therefore,, in this geographic area, the aggressive strain isolated that shows generation A and N-type OspC is preponderated.These data with only have 4 kinds of OspC types and the mankind in the relevant hypothesis of invasive infection run counter to.Also need the strain isolated that the larger patient crowd from different geographic regions is reclaimed to carry out more analyzing further to assess the verity of OspC type-invasive infection dependency, and determine the difference that whether has specific OspC type popularity degree in the geographic area limiting.

Use OspC to carry out variable provide protection that preventive vaccination provides in conjunction with describe (Seinost etc., 1999) of different OspC types, having increased antibody response may be the possibility of type specific.This hypothesis has obtained true support, with OspC preventive vaccination, is found only to provide the provide protection for same bacterial strain (Bockenstadt etc., 1997; Gilmore etc., 1999a; Probert and LeFebvre, 1994).Until before this report, the type specific for the antibody response of OspC in course of infection was not also directly assessed.In order to illustrate this problem, use the infection serum producing in the mouse of clonal population of expressing known OspC type, screened a series of A, B, C, D, H, K and N-type total length detection from Borrelia burgd albumen.The use of infecting serum is important, because this allows the antibody response of the central evaluation directed toward bacteria epi-position that specificity is presented in vivo.These analyses demonstrate, although there is strong sequence conservation in the N of OspC and C-terminal structural domain, for the antibody response of analyzed OspC type, are type specifics.For example, the serum of the mouse of use by oneself A or D type strain infection is immunoreactive in type specific mode, with other OspC type cross-immunity seldom or do not have.Although there is no to analyze the antibody response to all 21 OspC types, but it is not immunodominance that the data sheet showing above understands conservative structural domain, and the linear epitope of the OspC being presented by bacterium in course of infection is comprised in the variable domains of albumen (being type specific structural domain).

Up to the present several the location of epi-position or the researchs of evaluation of exploring OspC have only been delivered.Linearity and conformational epitope are all identified.Gilmore and Mbow have proved respectively that outside the leading peptide short independent N-end that reaches 6 residues is deleted and C-end cuts out 13 residues eliminations and knows clearly and the combination of the monoclonal antibody B5 (1999a such as Gilmore; Gilmore, 1998).From this result can inference B5 monoclonal antibody the epi-position that limits of identification conformation (Gilmore etc., 1999b).In the epi-position limiting in this conformation, do not identify the accurate residue that comprises antibody recognition site.With with monoclonal antibody B5, observe contrary, analysis for the Anti-TNF-α precursor reactant of the natural OspC of cell association shows, deletes the identification that the IgG causing in the infected process of OspC can not eliminated in last 10 residues of C-terminal of OspC or the extension area of N-terminal.Difference in this result infers it is to pay close attention to polyclonal antibody or the reflection of monoclonal antibody.These data are not got rid of the existence of conformational epitope certainly, have clearly proved yet and in OspC, have had linear epitope simultaneously.In research early, Mathieson etc. have also reported the linear epitope in OspC (Mathieson etc., 1998).They find that 7 residues of C-terminal of OspC have formed by the linear epitope that IgM identified the serum of collecting from the neural burgdorferi patient in Europe.Although do not assess IgM combination in this report, delete 10 residues of C-terminal of OspC and can not eliminate IgG combination.The epi-position demonstration of the IgG identification of infected induction is positioned on several sites of albumen.But, this do not show that C-terminal epi-position does not exist or not infected during the antibody that causes identify, but be illustrated in, on other position in OspC, there is other epi-position.

The immunoblotting assay of shorter OspC fragment is allowed to the more accurately location to OspC epi-position.The antigenicity district of OspC is positioned to two regions.One strides across 136-150 position residue, and another strides across 168-210 position residue.The structural models that uses the coordinate generation of X-ray diffraction analysis acquisition, is mainly placed on 136-150 position residue in the ring of surface exposure, is called ring 5 (Kumaran etc., 2001).In ring 5 monomers at OspC and two body Models, be all that surface exposes, and be arranged in obvious turning.Although confirmed that detection from Borrelia burgd in fact forms disome, also do not determine whether natural OspC forms disome or larger oligomer in vivo.Two body Models of OspC have pointed out that one obviously by the interface of embedding, and it has comprised albumen more than >30%.The embedding interface of this degree has shown to interact closely between monomer, and the disome form that is considered to albumen is indication (Kumaran etc., 2001 of biologic activity form; Eicken etc., 2001; Zuckert etc., 2001).In OspC disome, encircle the part that residue in 5 is expected to be the ligand binding pocket that the conformation of inferring limits, may there is biology importance.This charged bag is arranged with the amino acid that contains carbonyl, for example L-glutamic acid and asparagicacid residue.For the OspC albumen of A, I (Kumaran etc., 2001) and E (Eicken etc., 2001) three types after measured crystalline structure.In all these albumen, the structure in solvent of the binding pocket that this is inferred is obvious high conservative.Encircle 5 pairs of accessibilities that infect the antibody in serum supported this structural domain may be surface expose with the hypothesis that may can be used for ligand binding.Although encircle 5 and the ligand binding pocket of inferring there is structure conservative property between strong type, the sequence of this structural domain is alterable height in level between type.The sequence that α crosses over 5 structural domain 168-210 positions residue is also variable in level between type, except last 20 residue high conservatives.In order to determine, in type, under level, whether exist enough conservative propertys can build the chimeric OspC vaccine being formed by a series of type specific epi-positions, the sequence of OspC to be compared, and built tree derivation.By these, analyze, determined the OspC type (data do not show) of 227 sequences.Found to encircle 5 and α 5 epi-positions in type, in level, be all high conservative.For example, in 54 in 57 sequences, ring 5 epi-positions of A type OspC albumen are consistent, and in 42 of 43 A type sequences, α 5 epi-positions are guarded.In other OspC type, also notice the remarkable conservative property of these structural domains, at C, in I, M, N and O type, in ring 5 and α 5 epi-positions, shown conservative property in absolute type.

Originally studies confirm that in aggressive strain isolated and existed than the larger OspC diversity of recognizing in the past.This research has also confirmed that the antibody response for OspC is mainly type specific in mouse, is to be limited by the ring 5 and α 5 epi-positions that do not characterize in the past.The data of research early and submission here have clearly confirmed that single OspC albumen can not give the provide protection for diversity bacterial strain (Bockenstedt etc., 1997).A kind of possible preventive vaccination method is to utilize the chimeric OspC vaccinogen of epi-position exploitation restructuring of identifying in this report.Encircle 5 epi-positions or encircle 5 and the combination of α 5 epi-positions, if be proved to be, there are consistent antigenic words in the mankind, maximum hope can be provided.These epi-positions are relatively short in length, are linear, and in type high conservative in level.According to these features, can prove, ring 5-α 5 chimeric immunogens that structure can be given for the provide protection of highly diversified Lyme disease strain isolated are feasible technically.

In embodiment 2, the mankind for A type OspC encircle 5 structural domains antibody response analysis and encircle 5 epi-positions can applicablely assess in ImuLyme exploitation

The outer surface protein C (OspC) of lyme disease spirochete is advantage immunity (Fuchs etc., 1992) antigen of 22kDa, when tick take food and infection early expression (Schwan etc., 1995).Although produce for the strong antibody response that produces OspC in natural infection process, this reaction can not cause bacterium to be eliminated, because (Schwan etc., 1995) have just been closed in being created in after infection is established of OspC soon.OspC occurs as the potential candidate of important virulence factor and ImuLyme exploitation.But the effort of the vaccine of exploitation based on OspC has been subject to its heterogeneous obstruction (Theisen etc., 1993 between bacterial strain; Wilske etc., 1996; Wilske etc., 1993).Although caused the reaction with height protectiveness with OspC preventive vaccination, what great majority research was reported is only the specific provide protection of bacterial strain (Bockenstedt etc., 1997; Gihnore etc., 1996; Mbow etc., 1999; Probert etc., 1997; Rousselle etc., 1998; Scheiblhofer etc., 2003).Nearest analysis is for we provide important understanding to the basic understanding of the antigenic structure of OspC and the provide protection of bacterial strain specificity.21 kinds of OspC types have been defined, name (Lagal etc., 2003 from A to U; Seinost etc., 1999; Wang etc., 1999).The Borrelia burgdoyferi clone population infecting mouse that produces specific OspC type by use, has confirmed that infecting early antibody reaction be mainly (referring to the embodiment 1) of OspC type specific.This shows that the Dominant Epitopes of presenting in early days in infection may be positioned at the type specific structural domain of OspC.Although research early shows to only have 4 (Seinosts etc. relevant to invasive infection in 21 OspC types, 1999), but the nearest strain isolated that produces other OspC type that studies confirm that also can be set up invasive infection (Alghaferi etc., 2005; Embodiment 1).But A type OspC is obviously main in the bacterial strain that causes mankind's invasive infection.One of advantage linear epitope that the epitope mapping analysis of A type OspC shows initiation reaction in mouse is arranged in ring 5 structural domains (referring to embodiment 1).Encircle 5 structural domains is alterable height in level between type, but in the sequence of given type, is (referring to the embodiment 1) guarding.In this research, we have refined table bit position, have confirmed that its surface on intact bacterial exposes, and have confirmed that it has caused bactericidal properties antibody.

The research that great majority are sought the immunodominant epitope of definite OspC is (Bockenstedt etc., 1997 of using mouse to carry out; Gilmore etc., 1996; Mbow etc., 1999; Probert etc., 1994).But the verified antibody response for some epi-position is different (Lovrich etc., 2005) the mankind in respect to mouse and other Mammals.First object of this research is that the antibody whether ring 5 structural domains of definite OspC are caused during human infection is identified.Ideally, the serum of collecting the individuality that these analyses can be infected with the clone population from A type producing bacterial strain carries out.Because people can not determine blood surely individuality by allos or by the population of homology, infected, so we seek to identify that to A type specificity sequence table reveals the patients serum of reaction.In order to complete this task, by Enzyme Linked Immunoadsorbent Assay (ELISA), one group of serum sample collecting from suffer from the patient of migratory erythema (Lyme disease commitment) is screened.Restructuring A type OspC and the subfragment that contains the restructuring A type OspC that encircles 5130 to 150 residues are used to coated 96 orifice plates (250ng recombinant protein/hole; 0.1M Na ₂hPO ₄; 4 ℃ are spent the night).By plate sealing (10% skim-milk in phosphate buffered saline (PBS), 0.5% Tween 20; 37 ℃ 2 hours) and clean, in each hole, add (37 ℃ of mankind's Lyme disease patients serums (1:400 dilution); 1 hour).Anti-human immunoglobulin G (the IgG of goat that adds horseradish peroxidase; Sigma) (50 μ l, 1:40,000 dilution) (1 hour; 37 ℃), then according to supplier's (Sigma) explanation, add tmb substrate (3,3,5,5-tetramethyl benzidine).The optical density value that plate device is measured 450nm is read in use.With bovine serum albumin, be coated with other hole as negative control.All analyses are all carried out three parts.Average A450 value and standard deviation have been shown.As shown in Figure 6, find that several serum samples all have strong IgG reaction to total length A type OspC and ring 5 fragments.Serum sample 8 and 44 and ring 5 fragments demonstrate the strongest immunoreactivity, be therefore selected for further analysis.

For the residue of determining that more accurately the antibody of infected induction in ring 5 structural domains is identified, use from the serum of patient 8 and 44 and from A type OspC, produce the mouse that the clone population of strain B13 MI infects serum screening PepSpot array (referring to embodiment 1).PepSpot array forms (2 amino acid steps) by 12 to 13 the overlapping peptides of residue crossing over ring 5 structural domains of A type OspC, and they are spotting (150nmol/cm on Whatman 50 cellulose membranes ²; JPT Peptide Technologies GmbH, Berlin, Germany).By PepSpot film blocking-up (5% skim-milk-0.5% Tween 20 in phosphate buffered saline buffer), clean, with mouse and serum human sample (respectively with blocking-up liquid 1:1000 and 1:400 dilution), screen, use kind of paraspecific anti-IgG antiserum(antisera) to detect the combination of antibody.Although the concrete residue that forms immunoreactivity structural domain some difference slightly in mouse and people, main epi-position is positioned at (Fig. 7) among 130 to 146 residues.In A type OspC sequence, the C-terminal region of α spiral 3 and the N-terminal portions of ring 5 have been contained in this region.

The crystalline structure of OspC is spatially placed on (Eicken etc., 2005 in the obvious bending of albumen by ring 5; Kumaran etc., 2001; Figure 24).It is a part (Kumaran etc., 2001) for possible ligand binding pocket that this ring has been pushed off.In order to determine whether ring 5 is illustrated in the spirochete of cell surface and growth in vitro, can approach antibody, use anti-ring 5 antiserum(antisera)s to carry out immunofluorescence analysis (IFA).Use Borrelia burgdoyferi B31MI (A type OspC), the full cell pyrolysis liquid of B.parkeri and proved that with the immunoblotting assay that the restructuring A type OspC of S-label carries out ring 5 antiserum(antisera)s are specific, has set up the suitability of this antiserum(antisera) to IFA.The bacterial strain of analyzing by IFA is comprised of Borrelia burgdoyferi B31 MI (A type OspC) and LDP74 (K class OspC).Spirochete, 33 ℃ of growths, is transferred to 37 ℃ of growths expression with stimulation OspC in 3 days.The cell that IFA use is changed thoroughly (acetone is fixed) and the not cell of saturatingization (air dried) carry out, and standard method is according to former description (Roberts etc., 2002).Slide glass is used mouse ring 5 antiserum(antisera)s, the serum before mouse immune or the rabbit flagellin antiserum(antisera) of 1:1000 dilution to screen.Detect and use the goat α-mouse IgG of Alexa Fluor 568 couplings or the goat α-rabbit igg of Alexa Fluor 488 couplings (10 μ g/ml, in blocking-up damping fluid) to carry out.If suitable, slide glass is used rhodamine or fluorescein filter set to observe under Olympus BX51 fluorescent microscope, or observes by dark-field microscope, and uses Olympus MagnaFIRE photographic camera to take a picture.The mark of observing by IFA is high degree of specificity, and consistent with immunoblotting assay; The strain isolated that produces A type is surface markers, and the K type OspC of Borrelia burgdoyferi LDP74 is not (data do not show).In addition, consistent with the rise of OspC at elevated temperatures, it is obviously more that IFA demonstrates the cell that is grown in the spirochete surface markers of 37 ℃ than being grown in 33 ℃.The cell that α-FlaB antiserum(antisera) of the periplasm protein of identification inner membrance grappling can not mark not be changed thoroughly, but the cell (data do not show) of saturatingization of acetone for mark easily.It is in fact that surface exposes that this contrast has confirmed to encircle 5 epi-positions, and is used in the integrity that experiment condition in IFA does not destroy cell, thereby and artificially to have exposed be not the natural epi-position being presented on bacterium surface.

Encircling 5 antiserum(antisera)s has effectively increased in conjunction with the ability of the OspC of cell surface that to interact may be bactericidal possibility, (Bockenstedt etc., 1997 having proved as the antibody for for total length OspC; Ikushima etc., 2000; Jobe etc. 2003; Lovrich etc., 2005; Rousselle etc., 1998).In order to determine whether the antibody of target fixed ring 5 also shows fungicidal activity, use at 33 ℃ or shift to the Borrelia burgdoyferi strain isolated B31MI and the LDP74 that at the temperature of 37 ℃, cultivate and carried out sterilization analysis.By centrifugal results spirochete, cleaning, adjust to every 500 μ l 5x10 ⁵individual cell (in BSK-H substratum), then transfers to 12.5 μ l in the Eppendorf tube of aseptic 0.65ml.Then add (56 ℃ of 10 μ l heat inactivations; 30 minutes) ring 5 serum, add or do not add GPC (7.5 μ l; Sigma Chemical, St.Louis, Mo.), be incorporated in 33 or 37 ℃ and hatch 8 hours composition is mixed.Add the water of 70 μ l altogether, use Live/Dead BacLight dyestuff (Molecular Probes, Eugene, Oreg.) according to the specification sheets of manufacturers, spirochete to be dyeed.In simple terms, two kinds of dyestuffs are joined in cell; SYTO 9 and propidium iodide.These dyestuffs can be differentiated the bacterium of bacterium alive (having complete film) and film damage.The bacterium living is owing to being dyeed and sending green fluorescence by SYTO 9, and bacterium dead or that damage is owing to being dyeed and sending red fluorescence by propidium iodide.After the serum (containing or do not contain complement) of the heat inactivation with before immunity is processed, the baseline values of the cell with destroyed film of observing is 25%.On the contrary, being exposed to anti-α-encircle 5 sero-fast cells has about 70% to demonstrate film destroy.Fungicidal activity is determined to be complement-dependent.With after anti-ring 5 antibody treatment, consistent (Bockenstedt etc., 1997 that the Cavitation effect of here observing is reported when using other anti-OspC antibody; Escudero etc., 1997).The rise that is important to note that in addition OspC while raising with temperature is consistent, and in the spirochete growing at 37 ℃, the percentage of dead cell is same than the bacterium high (data do not show) being grown at 33 ℃.It is bactericidal from these data, having clearly stated anti-ring 5 antibody.

Several parts of reports are for exploitation ImuLyme has summarized clear strong reasonableness (summary is shown in Hanson and Edelman, 2004).But, up to the present, also there is no business-like vaccine.In exploitation, have in the effort of ImuLyme of extensive protectiveness, Baxter carries out the strategy (Hanson and Edelman, 2004) of the vaccine mixture of 14 kinds of different total length detection from Borrelia burgd albumen of a kind of generation.But this mixture is considered to have unacceptable reactionogenicity.Reactionogenicity may be by cause in mixture every type OspC albumen unique protective epitope enough reactions and a large amount of albumen of needing are caused.The potential problem of using the mixture vaccine of multiple full-length proteins be mistakenly guide pin to possibility conservative, antibody response irrelevant, non-protective epi-position.The chimeric recombinant immune that the advantage immunity linear epitope of being presented by every kind of advantage OspC type natural by exploitation forms is former, may can overcome this problem.This general concept is derived from the trial (Hanson and Edelman, 2004) of the epi-position exploitation malaria vaccine of the albumen that uses comfortable different infective stage expression.Same concept has been applied in the exploitation of the streptococcic sexavalence M protein vaccine of A type (Dale, 1999), and in the exploitation for the vaccine of several other pathogenic agent, and obtained great success (Apta etc., 2006; Caro-Aguilar etc., 2005; Fan etc., 2005; Horvath etc., 2005; Kotloff etc., 2005; McNeil etc., 2005; Wang etc., 2005).Along with the new understanding to the physics of OspC and antigenic structure, exploitation is now effective, restructuring OspC vaccine multivalence, chimeric is possible.New ring 5 structural domains of identifying are very suitable for being included in such vaccine.

Embodiment 3, the former exploitation of the restructuring chimeric of the tetravalence based on OspC

Lyme disease is the modal arthropod-borne disease in North America and Europe.At present, also do not have business-like vaccine to can be used for the mankind.Outer surface protein C (OspC) has antigenicity and expression characterization, makes it become attractive vaccine candidate person; But the heterogeneity of sequence has hindered it as vaccinogen.21 clearly defined OspC germline groups or " type " (called after A is to U) have been identified in sequential analysis.This research report the mapping of the linear epitope that B, K and D type OspC present in the mankind and Infection in Rodents, and utilize the tetravalence chimeric of these epi-positions (and A type OspC linear epitope of identifying in the past) exploitation restructuring former.Construct is found in the immunogenicity in mouse with height, the spirochete that derivative antibody can surface markers vitro culture.Importantly, vaccine inoculation induction is for the complement-dependent bactericidin of the bacterial strain of the every type of OspC mixing in expression construct.These results show, effective and extensive protectiveness, based on OspC, multivalence ImuLyme can be used as restructuring chimeric protein and produced.

Materials and methods

Borrelia burgdoyferi strain isolated and cultivation

The clone population [referring to embodiment 1] of Borrelia burgdoyferi strain isolated B31MI (A type OspC), LDP73 (Type B), LDP116 (D type) and LDP74 (K type) obtained [Sung etc., 2000] according to former description by bed board under liquid level.Each clone's OspC type by pcr amplification, (Promega) determine with the DNA sequencing of ospC, and assign type [referring to embodiment 1] by Phylogenetic Analysis by Taq polysaccharase.Spirochete is being cultivated in BSK-H substratum (Sigma) completely as indicated at 33 or 37 ℃.

Do not rely on the clone of ligase enzyme and the production of restructuring (r-) OspC albumen

The pcr amplification of the corresponding gene by each strain isolated, has produced B, K and D type OspC and a series of truncate and the fragment of total length.Primer is designed to have 5 ' protuberance, to allow not rely in pET-32 Ek/LIC carrier clone's (LIC) (table 3) [embodiment 1] of ligase enzyme.All LIC methods are carried out according to the explanation of manufacturers (Novagen) substantially.In simple terms, after amplification and regeneration strand tail, by amplicon and the annealing of pET-32 Ek/LIC carrier, be then transformed in NovaBlue (DE3) Bacillus coli cells and breeding.Reclaim plasmid and by DNA sequencing, confirm the sequence of Insert Fragment.For protein purification, the plasmid of purifying is used to transform e. coli bl21 (DE3) cell, and by add IPTG (1mM) inducible protein to express in substratum at logarithmic phase, then continues insulation 3 hours.By the N-end adding from pET-32Ek/LIC vector expression, merge and contain Trx-label, S-label and hexahistine (His-label) motif.His-label is used to allow by nickel affinity chromatography purification of recombinant proteins.In simple terms, by lysis, with benzonase nuclease and restructuring N,O-Diacetylmuramidase, nucleic acid and whole cell peptidoglycan are degraded respectively.According to the explanation of manufacturers (Novagen), soluble albumen centrifugal (16000xg, 15 minutes) clarification, by fixing nickel post, cleans and wash-out.The film (Slid-a-lyzer, Pierce) that eluted albumen use molecular weight cut-off is 10kDa is to phosphate buffered saline buffer (PBS; PH7.4) carry out degree of depth dialysis, final protein concentration is analyzed (Pierce) by BCA and is carried out quantitatively, and the purity of prepared product is assessed by SDS-PAGE.

Table 3, for generation of the PCR primer of all kinds OspC fragment.LIC afterbody represents with runic.

The epitope mapping of immunoblotting assay: B, D and K type OspC.

In order to allow that epi-position relevant between period of infection is mapped, with 104 spirochete clone populations of expressing B, K or D type OspC, infect C3H/HeJ mouse, at the 2nd, 4,6,8 and 12 weeks, by afterbody, get blood and collect blood.These serum are used to screen OspC albumen and the truncate of purifying according to the description in embodiment 1.Recombinant protein is carried out to SDS-PAGE, transfer to PVDF upper, with the type specific mouse infection serum (collecting at the 6th week) of 1:1000 dilution, screen.Equally, recombinant protein is used for screening from the patient's of the Borrelia burgdoyferi strain infection of the known B of being expressed, K or D type OspC serum (1:400) (being provided by Dr.Allen Steere kindness).The second antibody of using applicable IgG specificity horseradish peroxidase (HRP) coupling, result shows by chemoluminescence.

The structure that tetravalence chimeric is former and expression.

Select ring 5th district (131 to 149 amino acids) of A type and α spiral 5th district of B (160-201 amino acids), K (161-201 amino acids) and D (161-201 amino acids) type be included in tetravalence experimental vaccine former in.Use each region of containing epi-position of primer pair of listing in above-mentioned recombinant plasmid and table 4 to carry out pcr amplification.PCR condition is standard, and starting is 2 minutes denaturing steps of 94 ℃, and 15 seconds, the 50 ℃ primer annealings of 94 ℃ of sex change that then carry out 35 circulations extend 60 seconds for 30 seconds and 72 ℃, and last 72 ℃ are extended 7 minutes.The protease resistant joint sequence that primer is designed to have the LIC afterbody of carrier specificity or has structure is not as 5 ' protuberance (Fig. 9 A) [Crasto and Feng, 2000].All amplicons in the sepharose that uses TAE damping fluid, by electrophoresis, analyze and carry out gel-purified (QiaQuick gel extraction kit, Qiagen).Then in the PCR of subsequent rounds, use the product being purified as template.In second takes turns, the amplicon of A type ring 5 and Type B α spiral 5 is merged as template.After sex change, amplicon is annealed by their acomplementary connector sequence, to allow using A type ring 5 forward primers and Type B α spiral 5 reverse primers to carry out overlapping extension and amplification subsequently.K and D type α spiral 5 sequences are added in construct with the same manner, except annealing temperature being elevated to 60 ℃ after front 10 circulations to increase annealing specificity.Final product is annealed to coding N-end hexahistine label and merges in the pET-46Ek/LIC expression vector of (Novagen), transforms NovaBlue (DE3) Bacillus coli cells.By the plasmid of purifying is carried out to the sequence that DNA sequencing confirms vaccinogen.The expression of albumen and purifying complete according to description above.

Table 4, for generation of the former PCR primer of ABKD chimeric.LIC tail represents with runic, and joint sequence is by underscore.

Use the former immune mouse of tetravalence ABKD chimeric.

The male C3H/HeJ in 26 week age is that complete Freund's adjuvant for mouse (CFA) is by the former immunization of carrying out of 50 μ g chimeric of 1:1 ratio emulsification.The cumulative volume of the vaccinogen of administration is 200 μ L, is separated in intraperitoneal and subcutaneous lipids.The PBS that three control mice are used in CFA carries out false immunization.At the 2nd and the 4th week, the 50 μ g albumen that mouse is used in Freund's incomplete adjuvant were strengthened.The mouse of false inoculation is accepted the PBS in adjuvant.Before injection first and the 6th week time, by cutting tail, all mouse are got to blood.

The immunogenic assessment that ABKD chimeric is former

By the immunogenicity of immunoblotting assay and ELISA assessment vaccinogen.Immunoblotting produces and screening according to description above.By immunoblotting, the recombinant protein of every kind of purifying (A, B, K, D type OspC and chimeric are former) 1 μ g is analyzed.The BBN39 of restructuring, a kind of irrelevant albumen with His-label (paralog protein family 163) from Borrelia burgdoyferi, is used as negative control.In order to confirm same albumen carrying capacity, by the monoclonal antibody (mAb) (1:2000, Novagen) of anti-His label, screen a spot.In order to assess the reaction to immunization, same spot screens with the anti-ABKD antiserum(antisera) of mouse of 1:500 dilution.The goat anti-mouse IgG of HRP coupling (1:40000 dilution) is used as second antibody, by chemoluminescence, observes combination.Elisa assay is used 96 orifice plates, and (Costar 3590; Corning) carry out, each for hole the 100ng vaccine constructs in carbonate buffer solution (pH9.6) or detection from Borrelia burgd (A, B, K or D type) be coated with (4 ℃ 16 hours).By plate sealing, (PBS of 1% BSA, contains 0.2% Tween-20 (PBS-T); 2 hours), with PBS-T, wash 3 times, in the hole of double plate, add anti-ABKD antiserum(antisera) (the 100 μ L of serial dilution; 1:50 is to 1:109350 dilution) (1 hour).The goat anti-mouse IgG of HRP coupling (1:20000) is used as second antibody, and ABTS is as chromogenic substrate.(ELx 808 at ELISA, to read plate device; Biotek) upper, when speed of response is linearity, at 405nm, read absorbancy.By to light absorption value curve with four parameter logical equatiion (SigmaPlot) matching sigmoid curves and calculate 50% the dilution inverse corresponding to high absorbance platform, calculate titre.

The Immunoglobulin Isotype of anti-ABKD antibody response distributes

By distributing with the isotype that 100ng chimeric construct body coated double elisa plate in every hole is assessed anti-ABKD antibody response.According to description above, wash plate and sealing.Double has been analyzed anti-ABKD antiserum(antisera) (the 100 μ L that collect from 12 immune mouse of quilt; 1:10000 doubly; 1 hour).Combined vaccinogen specificity Ig is by hatching (1 hour with the special biotinylated second antibody of isotype; Mouse isotype test kit; Zymed) detect.In conjunction with streptavidin (30 minute) and the chromogenic substrate ABTS of biotinylated antibody by HRP coupling detect.All hatching all completes in room temperature.

Indirect immunofluorescence analysis (IFA)

For the surface of determining whether epi-position that ABKD chimeric comprises in former presents the Borrelia burgdoyferi of cultivating in vitro, carried out IFA analysis.In order to maximize the production of OspC, the temperature of the culture of the clone population of generation A, B, K and D type OspC is changed into 37 ℃ from 33 ℃.By centrifugal (7000xg, 15 minutes), collect the dense culture of 5mL (about 10 ⁷-10 ⁸individual cell/mL) spirochete in, washes 3 times with PBS, and Eddy diffusion, in 5mL PBS, is layered on 100 μ L the 2cm of charged slide (Superfrost Plus, Fisher Scientific) ²on area.One group of slide dry air, another group is fixed with acetone.By slide sealing (1 hour, the PBS-T of 3% BSA), the anti-flagellin antiserum(antisera) of rabbit of the anti-ABKD antiserum(antisera) then diluting with 1:100, preimmune serum or 1:1000 dilution screens (1 hour).Combined antibody detects by the goat anti-mouse IgG of Alexafluor 568 couplings or the goat anti-rabbit igg of Alexafluor 488-coupling (10 μ g/mL sealing damping fluid).Between each step, slide is washed 3 times with PBS-T, and all hatching is all in room temperature, to carry out 1 hour in the darkroom of humidification.Slide is with Fluoromount-G (Electron Microscopy Sciences), optionally under Olympus BX51 fluorescent microscope, by rhodamine or fluorescein filter set, observe, or by dark-field microscope, observe, use Olympus MagnaFire digital camera to take a picture.

The assessment of fungicidal activity

The anti-ABKD antiserum(antisera) of external assessment kills the ability of Borrelia burgdoyferi.As mentioned above, temperature is washed 3 times with BSK-H substratum from 33 ℃ of spirochetes that move on to 37 ℃, cell density is adjusted to about 10 ⁶cell/mL.8 μ L cells are mixed (at 56 ℃ of heat inactivations with 8 μ L GPCs (Sigma) and every kind of test serum of 4 μ L; 30 minutes).Contrast comprises the anti-ABKD antiserum(antisera) of heat inactivation that does not contain complement, the preimmune serum that only has the heat inactivation that contains complement of complement and merging.Optionally, by adding BSK-H substratum that total reaction volume is adjusted to 20 μ L, sample is hatched 18 hours at 37 ℃.Use BacLight LIVE/DEAD to analyze (Molecular Probes), with use the OlympusBX51 fluorescent microscope with fluorescein or rhodamine filter set to carry out artificial counting to the cell with dead/damage of living in 5 high magnification visuals field, situation is killed in assessment.

Result

B, the D presenting in evaluation mouse and human infection's process and the epi-position of K type OspC

Up to the present, from determined the patient who suffers from invasive infection, A, B, C, D, H, I, K and N-type OspC[Seinost etc. have been reclaimed, 1999; Embodiment 1; Alghaferi etc., 2005].From these OspC types, selected 4 (A, B, K and D) to come for using the principle of polyvalent chimeric OspC vaccine to set up checking.Because only identified the epi-position of the A type OspC presenting between period of infection, therefore in this research, the first step is to identify B, K and epi-position relevant to infection in D type OspC.In order to complete this task, use from the serum of the mouse being infected by the clone population of Borrelia burgdoyferi (B, K or D type OspC) or be determined to mankind's Lyme disease patient's the serum (personal comminication that small part is produced the Borrelia burgdoyferi strain infection of B, K or D type OspC, Dr.Allen Steere and Kathryn Jones), the immunoblotting of the truncate of every type or fragment is screened.Antibody response in mouse the 6th week time is type specific; But some serum human demonstrates the cross-immunoreactivity (data do not show) between type, the spirochete population mixing that shown these patient's PIs.For mouse infection serum, the epi-position of Type B OspC is arranged in α spiral 5 (between 175 and 200 amino acids).The mankind's infection serum fragment (164 to the 185 amino acids) reaction identical with position, shows that α spiral 5th district of Type B are antigenic.In mouse, the epi-position of K type OspC is mapped between 148 to 160 amino acids, is plotted on (between 160 to 175 amino acids) in α spiral 5th district in the mankind.In mouse and the mankind, the epi-position of D type OspC is mapped in α spiral 5th district (between 167 to 180 amino acids), although serum human is identified other a plurality of epi-positions.These data show to be applicable to selecting α spiral 5th district of B, K and D type OspC to be included in tetravalence ABKD vaccinogen construct.

Structure, expression and the purifying of the chimeric OspC vaccinogen of tetravalence

A type ring 5 epi-positions of using [embodiment 1] definition in B defined above, K and D type α spiral 5 epi-positions and research early, the chimeric recombinant vaccine that has produced the multivalence consisting of 4 regions of containing epi-position is former.These epi-positions are by short, structureless, to have protease resistant joint sequence be connected (Fig. 9 B).Recombiant vaccine raw footage is 169 amino acid, and molecular weight is 18.0kDa, and iso-electric point is 6.49.Its structure be predicted to be substantially spiral [Gasteiger etc. 2005; Kneller etc., 1990], and there is high stable index [Guruprasad etc., 1990].After dialysing with PBS, the more original precipitations of recombiant vaccine; But about 500 μ g/mL keep dissolving, the albumen of this dissolving is used to all experiments.The SDS-PAGE of the vaccinogen albumen of purifying analyzes the single band that has confirmed molecular weight 18kDa, there is no contaminating protein.

The former immunogenicity in mouse of ABKD chimeric

In order to assess the antibody response of the former and individual composition epi-position of ABKD chimeric, C3H/HeJ mouse is used to the vaccinogen in freund's adjuvant.From being collected serum by mouse (n=3) of (n=12) of immunity and false (PBS+ adjuvant) immunity, and assessment and the former reactivity with A, B, K and D type total length detection from Borrelia burgd albumen of ABKD chimeric.Western engram analysis has confirmed anti-ABKD antiserum(antisera) and vaccinogen albumen and A, B and K type detection from Borrelia burgd generation strong reaction.On the contrary, with the reactivity of the C-terminal D type OspC epi-position of chimeric construct body quite weak (Figure 10).Any serum does not all have reactivity with negative control albumen (r-BBN39), from the serum of false immune mouse not with any albumen test.The quantitative titration based on ELISA of serum reactivity has been confirmed to the high titre IgG reaction for the former albumen of ABKD chimeric, and average titer is 27800 (Figure 11 A).Combination by the total length detection from Borrelia burgd albumen to fixing is assessed, and has completed the reactive titration to the epi-position for type specific.Observed the antibody titers significantly different (Figure 11 B) for individual epi-position.Titre that it should be noted that epitope specificity reduces along with approaching the C-terminal of vaccinogen.

The sero-fast Immunoglobulin Isotype of anti-ABKD distributes

It is crucial that Immunoglobulin Isotype distributes for the potential effect device function of assessing vaccine specific antibody.In order to assess the heavy chain immunoglobulin classification conversion of the former induction of ABKD chimeric, by ELISA, determine that isotype distributes.Main isotype is IgG1, and that more lower slightly is IgG2a and IgG2b.The serum of 6 weeks only demonstrates IgM, IgG3 or the IgA (Figure 12) of limited level.

Indirect immunofluorescence analysis

By indirect immunofluorescence microscopic evaluation the antibody that causes for each former epi-position of the ABKD chimeric OspC on the burgdorferi cell surface ability of being combined.On the cell that produces A, B, K and D type OspC, observed specific surface markers (data do not show).The intensity of fluorescent signal is consistent with type specific titre, and has for the fluorescence with A or the viewed maximum strength of Type B OspC cell.With the fluorescence intensity of K or D type cell a little less than, dye irregular, make cell there is mottled appearance.In the cell of surveying with the preimmune serum of coupling, do not observe reactivity.Anti-flagellin antibody does not have surface markers to the fixing cell of air, and this is used to confirm that the adventitia of cell is complete, and the natural surface that is presented to cell of detected epi-position.As expected, with the permeabilized cell of acetone by anti-flagellin antibody institute's mark (data do not show).

The former preventive vaccination induction bactericidin that carries out of ABKD chimeric for proof

Use LIVE/DEAD BacLight to analyze [TiIy etc., 2001; Ledin etc., 2005; Elias etc., 2000; Montgomaery etc., 2006; Elias etc., 2002; Shin etc., 2004] assessed the sero-fast fungicidal activity of anti-ABKD.Detected for the fungicidal activity with being included in the bacterial strain of all types of OspC in chimeric construct.Hatch together with anti-ABKD antiserum(antisera), induced obvious cell aggregation.The cell with dead of living is all present in aggregate.Due to the intrinsic degree of difficulty that cell in aggregate is counted, only the free cell that does not have to assemble has been counted to determine percentage ratio that live and dead cell.For all 4 kinds of OspC types, the background level of the culture dead cell of analyzing for sterilization is approximately 20-30%.The background level of this dead cell with in our laboratory, culture is being transferred to 37 ℃ from 33 ℃ to raise observe OspC expresses consistent.In sterilization is analyzed, kill in the mode of complement-dependent and carry out, the percentage of dead cell is increased on background significantly, to 56-90%.The quantity of dead cell is at least the twice of the dead cell quantity of seeing in any contrast in all cases.Only with complement, do not cause sterilization.The preimmune serum merging does not cause fungicidal activity, and it is essential by fungicidal activity to show for the specific immune response of vaccinogen.

Discuss

OspC may apply as potential vaccinogen of lyme disease spirochete explored in several researchs.Although caused the antibody response of protectiveness by OspC immunization, having been reported provide protection is mainly specific [Gilmore etc., 1996 of bacterial strain; Scheiblhofer etc., 2003; Wallich etc., 2001; Brown etc., 2005; Probert and LeFebvre, 1994; Gilmore etc., 2003].The trial of using the mixture of multiple OspC albumen to cause wider provide protection is proved to be unsuccessful.Baxter has tested the OspC mixture being comprised of 14 kinds of different total length OspC varients; But they can not cause the enough antibody titerss for the unique texture territory of each varient, and this is the prerequisite that produces extensive provide protection.In addition also having reported, can not acceptable response originality [Hanson etc., 2004].General worry for mixture vaccine is may mislead for be not the natural antibody response of presenting and can not causing the epi-position of protection antibody in infection.The generation of chimeric provides a kind of alternative method, can avoid the problem that these run into when using the mixture of simple recombinant protein.For malaria, [Hanson etc. 2004; Caro-Aguilar etc., 2005], category-A suis [McNeil etc., 2005; Dale etc., 2005; Hu etc., 2002; Dale, 1999; Kotloff etc., 2005; Horvath etc., 2005] and several virus [Apt etc., 2006; Fan and Mei, 2005; Wang etc., 2005; In the exploitation of vaccine Bouche etc., 2005], explored the chimeric being formed by a series of immunodominant epitopes.If multivalence OspC vaccine will have provide protection widely, must in vaccinogen, mix abundant epi-position to cause the aversion response for various bacterial strain.Phylogenetic analysis greatly facilitates the ability that the structure towards this vaccinogen advances, 21 different OspC classifications in phylogenetic analysis, have been described, name is from A to U[Seinost etc., 1999], wherein only have a subclass be associated with the invasive infection in the mankind [Seinost etc., 1999; Embodiment 1; Alghaferi etc., n2005].

The former exploitation of chimeric need to be to the epi-position structure of OspC detailed understanding.Existing several reports had been described OspC table bit position [Gilmore etc., 1996 in the past; Jobe etc., 2003; Lovrich etc., 2005].Two researchs have reported that the epi-position of responsible initiation bactericidal properties antibody is arranged in C-terminal structural domain [Jobe etc., 2003 of OspC; Lovrich etc., 2005]; But because this structural domain guards relatively, why therefore unclear do not have provide protection widely for the antibody of C-terminal.Matheisen etc. also advise that C-terminal is the major objective of antibody response, and the reactivity of having noticed neural burgdorferi patient obtains from Europe serum and total length OspC is higher than having clipped the reactivity [Mathiesen etc., 1998] of 10 amino acid whose forms with C-terminal.From then on, deduction must be C-terminal epi-position; But because the antigen of test consists of the single OspC varient of UNKNOWN TYPE, the more extensively identification of C-terminal may be the higher conservative property due to this structural domain, and must not show that C-terminal is immunodominance.Gilmore etc. have confirmed with non-sex change, rather than sex change, detection from Borrelia burgd immunized mice has produced for the provide protection of the attack of homology strain isolated [Gilmore etc., 1996; Gilmore and Mbow, 1999], show that protective epitope may be that conformation limits.In another is analyzed, Gilmore etc. have analyzed the brachymemma OspC and the immunoreactivity [Gilmore and Mbow, 1999] that causes the anti-OspC monoclonal antibody of passive immunization of the limited quantity of the OspC (A type) from single type.Deletion N-end or C-end have been eliminated recombinant protein and have been detected by monoclonal antibody, have further pointed out the existence of conformation or discontinuous epi-position.Whether whether the epi-position of not clear monoclonal antibody identification be immunodominance, relevant from natural infection process or noly in different OspC types, guard.Recently, the linearity advantage immune epitope of A type OspC is mapped, find that it is positioned at ring 5 and α spiral 5th district [embodiment 1].The recombinant protein that contains A type ring 5 epi-positions has caused bactericidal properties antibody in mouse, and having improved individual type specific epi-position can be for the possibility in vaccine development [referring to embodiment 2].In this report, to infecting B, K and the D type OspC epitope mapping of presenting in early days, and the tetravalence chimeric having built based on these epi-positions is former.This ABKD chimeric is former is hyperimmunization originality in mouse, causes the antibody in conjunction with the OspC of cell surface, and effectively kills in the mode of complement-dependent the bacterial strain that produces A, B, K and D type OspC.

At us, develop in the work of the chimeric experimental vaccine of tetravalence, the first step is to identify B, the K in mouse and human infection's process, occur and the linear epitope of D type OspC.These are analyzed is according to identifying in embodiment 1 that the ring 5 of A type OspC and the description of α spiral 5 epi-positions carry out substantially.In simple terms, use the mouse that infects from clone and separate strain and from be expressed at least partly corresponding OspC type strain infection the mankind serum screening B, K and D type OspC truncate and slice groups widely.It is possible that use is carried out accurate epitope mapping from the serum of experimental infection mouse; But in the mankind of natural infection, antibody response will be for epi-position array widely.This is not amazing, may reflect the expansion for the antibody response of the OspC epi-position of conventionally not presenting on bacterial cell surface in early infection process.New epi-position, wherein some may for example, from the conserved domain (α spiral 1) of OspC, dead and discharge OspC from film once bacterial cell, can become and can approach.This has shown to use the warning of mankind's serum sample in epitope mapping; In general the accurate process infecting is unclear, and the clone's property that infects population is suspectable [Wang etc., 1999; Ruzic-Sabljic etc., 2006; Hofmeister etc., 1999; Guttman etc., 1996; Rijpkema etc., 1997].In any case, from the analysis of serum human sample, can be clear that, the epi-position in α spiral 5th district is produced the bacterial strain of A, B, K and D type OspC and is identified in course of infection.In addition in several dissimilar OspC producing bacterial strains, to the consistence of the reaction of α spiral 5, may be the indication of this OspC structural domain functional dependency.

Although α spiral 5 and to encircle 5 sequence be variable between OspC type, these regions are [referring to the embodiment 1] of high conservative in each type.This shows for chimeric to only have the OspC epi-position of limited quantity to need for producing provide protection widely.As the first step of vaccinogen of the extensive protectiveness of exploitation, ring 5 epi-positions of A type and be used to development experiments vaccinogen from α spiral 5 epi-positions of B, K and D type OspC.The primer that use is designed to encode joint sequence, carries out pcr amplification to the region of containing these epi-positions.This allows to use the overlapping extension of PCR in producing chimeric construct, and provides epi-position and method [Crasto and Feng, 2000] short, that aminoacid sequence structureless, that have protease resistant is separated.In this research exploitation experimental based on OspC, tetravalence ABKD chimeric is former in all test mices (n=12), has caused IgG antibody response consistent, high titre.In addition, vaccinogen has caused the antibody for every kind of epi-position being impregnated in.What is interesting is, epitope specificity titre seems and is subject to the impact of the position of epi-position in construct.Epi-position from N-end epi-position (ring 5 of A type) to C-end (the α α spiral 5 of D type), titre reduces gradually.The phenomenon that C-end epi-position titre reduces also has report [Dale etc., 1993 in the research of the chimeric based on streptococcal M protein in early days; Dale etc., 1996].The basis of the location specific impact of this titre is not clear, but may be vivo degradation or the change [Dale etc., 1999] due to C-end structure.

Although the distribution for the necessary Th cytokine reaction of the provide protection of borrelia infection and relevant Immunoglobulin Isotype is also solved [Kraiczy etc., n2000 completely; Widhe etc., 2004; Keane-Myers etc., 1995; Keane-Myers etc., 1996; Keane-Myers and Nickell, 1995], but this pattern definite is the important step in vaccinogen exploitation, and the important information about the potential protective capability of different vaccinogen constructs can be provided.The isotype of reaction distributes and to determine by ELISA, has observed with the Th1 of mixing and Th2 cytokine and has reacted relevant heavy chain Ig isotype.The type conversion of noticing in this research has pointed out sufficient T cell auxiliary, even in the situation that do not mix definite T-cell epitope in vaccinogen.Use predictability peptide combination algorithm to carry out vaccinogen sequential analysis to mouse (H2 Ak/H2Ek) and the mankind (HLA-DRB 1) II class MHC subgroup, having found to estimate in vaccinogen can be in conjunction with all available allelic possible T-cell epitope [Rammensee etc., 1999; Zhang etc., 2005].One of binding peptide of prediction, LANSVKELT has repeated 3 times in construct, and this repetition may be important [Jiang etc., 1999 for causing Th reaction; Ahlborg etc., 1998; Kjerrulf etc., 1997; Theisen etc., 2000].Although be not exhaustive to the analysis of possible T-cell epitope, our data have been supported in prediction, and showing chimeric, former can in mouse, to cause T-lymphocyte auxiliary.In addition, it points out this construct may also do like this in the mankind, does not need to merge the T-cell epitope sequence mixing.The importance of freund's adjuvant in producing this isotype distribution is not also understood, but is suitable for the mankind's adjuvant for alum and other, needs assessment reaction and isotype distribution [ten Hagen etc., 1993; Lindblad, 2004; Petrovsky and Aguilar, 2004; Brewer etc., 1999; McNeela and Mills, 2001].In addition, the epi-position order that chimeric is former or the change of structure can provide a kind of mechanism, can customize immunne response, to maximize Protective effect [Tongren etc., 2005 by it; Cai etc., n2004].

For vaccine development, because be productive to the reaction of vaccinogen, the antibody being initiated must be able to be attached to the surface of complete Borrelia burgdoyferi cell and cause sterilization.IFA analyzes the cell surface demonstrating producing the bacterial strain of A, B, K and D type OspC very strong mark.Even if the antibody titers for D type epi-position is obviously lower than the titre of the epi-position initiation of more close N-end, but the surface markers of D type producing bacterial strain is also quite obvious.In cell subsets in the culture of every kind of OspC type, observe not by anti-ABKD Antiserum Labeled, show that those cells do not express OspC.But, in vivo, verified from tick to mammiferous communication process and mammalian infections early stage, even if most cell not every, all express OspC[Gilmore etc., 2001; Zhong etc., 1997].The ability of the effective cell killing of anti-ABKD antibody is also evaluated.Serum from the mouse of vaccine inoculation effectively kills the spirochete of expressing A, B, K and D type OspC albumen in complement-dependent mode.Although for all OspC types, kill lower than 100%, this may be in the cell of population, the heterogeneous effect that external OspC expresses, and this phenomenon has been recorded [Schwan etc., 1995 in vivo well; Schwan and Piesman, 2000; Hu etc., 1996].

That the present embodiment has been described is new, restructuring, structure and principle thereof chimeric, multivalence, the ImuLyme based on OspC prove.The restructuring chimeric protein of use based on epi-position allows in same construct, to cover multiple OspC type, and avoids misleading the potential problem for the immunne response of irrelevant protein structure domain.The key that is chimeric exploitation to the mapping of the linear epitope being identified in active course of infection forms, and for 4 kinds of OspC types relevant with mankind's invasive infection, is successfully completed.Be included in the IgG antibody that epi-position in vaccinogen has caused type specific, they can be in conjunction with the OspC of burgdorferi cell surface, and the sterilization of exercising complement-mediated.

Embodiment 4, for object, be to improve the immunne response of immunogenic chimeric multivalence ImuLyme varient

In this research, we have explored the solvability of improving construct, and have assessed epi-position layout, epi-position repeats and comprised the C-end stabilization label of the inferring potential impact to immunne response.These are analyzed as building the layout strategy of chimeric in the OspC vaccine of extensive protectiveness and universal significance new understanding are provided.

Materials and methods

The expression of detection from Borrelia burgd and purifying

A, B, K and D type recombinant full-lenght OspC albumen produced [referring to embodiment 1 and 2] according to former description.In simple terms, from the ospC gene of the clone population of Borrelia burgdoyferi strain isolated B31 MI (A type OspC), LDP73 (Type B), LDP74 (K type) and LDP 116 (D type) by pcr amplification, use has 5 ' outstanding primer, to allow not rely on the clone (LIC) [embodiment 1] of ligase enzyme in pET-32 Ek/LIC carrier (Novagen).After amplification and regeneration strand afterbody, by amplicon and the annealing of pET-32 Ek/LIC carrier, carrier is transformed in NovaBlue (DE3) Bacillus coli cells and breeds.After confirming insertion sequence by DNA sequencing (MWG-Biotech), use the expression of IPTG (1mM) inducible protein.By nickel affinity chromatography, the hexahistine motif (Novagen) that uses pET-32 Ek/LIC to express label coding carrys out purifying protein.The film (Slid-a-lyzer, Pierce) that is 10kDa through molecular weight cut-off by the albumen of imidazoles wash-out is to phosphate buffered saline (PBS) (PBS; PH7.4) degree of depth dialysis, protein concentration is analyzed (Pierce) by BCA and is carried out quantitatively, and the purity of prepared product is assessed by SDS-PAGE.

Structure, expression and the purifying of ABKD vaccine varient

For mechanism and the solution that the IgG titre of studying across the concrete epi-position of vaccine constructs reduces, built the multiple varient of original vaccine.All vaccine varients are all the sequences [embodiment 3] of the ABKD vaccinogen based on former description, and contain the same sequence that contains epi-position.These comprise ring 5th district (131 to 149 amino acids) of A type and α spiral 5th district (the little figure of Figure 13) of Type B (160-201 amino acids), K type (161-201 amino acids) and D type (161-201 amino acids).ABKDppa and ABKDgg have added Pro-Pro-Ala or Gly-Gly motif at the C-end of original ABKD construct respectively.These two kinds of constructs are all prepared by the ABKD construct by using reverse primer (table 5) to increase original, and these primers add motif (Figure 13 A) by the suitable amino acid whose 5 ' protuberance of coding.Other vaccine varient is prepared by overlapping annealing and elongation technology, with in the structure [embodiment 3] of original ABKD vaccinogen, use identical.ABKDD construct is by being used the reverse primer of 3 ' afterbody sequence of structureless with coding, to have protease resistant joint, again increases ABKD construct and prepares.In this sequence that PCR product is annealed to contain D type OspC epi-position, described sequence has been used the sequence amplification at 5 ' end coding acomplementary connector, and the construct of annealing is carried out to follow-up overlapping extension and amplification (Figure 13 B).ADBK construct, by being annealed each other in the region of containing A and D type epi-position of amplification respectively, then with from B and K type α spiral 5 epitope regions of original ABKD construct amplification is annealed and prepares (Figure 13 C).ADBKD construct is by preparing (Figure 13 D) by the recon annealing from ABKD and ADBK sequence.In all cases, pcr amplification completes with GoTaq Green (Promega), use 94 ℃ of denaturing steps of initial 2 minutes, be then 94 ℃ of 15 seconds, primer of 35 circulations 50 ℃ of annealing 30 seconds, extend 60 seconds at 72 ℃, last 72 ℃ are extended 7 minutes.All primers that use in the structure of these vaccinogens are listed in table 5.All PCR products before the template as follow-up PCR reaction by gel-purified (Qiagen).Last amplicon is annealed in pET-46 Ek/LIC carrier by not relying on the clone of ligase enzyme, and is transformed in Novablue (DE3) Bacillus coli cells.Use T7 primer screening to there is the bacterium colony of Insert Fragment, reclaim plasmid (Qiafilter Midi, Qiagen), by DNA sequencing, confirm Insert Fragment.Recombinant protein is according to description expression and purifying above.After purifying, film (the Slid-a-lyzer that is 10kDa by molecular weight cut-off by vaccine protein, Pierce) to PBS (pH7.4) or pH8 damping fluid (Arg/Glu damping fluid) [the GoIo vanov etc. that contain 100mM phosphoric acid salt, 100mM NaCl, 50mM arginine, 50mM L-glutamic acid, 2004] dialyse, change damping fluid 3 times.The purity of construct is assessed by SDS-PAGE.

Table 5, for building the former primer of chimeric.LIC afterbody represents with runic, and joint sequence is by underscore.

With vaccine varient immune mouse

With the male C3H/HeJ mouse in 6 week age of every kind of immunity of 6 kinds of vaccinogen varients (every kind construct 3).Because the immunogenicity of varient will be compared with each other, therefore wish to use albumen with mole foundation, to compensate the difference of the epi-position number of per unit vaccinogen quality.Every each immunization of mouse is accepted about 2.8 nmole albumen, is 50 μ gABKD, ABKDppa, ABKDgg and ADBK construct or 62.5 μ gABKDD and ADBKD construct.Mouse is used in the vaccine immunity in Freund's complete adjuvant, then at the 2nd week and the 4th week, with Freund's incomplete adjuvant, strengthens.Before inoculation for the first time, with the 6th week, by cutting tail, from all mouse, collect serum.In order to determine that adjuvant is on impact and the impact on isotype distribution total and titre epitope specificity antibody, with as mentioned above in freund's adjuvant emulsification or be adsorbed on alum (ImiectAlum, Pierce) the ABKD vaccinogen immune mouse on (every kind adjuvant 6), collected serum at the 6th week by cutting tail.

The assessment of the epitope specificity IgG titre of vaccine varient induction

By Western trace and ELISA, the immunogenicity of every kind of vaccinogen is assessed.In western trace, A, B, K, D type detection from Borrelia burgd be with per pass 500ng loading in reductibility sample buffer, SDS-PAGE gel C riterion, Biorad 12.5%) upper electrophoresis, then electricity is transferred to PVDF (Immobilon-P, Millipore).With the 1%BSA sealing trace in the salt solution (PBS-T) of the phosphate buffered that contains 0.2%Tween-20.Use every kind of antiserum(antisera) of PBS-T1:2500 dilution to detect trace 1 hour, then wash 3 times.In order to confirm same albumen carrying capacity, the monoclonal antibody (1:2000 of anti-His label for trace; Novagen) screen.The second stage is detected goat anti-mouse IgG and the chemoluminescence (Super Signal Pico, Pierce) of the peroxidase coupling of using 1:40000 dilution and is carried out.For quantitative analysis, by elisa assay, determine the titre of OspC type specific IgG.A, B, K or D type detection from Borrelia burgd are coated on that on 96 orifice plates, (Costar 3590; Corning), carry out 16 hours in 4 ℃ in carbonate buffer solution (pH9.6) in 100ng/ hole.By plate sealing (1% BSA in the PBS that contains 0.2% Tween-20 (PBS-T); 2 hours), with PBS-T, wash 3 times, then in the hole of double plate, add the anti-vaccinogen antiserum(antisera) (100 μ L) (1 hour) of serial dilution.The goat anti-mouse IgG of HRP coupling (1:20000) is as second antibody, and ABTS is as chromogenic substrate.When speed of response is linearity, (ELx 808 at ELISA, to read plate device; Biotek) above in 405nm, read absorbancy, by four parameter logical equatiions (SigmaPlot), sigmoid curve is fitted to absorbancy fitting of a curve and calculate titre.Titre is reported as the dilution inverse corresponding to 50% maximum absorbance platform.

What epitope specificity Immunoglobulin Isotype distributed determines

The isotype of having assessed for the antibody response of ABKD, ABKDD and ADBKD vaccine varient construct by ELISA distributes.96 orifice plates are coated by A, B, K and the D type detection from Borrelia burgd of every hole 100ng.According to description above, plate is sealed and cleaned.The antiserum(antisera) of anti-vaccinogen is added in plate, carries out double analysis (100 μ L; 1:10000; 1 hour).In conjunction with epitope specificity Ig by hatching (1 hour with the specific biotinylation second antibody of isotype; Mouse isotype parting kit; Zymed) detect.Second antibody detects by streptavidin (30 minutes) and the chromogenic substrate ABTS of peroxidase coupling.All hatching all at room temperature carried out.

Vaccine specific T lymphocyte produces the mensuration of IFN-γ and IL-4

By the reaction of the cytokine of the splenocyte of the mouse of immunization, by use vaccinogen, according to the method for [1999] such as Abuodeh of revising, stimulated and assess again in vitro.The mouse being vaccinated carrys out euthanasia by carbon dioxide narcosis, and aseptic taking-up spleen is also placed on (Sigma) in RPMI substratum.The spleen of 3 mouse with the inoculation of every kind of vaccine constructs is merged, by repeatedly carrying out harvested cell to injecting RPMI in splenic capsule with No. 22 syringe needles.Cell suspending liquid is transferred in 50mL centrifuge tube to 5 minutes harvested cells of 200xg.By exposing and within 1 minute, carry out splitting erythrocyte in the ammonium chloride (R-7757, Sigma) at 3mL 8.3mg/mL.Then use 20mL RPMI (Sigma) dilution ammonium chloride, by cell centrifugation and clean 3 times.In the RPMI that cell Eddy diffusion is contained to 10% FCS, 100 μ g/mL Streptomycin sulphates, 100U/mL penicillin, 2.5 μ g/mL amphotericin Bs at 10mL.Cell, is counted with hematimeter with assessment viability with trypan blue staining, and all cell suspending liquids are adjusted to 10 ⁷individual cell/mL.With 10 ⁷the amount of individual cells/well is distributed to (2 holes of every kind of vaccinogen Class1) in 24 orifice plates (Costar 3526) by cell.Use three parts of holes of immune vaccine primary stimuli of 5 or 10 μ g/mL.Contrast comprises the irrelevant albumen of three parts of use, 10 μ g/mL---the hole that bovine serum albumin stimulates and the hole (without albumen) not stimulating.All plates are hatched 96 hours under 37 ℃, 5% CO2, then gather in the crops supernatant liquor and are chilled at-80 ℃, wait that cytokine is carried out to ELISA is quantitative.

For the level of Th1/Th2 cytokine IFN-γ and IL-4 is carried out quantitatively, according to the specification sheets of manufacturers, use the analysis (ELISA-Max based on ELISA; Biolegend).In simple terms, capture antibody is coated on 96 hole elisa plates, by plate sealing, every kind of culture supernatant of double 100 μ L is hatched 2 hours in plate.For IL-4, detect, use undiluted culture supernatant, and for IFN-γ, with undiluted and test with the supernatant liquor that PBS 1:20 dilutes.With the sample of the every kind of cytokine that contains concentration known, produce typical curve.By biotinylation second antibody, the streptavidin by HRP coupling and detect the cytokine of combination with adding lustre to of tmb substrate then.

Result

The structure of varient vaccine constructs, expression and purifying

Use has primer and overlapping annealing and the extension PCR technology of 5 ' protuberance, has produced 5 kinds of varients (Figure 13) of original ABKD vaccine.The DNA sequence dna of all these constructions is confirmed.The physico-chemical property of the selection of vaccinogen is presented at [Gasteiger etc., 2005] in table 6.By nickel dam, analyse after purification of Recombinant vaccinogen, noticed and had the recombinant protein of significant proportion to precipitate in for PBS dialysis procedure.This has also been noted [embodiment 3] in the Initial Report of ABKD vaccinogen.Although construct ABKDD and ADBKD that molecular weight is higher have higher solubleness in PBS, the precipitation of recombinant protein is still obvious.Therefore, the dialysis buffer liquid (Arg/Glu damping fluid) of revising has been developed in the work based on [2004] such as Golovanov.The pH of damping fluid is increased to pH8.0 from PBS (pH7.4), to increase the difference between the iso-electric point (pI 6.49 or 6.85) of pH and recombinant protein of damping fluid.In addition, salt concn is reduced to 100mM from 150mM, and has added 50mM arginine and 50mM L-glutamic acid.Use this damping fluid, do not notice the precipitation of any recombinant protein, the concentration of soluble proteins obviously increases.As visible by SDS-PAGE, recombinant protein is pure, does not contain degraded product (Figure 14).

The physico-chemical property of table 6, vaccinogen

Construct	Amino acid	Molecular weight (Da)	Iso-electric point	Instability index
					ABKD	169	18014.4	6.49	10.14
ABKDppa	172	18279.7	6.49	14.93
					ABKDgg	171	18128.5	6.49	10.86
ABKDD	214	22632.7	6.85	15.49
					ADBK	170	18027.4	6.49	8.51
ADBKD	215	22645.7	6.85	14.18

The immunogenicity of vaccinogen varient

In order to assess the premunition originality of ABKD vaccine varient, mouse is carried out to immunity with every kind of varient in freund's adjuvant.Epitope specificity by western trace assessment serum is reactive, and wherein serum is used to survey in 4 types the fixing detection from Borrelia burgd of PVDF of each.Albumen is proved on trace with same carrying capacity, as by with the reactivity of the anti-His tag monoclonal antibody of the specific mouse of label assess.Serum from the mouse by immune has confirmed to exist vaccinogen dependency difference (Figure 15 A) in the level of reactivity with every kind of detection from Borrelia burgd albumen.It should be noted that in the mouse with ABKDppa and the inoculation of ABKDgg varient and reduced with the reactivity of D type α spiral 5 epi-positions, be apparent that most and use ADBK varient.In order to assess these in quantitative mode, change, by ELISA, then every kind of total length detection from Borrelia burgd passing through 4 types of uses is as fixing antigen, has completed IgG and every kind of reactive titration that is included in the OspC type specificity epi-position in construct.Titre has been simulated the discovery of qualitative western trace substantially, has confirmed the vaccine specific difference (Figure 15 B) in the reactivity of immune serum and individual epi-position.Significant difference is seen in the reactivity with D type epi-position, for ABKDppa, ABKDgg and the visible low especially titre of ADBK varient.

The isotype of vaccine varient specific immunoglobulin distributes

In order to understand in more detail the immunne response of varient vaccinogen induction, by epitope specificity titer determination, completed 3 epitope specificity Immunoglobulin Isotypes with the varient (ABKD, ABKDD, ADBKD) of best vaccine potentiality and distributed.Generally speaking, in antigen specific immune sphaeroprotein, exist dominant IgG1, a small amount of IgG2a and IgG2b, and considerably less IgG3 or IgM, this pattern had been noted [embodiment 3] (Figure 16) in the past.For all epi-positions and all vaccine varients, the pattern of Ig isotype is similar, only has an exception.In the mouse with ABKDD immunity, the reactivity of K and D type epitope specificity IgG2a and IgG2b is than high with the mouse of ABKD or the immunity of ADBKD varient.

The production of the lymphocytic Thl/Th2 cytokine of vaccine specific T

In order to be evaluated at the cytokine environment of variation induction in ABKD vaccinogen and the potential impact of Th1/Th2 balance, use stimulates the splenocyte of mouse in vitro again for the vaccinogen of immune mouse.Between the mouse of different immunity, noticed the significant difference of the induction level of IFN-γ.All vaccine varients are all relevant with the increase of IFN-γ level in culture supernatant, although the level that ADBK and ADBKD have is than high 2 to 3 times (Figure 17) of other vaccinogen induction.In all cases, concentration is that the antigen of 5 μ g/mL and 10 μ g/mL is all induced IFN-γ, and its level arrives in the scope of 8.6ng/mL 0.5.From the splenocyte not stimulating or the splenocyte that stimulates with bovine serum albumin cell culture supernatant all there is the IFN-γ level lower than the analyzing and testing limit of 15.6pg/mL.In boosting vaccine and culture supernatant contrast, IL-4 all do not detected, show that concentration is lower than the analyzing and testing limit of 2.0pg/mL.

The impact that adjuvant type antagonist titre and isotype distribute

In order to determine that adjuvant type, on the impact for vaccinogen reaction, is used in ABKD albumen emulsification or that be adsorbed in alum in freund's adjuvant mouse is carried out to immunity.In the mouse with adsorbed onto alum adjuvant immunity, for whole vaccinogen and slightly low for the IgG titre of each component epi-position, although the aggregated model reacting between two kinds of adjuvants similar (Figure 18 A).Although IgG1 level is similar, with the mouse of alum immunity, there is vaccinogen specific IgG 3, IgG2a and the IgG2b (Figure 18 B) of minimizing.The distribution closely similar (data do not show) of observing when epitope specificity isotype distributes with the whole vaccinogen of use.

Discuss

The chimeric protein that use contains a plurality of B cell epitopes, compares with peptide conjugate with whole protein polyvalent vaccine is former, has potential advantage in vaccine development.Only comprise protective epitope's sequence and reduced to mislead the possibility for the reaction of irrelevant epi-position in parent's molecule or peptide carrier.If as OspC, having in recombiant vaccine is former is that [embodiment 1 for large conserved domain advantage immunity, that still do not presented by bacterium in course of infection; Kumaran etc., 2001; Eicken etc., 2001], this will be important.Such epi-position and the generation of protective immune response are irrelevant.But the needs that produce new albumen are considered stability and the deliquescent intermolecular and intramolecular interaction that may seal epi-position or affect albumen.In the present embodiment, our former conducting in-depth research of polyvalent chimeric ImuLyme to the restructuring based on OspC.Original ABKD vaccine is hyperimmunization originality in mouse, and derivative IgG is in conjunction with the lip-deep natural OspC of bacterial cell and cause the germicidal action [embodiment 3] of complement-dependent.Although this success, ABKD construct also has two factors to need to improve, its poorly soluble in PBS, and this has hindered the production of recombinant protein and may affect stability in storage, and for the difference of the IgG titre of the individual epi-position in albumen.Specifically, titre reduces along with the approaching of C-end of epi-position and vaccine.The object of this research is to improve the solvability of vaccinogen, and uses in epi-position position, epi-position and repeat and comprise the vaccinogen of stablize modifications different aspect motif to improve integral body and immunne response epitope specificity.

In research early, have been noted that recombiant vaccine is former has obvious precipitation [embodiment 3] after for PBS dialysis.This poor solvability has not only limited the production of vaccinogen, and influential to the immunogenicity of vaccine.The peak concentration that ABKD construct can reach after PBS is dialysed is 0.5mg/mL, conventionally lower.The crystalline structure of OspC shows that spiral 5 epi-position districts have participated in bunchy [Kumaran etc., 2001 of 4 spirals in monomer in natural OspC albumen; Eicken etc., 2001].Within this can cause vaccinogen albumen and between the hydrophobic helicoidal surface of exposure between interaction, and then cause precipitation.In dialysis buffer liquid, add arginine and L-glutamic acid to be found to make the solubleness of the former albumen of all recombiant vaccines to increase by 4 to 100 times (table 7).The deliquescent basis of this increase may be by the interaction of the aliphatic portion with arginine and L-glutamic acid side chain, arginine and L-glutamic acid and with the residue of the exposure of opposite charges and with the interaction [Golovanov etc., 2004] of hydrophobic residue.The mouse of the ABKD construct immunity that use is dialysed in Arg/Glu damping fluid has than the obvious higher titre [embodiment 3] of the mouse of the ABKD vaccinogen immunity with PBS dialysis.Arg/Glu damping fluid can cause the folding mode that more has superiority or intermolecular or intramolecular interaction still less, thereby epi-position can be approached with B-cell receptor better.Epi-position identification is not obviously disturbed in the absorption of arginine and L-glutamic acid and recombinant protein.Arg/Glu damping fluid has been reported in the external activity for proteolytic enzyme provide protection [Golovanov etc., 2004].Although all there is no obvious proteasome degradation phenomenon in the sample of PBS and Arg/Glu dialysis, can not get rid of the interior provide protection for proteolytic cleavage of body.Dialysis for the damping fluid that contains arginine and L-glutamic acid may be a kind of useful instrument, can have for other new chimeric protein of obvious molecular interaction.

Table 7, for the soluble proteins concentration of the vaccinogen of PBS or Arg/Glu damping fluid dialysis

In chimeric streptococcal M protein vaccinogen, reported in the past the poor relation with C-end epi-position position of immunne response, this may be due to the structure problem relevant with C-end or by the proteolytic degradation of carboxypeptidase [Dale etc., 1993; Dale etc., 1996; Dale etc., 1999].Propose many method protection peptides and recombinant protein and exempted from protease activity, comprised amidation or PEGization, the acetylize of amino (N) end and amino acid motif [Brickerhoff etc., 1999 of adding protectiveness of C-end; Powell etc., 1992; Lee etc., 2005; Alvarez etc., 2004; Kawarasaki etc., 2003; Walker etc., 2003].Amino acid motif be also in the news can be by suppressing the effect of carboxypeptidase the C-end of stabilize proteins; But their protective capability is only assessed several albumen.Assess two stability motifs and increased the ability for the antibody response of ABKD vaccinogen.Add two neutral hydrophilic Gly residues can reduce the activity of cathepsin A and D, these two kinds of enzymes have specificity [Alvarez etc., 2004 to hydrophobic and alkaline C-end amino acid respectively; Kawarasaki etc., 2003; Remington and bredam, 1994].Add Pro-Pro-Ala motif can spatially hinder by proline residue arranged side by side, large volume advance [Walker etc., 2003] of carboxypeptidase.

In order to assess, add these motifs on the impact for the former antibody response of ABKD chimeric, with ABKDgg or ABKDppa construct immune mouse.In both cases, compare with the mouse of ABKD construct immunity with unmodified, serum has lower average IgG titre for a kind of in multiple epi-position.ABKDppa construct has reduced the titre of D type specificity IgG, although this is mainly due to single outlier.ABKDgg construct has reduced the titre of K and D type epi-position.On the basis of IgG titre, any one that use any these motifs all do not have advantage.To the reduction of the titre of C-end epi-position show as be not to have been given by these motifs by those resistance carboxypeptidase effect mediated; although [Golovanov etc., 2004] are covered in the similar provide protection that likely any advantage causing due to proteolytic enzyme provide protection may be provided by Arg/Glu damping fluid.

In order studying, to relate to the possible textural factor poor to the immunne response of C-end epi-position, to have produced several other constructs.In chimeric suis vaccine, C-terminal repeat N-end epi-position by unknown mechanism " protection " previous C-end epi-position [Dale etc., 1999; Dale etc., 2005; Hu etc., 2002; McNeil etc., 2005].Based on this success, developed the similar varient of ABKD vaccinogen.Whether produced ABKDD construct can be protected by second C-end D type epi-position for the reaction of D type epi-position with assessment.In order to assess whether the reduction of titre is mainly due to C-end table bit position, D type epi-position is moved on to the position (ADBK) of the most close N-end of second.Finally, with ADBKD construct, assess the provide protection of the C-end epi-position of repetition, and use ABKDD, the impact of the epi-position that assessment repeats on specific immune response.In ABKDD vaccinogen, epi-position repeats to make D type specificity IgG titre to double, but has caused that the titre for contiguous K type epi-position reduces simultaneously.In ADBK construct, when D type epi-position is placed on the position of N-end more, D type specificity IgG titre has been significantly reduced.In addition, in ADBK construct, the reactivity for the K type epi-position of C-end has been enhanced with comparing in ABKD.Add that C-end D type epi-position (ADBKD) does not increase K type specificity titre; But, although it produced obvious increase be not double, for the titre of D type epi-position.These results show that the C-terminal position of D type epi-position is more preferred than interior location, and " protectiveness " C-end epi-position separately adding does not have significant protective effect to C-end epi-position.In this vaccinogen, the main determining factor of epitope specificity titre is not the proximity of it and C-end, and may be more the tertiary structure of chimeric protein.

The Ig isotype of vaccinogen induction may be significant to endogenous protective effect.By changing epi-position or their order, may be able to change the distribution [Tongren etc., 2005] of isotype, thereby change the effector function of antibody.In order to measure the isotype of epitope specificity, distribute, antiserum(antisera) for most promising vaccinogen (ABKD, ABKDD, ADBKD) is combined with fixing A, B, K or D type detection from Borrelia burgd, and use the specific antiserum(antisera) of isotype to detect the antibody of combination.As in the past ABKD vaccinogen being reported [embodiment 3], main isotype is the lower slightly IgG2a of IgG1 and level and IgG2b, and this depends on construct, and low-level IgM and IgG3.Between ABKD and ADBKD antiserum(antisera), the distribution of epitope specificity Ig isotype is that similarly the level of IgG2a and IgG2b reduces from N-to C-end epi-position, has simulated total IgG titre.ABKDD has consistent IgG2a and IgG2b level for all epi-positions, although K type specificity total IgG titre is lower.

ABKD vaccinogen causes the bactericidin [embodiment 3] of complement-dependent.In mouse, IgG1 is activating complement [Dangl etc., 1988 not; Miletic and Frank, 1995], show that most of derivative isotype may not have provide protection.Although reported that OspA specific IgG 1 can kill burgdorferi [Munson etc., 2000] by not relying on the mechanism of complement, the germicidal action of the antibody of ABKD vaccinogen induction is complement-dependent [embodiment 3].The isotype being initiated distributes and may be subject to using the impact of C3H/HeJ mouse---standard animal model of a kind of Lyme disease research---.Between Borrelia burgdoyferi period of infection, had been noted that C3H/HeJ and BALB/c are the difference of the humoral immunization between mouse, particularly in total IgG level, particularly in the level of IgG2a, these two all higher [Yang etc., 1992 in C3H/HeJ mouse; Keane-Myers and Nickell, 1995].In addition, C3H/HeJ mouse germline lacks TLR-4, although estimate that it is not crucial for the provide protection for Lyme disease between preventive vaccination or period of infection, because burgdorferi does not produce lipopolysaccharides [Takayama etc., 1987; Barthold etc., 1990].

Because generally accepted, be body fluid to kill burgdorferi activity be complement-dependent, it may be favourable therefore causing the reaction of Th1 cytokine, because it appears in many bacteriosises (summarizing in [Spellberg can Edwards, 2001]).In active course of infection, cytokine participates in the development of Lyme disease and sequela thereof and disappears.Several research has been found that IL-4 is not crucial cytokine [Munson etc., 2000 for the antibody response of burgdorferi is killed in development; Potter etc., 2000; Christie etc., 2000; Satoskar etc., 2000-64], mean that the reaction of Th1 type may be relevant with provide protection.In addition, the Th1 cell of secretion of gamma-IFN has promoted [Bockenstedt etc., 2001 of disappearing of the carditis relevant with Lyme disease; Kelleher etc., 1998].On the contrary, in course of infection, the spirochetal carrying capacity of arthritic seriousness and skin reduces by administered recombinant IL-4, by using α-IL-4 antibody, increases [Keane-Myers and Nickell, 1995; Keane-Myers etc., 1996].In natural infection process, the production of IFN-γ relevant with the development of chronic Lyme disease [Widhe etc., 2004], and relevant with the degree of arthroncus in Lyme arthritis [Gross etc., 1998].IFN-γ also relates to the generation relevant [Munson etc., 2002] that suppresses to induce the anti-OspA antibody that kills burgdorferi from external lymphoglandula culture.In order to study the Th cytokine environment of immunization induction, use in vitro vaccinogen, Th1 (IFN-γ) and Th2 (IL-4) cytokine to carry out again stimulating to mouse boosting cell, and quantitative by ELISA.IFN-γ in the supernatant liquor of the cell again stimulating with vaccinogen, detected, although its concentration is according to construct and difference.On the contrary, IL-4 in any Activity in Supernatant of Spleen Cell, do not detected.ABKD, ABKDppa and ABKDD construct all have similar IFN-γ concentration.ADBKD has almost double IFN-γ concentration, and the level of ADBK is even higher.In the supernatant liquor of the cell again stimulating, between IFN-γ level and total epitope specificity serum IgG titre or isotype distribution, there is no obvious dependency.

Cytokine and relevant Ig isotype distribute can be by selecting immunological adjuvant to change.Freund's complete adjuvant and the reaction of Th1 cytokine relevant [Cribbs etc., 2003; Shibaki and Katz, 2002], it can increase the level of IgG2a.Unique adjuvant that is approved at present the mankind is alum, secretion [Cribbs etc., 2003 of its known increase Th2 cytokine; Brewer etc., 1999; Lindblad, 2004; Petrovsky and Aguilar, 2004].In the mouse with alum immunity, noticed with using freund's adjuvant and compared, for the expected appropriateness reduction of IgG titre of vaccinogen and composition epi-position thereof.In addition, compare IgG3, IgG2a and the proportional reduction of IgG2b isotype with IgG1.The lower Th1 cytokine reaction of having expected when this has confirmed to use this adjuvant.But vaccinogen continue to cause antibody that can conjugated complement, show for the obvious change of construct or concerning the modification of adjuvant for may be optional effective reaction.

In the present embodiment, we have studied the potential former change of chimeric multivalence ImuLyme, its objective is and optimize derivative humoral immunoresponse(HI).By using, Arg/Glu damping fluid is dialysed to increase its solvability, realized that construct is immunogenic to be significantly improved.This may reduce protein-interacting in vivo, makes more epi-position expose [Theisen etc., 2000].Add the C-end motif of proteolytic enzyme protectiveness or add that " protectiveness " C-end epi-position all can not improve the immunne response for vaccinogen.Epi-position is resequenced and caused the immunne response for the epi-position being moved significantly to reduce.Shown for the difference of the immunne response of the composition epi-position in this vaccine constructs is mainly the structure that depends on albumen, rather than the resistance of albumen to protease digestion.In addition, evidence suggests the Th cytokine that vaccinogen causes and IgG isotype can be fitted the structure of construct and the preparation of adjuvant changes.This research to for the former Asia of chimeric the suitableeest immunne response basis and improve aspect the method for these reactions, important information is provided.

Embodiment 5, available OspC sequential analysis have confirmed the feasibility of extensive protectiveness polyvalent chimeric ImuLyme

For the ease of further developing the chimeric construct body of extensive protectiveness, we have carried out Phylogenetic Analysis to the OspC sequence that can obtain from database.Analyzed OspC fragment is crossed over the residue (using the numbering to B31MI sequence) of 20 to 200.In database, shorter sequence is excluded from these and analyzes, and stays sequence from 280 burgdorferi bacterial strains for analyzing.The OspC type of assigning each sequence is determined by comparison (PAM40 score matrix) and consistency matrix analysis between two.Consistent with former research, show 95% or the sequence of higher sequence identity be considered to belong to same OspC type (Attie etc., 2006; Wang etc., 1999) (Figure 19).Observed the clear and definite bimodal distribution of sequence comparison, between different OspC type sequences, mean sequence consistence is 65%, and the consistence of type inside is greater than 97%.Except 21 types being described by (1999) such as Wang, other 17 bunches of groups have also been defined.For comprise be less than 3 sequences bunch group we do not assign OspC type.In the name of new OspC type, we have selected to maintain existing OspC type name (Wang etc., 1999) from A to U, according to the prototype bacterial strain being included in each bunch of group, other type are named.In 280 analyzed sequences, there are 202 to be assigned OspC type, they are all from the kind that causes Lyme disease.78 sequences that are not assigned to OspC type comprise spirochete (51 strain isolateds) and other the burgdorferi kind (27 strain isolateds) that causes Lyme disease.Obtaining the geography of strain isolated of each OspC sequence and biological origin is presented in Figure 20 with the form of form.Most of Borrelia burgdoyferi strain isolated is from North America (80%), and minority is from Europe (16%) and Asia (4%).The strain isolated that 53% Borrelia burgdoyferi, 48% Erichsen burgdorferi and 79% Borrelia garinii OspC sequence are collected since the mankind.It should be noted that the Borrelia garinii OspC sequence from mankind's strain isolated is mainly (68%) in cerebrospinal fluid (CFS) source, and Erichsen burgdorferi strain isolated is mainly from skin (83%).On the contrary, the OspC sequence in Borrelia burgdoyferi source is carried out the strain isolated reclaiming since human skin (51%), blood plasma (30%) and CSF (19%).These find that the known disease type cause with these organisms is consistent, and the sample that shows the OspC sequence assessed in this report has represented the true population of lyme disease spirochete.

For the ease of further Phylogenetic Analysis, by deleting consistent sequence, analyzed sequence set is reduced to 74.Then use Phylip (v.3.66) Phylogenetic Analysis software package to use bootstrap (n=1000) that these sequences are compared and analyzed.The distance of using Dayhoff PAM matrix computations to cross over 20 to 200,20 to 130 and 131 to 200 regions, by being close in conjunction with generation tree.B.hermsii OspC ortholog (Vmp33) sequence is used as outgroup (Margolis etc., 1994).By plurality rule (boundary is 50% for the comprising of group), produce stric consistency tree (consensus tree).Under Dayhoff PAM model, by maximum likelihood method, be stric consistency tree computed range (Figure 21) again.

Use the stric consistency tree of the 20-200 amino acids fragment generation of OspC well to be supported at terminal node, all definite OspC types are according to expection sub-clustering.Although the support less (Figure 21 A) that bootstrap analysis has obtained several darker branches, this is not expect, because the consistent region of expanding between sequence makes their phylogeny difference very meticulous.Use the 20-200 of OspC and the stric consistency tree of 20-130 amino acids fragment generation to demonstrate similar phylogeny sub-clustering (Figure 21 A, 21B), this is the identity based on species mainly.But, use the stric consistency tree (Figure 21 C) that 131-200 amino acids produces to produce visibly different sub-clustering pattern, do not obtain the strong support of bootstrap analysis.This observation meets the hypothesis of the restructuring between the short-movie section that ospC gene has occurred between the bacterial strain of different OspC types.Between OspC type, the evidence of OspC short-movie section restructuring can be seen in specific sequence.For example, the sequence of the OspC type PLj7 of Erichsen burgdorferi, the region having in the structural domain of 20-130 amino acids with in forming the OspC sequence of Borrelia garinii of Pki bunch, see identical.In the region, 131-200 position of PLj7, the motif that ring 5 He Huan 6th district of hypermutation have respectively with in F and M type Borrelia burgdoyferi OspC, see identical.Other evidence of restructuring is from the startup scanning (Lole etc., 1999) of using SimPlot (v.3.5.1).In starting scanning, by moving window (window of 40 bases, 10, every step interval base) produce phylogenetic tree (Kimura model, Ts/Tv ratio=2.0, contiguous combination) for sequence fragment, assess potential restructuring.These trees are bootstrapped (n=100), have reported the quantity of the tree of the schedule of supporting sequence of packets in this window.When being greater than the tree of 70% schedule sequence is collected in window, it is generally acknowledged that the sign of restructuring has obtained support.In the type of describing (Figure 22) and (data do not show) in other type in a large number, found the sign of possible restructuring in the above.

OspC variability be by the exchange between existing OspC type rather than by the evidence of super sudden change, for the absolute quantity of the OspC type specific epi-position that needs to comprise in extensive protectiveness vaccinogen exists limit that evidence is provided.Because the linear epitope of mapping is included in the C-stub area (131-200 amino acids) of OspC at present, determine that the theoretical quantity of the former required epi-position of chimeric is possible.By check this region in 74 above-mentioned representative series, the quantity in the region of containing unique epi-position has been reduced to 34 (Figure 22) by eliminating sequence consistent or that only have an amino acid to change.Some epi-position by epitope mapping, this quantity may can further be limited, because can be given the provide protection for two or more OspC types.Consider those and human diseases or the relevant OspC type of aggressive human diseases more particularly, required epitope number also can further reduce (referring to embodiment 1).For carrying out premunitive a kind of theoretic misgivings for OspC epi-position subclass, are the selectivity that possible drive for not being included in the type in vaccinogen, thereby increased the ratio with the population of those rare alleles.But because the mankind are only occasional hosts, immunization obviously changes the storage of tick carrier or Mammals, and to express that the population of the bacterial strain of specific OspC type distributes in main be impossible.

In short, the deep essence of OspC database makes to analyze and carried out completely, and it has defined new OspC type, and provide about they separated frequencies and with the information of the dependency of human diseases.Data show, in the quantity of the sequence that contains OspC epi-position that extensively the former middle needs of chimeric of protectiveness comprise, are limited, and the former exploitation of chimeric is feasible.

Embodiment 6, the chimeric structure of octavalence

Below ENICABKD octavalence construct is presented at, use several character of the construct of PROTPARAM program calculating to be listed.The fragment that is named as " L# " is joint sequence." RS " indicated the position of the restriction site using in manufacturing construct.

<----TAG----->RSLEssT.LTssT.L T----------------------E type---------------

1 AHHHHHHVDDDDKITGLKSEHAVLGLDNLTDDNAQRAILKKHANKDKGAAELEKLFKAVE

------------<L9>-----------------N type-----------------><L

61 NLSKAAQDTLKNAPGVGATTDEEAKKAILRTNAIKDKGADELEKLFKSVESLAKAAQDAT

6><----------------I type----------------><L7>LEssT.LTss T.LT-------------

121 QMLKTNNDKTKGADELEKLFESVKNLSKAAKEMLTNSVKELTSTEPSEEFTKKLKEKHTD

------C type---------------------><L8RS<------A type-----><-

181 LGKKDATDVHAKEAILKTNGTKDKGAAELEKLFESGEDVSETFTNKLKEKHTDLGKEGSM

L1><----------------B type-----------------><L2>LEssT.LTss T.LT-----------

241 GMLKANAAGKDKGVEELEKLSGSLESLSKAAKEMLANSVKELTSTNGNLITDAAKDKGAA

---K type------------------><L3>LEssT.LTss T.LT---------------D type-----

301 ELEKLFKAVENLAKAAKEMLANSVKELTSSMSVLKTHNAKDKGAEELVKLSESVAGLLKA

----------------------->

361 AQAILANSVKELTSPVVAESPKKP(SEQ?ID?NO：249)

Amino acid whose quantity: 384

Molecular weight: 41263.7

Theoretical iso-electric point: 6.52

Amino acid forms:

Ala(A) 51 13.3％

Arg(R) 2 0.5％

Asn(N) 20 5.2％

Asp(D) 25 6.5％

Cys(C) 0 0.0％

Gln(Q) 5 1.3％

Glu(E) 42 10.9％

Gly(G) 21 5.5％

His(H) 12 3.1％

Ile(I) 7 1.8％

Leu(L) 46 12.0％

Lys(K) 62 16.1％

Met(M) 7 1.8％

Phe(F) 7 1.8％

Pro(P) 5 1.3％

Ser(S) 27 7.0％

Thr(T) 26 6.8％

Trp(W) 0 0.0％

Tyr(Y) 0 0.0％

Val(V) 19 4.9％

The sum of electronegative residue (Asp+Glu): 67

The sum of positively charged residue (Arg+Lys): 64

Atom forms:

Carbon C 1787

Hydrogen H 2983

Nitrogen N 501

Oxygen O 597

Sulphur S 7

Molecular formula: C1787H2983N501O597S7

Total atom number: 5875

The transformation period of estimating:

The N-end of considering sequence is A (Ala).

The transformation period of estimating is: 4.4 hours (Mammals reticulocyte, external)

>20 hour (yeast, in body)

>10 hour (intestinal bacteria, in body)

Unstable index:

Instability index (II) is calculated as 12.58

This is stable by Protein classification

Aliphatics index: 81.46

Overall average wetting ability (GRAVY) :-0.668

When being applied to experimental animal, finding that this chimeric protein construct causes strong immunne response, and provide protection for the development of Lyme disease.

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Although the present invention is described with reference to its preferred embodiment, those skilled in the art will recognize that, after modifying in the spirit and scope of the claim of enclosing, the present invention still can be put into practice.Therefore, the invention is not restricted to above-described embodiment, but should further be included in all modifications and Equivalent thereof within the spirit and scope of description provided herein.

Sequence table

<211>571

<212>PRT

<213>Artificial

<220>

<223>Chimeric?protein

<220>

<221>misc_feature

<222>(73)..(73)

<223>Xaa?can?be?any?naturally?occurring?amino?acid

<220>

<221>misc_feature

<222>(146)..(146)

<223>Xaa?can?be?any?naturally?occurring?amino?acid

<220>

<221>misc_feature

<222>(217)..(217)

<223>Xaa?can?be?any?naturally?occurring?amino?acid

<220>

<221>misc_feature

<222>(288)..(288)

<223>Xaa?can?be?any?naturally?occurring?amino?acid

<220>

<221>misc_feature

<222>(359)..(359)

<223>Xaa?can?be?any?naturally?occurring?amino?acid

<220>

<221>misc_feature

<222>(430)..(430)

<223>Xaa?can?be?any?naturally?occurring?amino?acid

<220>

<221>misc_feature

<222>(501)..(501)

<223>Xaa?can?be?any?naturally?occurring?amino?acid

<400>126

<210>129

<211>569

<212>PRT

<213>Artificial

<220>

<223>Chimeric?protein

<220>

<221>misc_feature

<222>(73)..(73)

<223>Xaa?can?be?any?naturally?occurring?amino?acid

<220>

<221>misc_feature

<222>(144)..(144)

<223>Xaa?can?be?any?naturally?occurring?amino?acid

<220>

<221>misc_feature

<222>(215)..(215)

<223>Xaa?can?be?any?naturally?occurring?amino?acid

<220>

<221>misc_feature

<222>(286)..(286)

<223>Xaa?can?be?any?naturally?occurring?amino?acid

<220>

<221>misc_feature

<222>(357)..(357)

<223>Xaa?can?be?any?naturally?occurring?amino?acid

<220>

<221>misc_feature

<222>(428)..(428)

<223>Xaa?can?be?any?naturally?occurring?amino?acid

<220>

<221>misc_feature

<222>(499)..(499)

<223>Xaa?can?be?any?naturally?occurring?amino?acid

Claims (28)

1. chimeric recombinant protein, comprises ring 5th district or α spiral 5th district or the epi-position of these two from the outer surface protein C (OspC) of two or more types.

2. the chimeric recombinant protein in claim 1, wherein said OspC type is selected from 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.

3. the chimeric recombinant protein in claim 2, wherein said chimeric recombinant protein comprises the epi-position from A, B, K and D type OspC.

4. the chimeric recombinant protein in claim 2, wherein said chimeric recombinant protein comprises the epi-position from E, N, I, C, A, B, K and D type OspC.

5. the chimeric recombinant protein in claim 1, wherein said chimeric recombinant protein has the one-level aminoacid sequence showing in SEQ ID NO:75 or SEQ ID NO:249.

6. the chimeric recombinant protein in claim 1, wherein said OspC type is relevant with aggressive borrelia infection.

7. in the individuality of needs, cause the method for the immunne response of burgdorferi, comprise the steps:

Use ring 5th district of outer surface protein C (OspC) or the chimeric recombinant protein of α spiral 5th district or the epi-position of these two comprising from two or more types to described individuality.

8. the method in claim 7, wherein said OspC type is selected from 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.

9. the method in claim 7, wherein said chimeric recombinant protein comprises the epi-position from A, B, K and D type OspC.

10. the method in claim 7, wherein said chimeric recombinant protein comprises the epi-position from E, N, I, C, A, B, K and D type OspC.

Method in 11. claims 7, wherein said chimeric recombinant protein has the one-level aminoacid sequence showing in SEQ ID NO:75 or SEQ ID NO:249.

Method in 12. claims 7, wherein said OspC type is relevant with aggressive borrelia infection.

13. determine that whether individuality has been exposed to or has infected burgdorferi or the method for these two, comprises the following steps:

From the described individual biological sample that obtains;

Described biological sample is exposed to at least one restructuring chimeric protein, and wherein said at least one chimeric protein comprises from ring 5th district of the outer surface protein C (OspC) of two or more types or α spiral 5th district or the epi-position of these two; And

Determine whether antibody is combined with described at least one chimeric protein in described biological sample, wherein the detection of antibodies is exposed to or has infected burgdorferi before having indicated.

Method in 14. claims 13, wherein said OspC type is selected from 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.

Method in 15. claims 13, wherein said chimeric recombinant protein comprises the epi-position from A, B, K and D type OspC.

Method in 16. claims 13, wherein said chimeric recombinant protein comprises the epi-position from E, N, I, C, A, B, K and D type OspC.

Method in 17. claims 13, wherein said chimeric recombinant protein has the one-level aminoacid sequence showing in SEQ IDNO:75 or SEQ ID NO:249.

Method in 18. claims 13, wherein said OspC type is relevant with aggressive borrelia infection.

19. for containing from ring 5th district of outer surface protein C (OspC) of two or more types or the antibody of the chimeric recombinant protein of α spiral 5th district or the epi-position of these two.

Antibody in 20. claims 19, wherein said OspC type is selected from 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.

Antibody in 21. claims 19, wherein said chimeric recombinant protein comprises the epi-position from A, B, K and D type OspC.

Antibody in 22. claims 19, wherein said chimeric recombinant protein comprises the epi-position from E, N, I, C, A, B, K and D type OspC.

Antibody in 23. claims 19, wherein said chimeric recombinant protein has the one-level aminoacid sequence showing in SEQ IDNO:75 or SEQ ID NO:249.

Antibody in 24. claims 19, wherein said OspC type is relevant with aggressive borrelia infection.

Antibody in 25. claims 19, wherein said antibody is polyclonal.

Antibody in 26. claims 19, wherein said antibody is monoclonal.

27. the antibody in claim 19, wherein said antibody is bactericidal for Borrelia spirochete.

The immunogenicity mixture of 28. chimeric recombinant proteins, every kind of chimeric recombinant protein in wherein said mixture comprises from ring 5th district of the outer surface protein C (OspC) of two or more types or α spiral 5th district or the epi-position of these two.

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