CN104379164A - Vaccines and methods to treat lyme disease in dogs - Google Patents
Vaccines and methods to treat lyme disease in dogs Download PDFInfo
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- CN104379164A CN104379164A CN201380030342.1A CN201380030342A CN104379164A CN 104379164 A CN104379164 A CN 104379164A CN 201380030342 A CN201380030342 A CN 201380030342A CN 104379164 A CN104379164 A CN 104379164A
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A—HUMAN NECESSITIES
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The instant invention provides an immunogenic composition comprising an antigenic fragment of OspA protein of Borrelia burgdorferi and a chimeric protein containing antigenic fragments of different phylotypes of OspC protein of Borrelia burgdorferi. Vaccines incorporating the immunogenic composition of the invention, as well as methods of preventing Lyme disease in dogs and/or protecting dogs from Lyme disease using the vaccines are also provided.
Description
Technical field
The invention belongs to veterinary medicine field.More particularly, the invention belongs to the field of the vaccine of the Lyme disease (Lyme disease) in treatment or prevention Canis familiaris L..
Background technology
Lyme disease is by the microbial bacteriological infection of pathogenicity spiral of Borrelia (genus Borrelia).Infection can occur in the mankind, Canis familiaris L., deer, mice and other animal, and is propagated by arthropod carrier (it should be noted that hard Ticks belongs to the Ticks worm of (genus Ixodes) most).Borrelia burgdoyferi, the modal Lyme disease cause of disease in North America, cultivated in first time nineteen eighty-two.Burgdorferi is introduced into host at the position that Ticks insect bite is hindered and this position is also the position of initial characteristics dermatoses stove (erythema chronicum migrans (erythema chronicum migrans)).In Canis familiaris L., Lyme disease shows as limping that arthritis causes, apositia, heating, drowsiness, lymph node pathological change and lethal glomerule nephritis in some cases.Nearest research shows, in endemic area, the percentage ratio of the Canis familiaris L. of active infection can up to 11%.
Antibiotic (as penicillin, erythromycin, tetracycline and ceftriaxone (ceftriaxone)) can be used at any time to treat infect.But once occur infecting, medicine just may not remove the host of spirillum, and only can play the effect of the chronic form controlling disease.As the complication such as arthritis and fatigue can continue the several years after Diagnosis and Treat.
Exploitation canine lyme disease vaccine with by mainly induce OspA spirillum acid antibody protection is provided.Borrelia burgdoyferi (B.burgdorferi) OspC is another for the potential target of the antibody-mediated immunity of spirillum acid.This protein seems to have the epitope of responsible inducing cycloidic body acid antibody, and is not conservative in pathogenic Borrelia.Although the concrete function of OspC protein is not yet known, mammalian infections is proposed but not Ticks insect infection needs OspC to express.Burgdorferi just expresses OspC soon after Ticks worm starts feed, and must infect to be formed in mammal by continuous expression OspC.Therefore, compared with OspA spirillum acid antibody, " the effect window " of OspC spirillum acid antibody significantly increases.
People's (United States Patent (USP)s the 6th such as Callister (Callister); 210; No. 676 and the 6th; 464; No. 985, being incorporated herein by reference) suggestion uses the immunogenic polypeptide fragment (separately or with OspA polypeptides in combination) of OspC to prepare vaccine and exempts to suffer from Lyme disease protect the mankind and other mammal.People's (United States Patent (USP) the 6th, 872, is incorporated herein by reference by No. 550) such as Lai Fuli (Livey) also propose by the combination of recombinant type OspA, OspB and OspC protein for the preparation of vaccine Lyme disease being realized to immunity.
But, before the successful vaccine of generation, need to overcome at least two obstacles.First, exist more than 20 kinds of OspC system type, and it be unclear that in vaccine to comprise which system type.Secondly, the suitable antigen of the development determining the anti-OspC antibody of spirillum acid is needed to determine base.
Therefore, still need the vaccine through improveing for protecting mammal in the art for a long time, and get on very well especially, dog, exempts to suffer from Lyme disease.
Summary of the invention
In an aspect, the present invention solves these and other demand by providing immunogenic composition, described immunogenic composition comprises: the first protein, it comprises the aminoacid sequence consistent with SEQ IDNO:1 (MDPNTVSSFQVDSFLWHVRKRVADQELGDAPFLDRLRRDQKSLRGRGSTLGLDIET ATRAGKQIVERILKEESDEALKMTMGKQNVSSLDEKNSVSVDLPGEMNVLVSKEKN KDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFK EDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTEIKSDGSGKAK EVLKSYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAA WNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNA LK) at least 95%, with the second protein, its immunodominant antigen comprising OspC system type F and N determines base.
In one group of embodiment, second protein comprises and determines with from the ring 5 (cyclic peptide) of one or more OspC system type I, H, C, M and D and the immunodominant antigen of α spiral 5 (helical peptides) multiple peptides that base at least 95% is consistent, wherein in addition, from the cyclic peptide of often kind of system type and helical peptides adjacent one another are and wherein, cyclic peptide and helical peptides continuous arrangement; With at least one in following each: the cyclic peptide of OspC system type F adjacent one another are and helical peptides, or the aminoacid sequence consistent with SEQ IDNO:3295%.In one group of embodiment, if there is the described aminoacid sequence consistent with SEQ IDNO:3295%, so it is positioned at the c-terminus of described second protein.
In one group of embodiment, the first protein is SEQ ID NO:1.In another group embodiment, system type I, H, N, C, M, D are consistent with SEQ ID NO:4-17 at least 95% respectively with helical peptides with the ring of F.
In another group embodiment, immunogenic composition can also comprise extra loop from one or more OspC system type F, T, U, E, A, B and K and helical peptides, and it is consistent with SEQ ID NO:16-29 respectively in certain embodiments.
In another group embodiment, immunogenic composition can more comprise at least one additional antigens, and described at least one additional antigens has the protectiveness of the microorganism for the disease that can cause in Canis familiaris L..The group of the optional self-contained following each of described microorganism: distemper (CD) virus, hepatitis infectiosa canis virus 2 type (CAV-2), dog parainfluenza (CPI) virus, dog small virus (CPV), canine coronavirus (CCV), canine alphaherpesvirus and rabies virus.Can be modified live virus preparation or nonactive virus formulation form for the antigen from these pathogen in vaccine combination of the present invention.Other pathogen also comprises leptospira bataviae (Leptospira bratislava), leptospira biflexa (Leptospira canicola), leptospira grippotyphosa (Leptospiragrippotyphosa), icterohemorrhagic form leptospira (Leptospiraicterohaemorrhagiae), leptospira pomona (Leptospira pomona), Ha Boweisi leptospira (Leptospira hardjobovis), Porphyromonas Pseudomonas (Porphyromonasspp.), Bacteroides (Bacteriodes spp.), leishmaniasis (Leishmania spp.), Ai Lixi body belongs to (Ehrlichia spp.), mycoplasm hyopneumoniae belongs to (Mycoplasma spp.) and Sabouraudites lanosus (Microsporum canis).
In a particular embodiment, immunogenic composition comprises SEQ ID NO:1 and SEQ IDNO:30 or SEQ ID NO:31.
On the other hand, the invention provides the vaccine combination comprising immunogenic composition as described above.Vaccine can also comprise adjuvant and pharmaceutically acceptable supporting agent.In different embodiments, adjuvant includes, but is not limited to mineral salt, surfactant and micron particle, bacterial product, cytokine and hormone, supporting agent, O/w emulsion and water-in-oil emulsion.
On the other hand, the present invention also provides the method for the Lyme disease in preventing canine, and it comprises the vaccine combination cast its dog immunity effective dose in need.
Accompanying drawing explanation
The adjacent bond tree of the OspC type that Fig. 1 explanation is undertaken cloning by the skin biopsy sample obtained from Canis familiaris L. and differentiated.
Fig. 2 illustrates the adjacent bond tree of the OspC type differentiated by carrying out sequencing to the Borrelia burgdoyferi pure lines of the skin biopsy body separation obtained from Canis familiaris L..
Fig. 3 A and Fig. 3 B illustrates chimeric sequences A12CF and A10CF (being SEQ IDNO:31 and 30 respectively), and it is suitable as the second protein of immunogenic composition described herein.
Fig. 4 illustrates the protein sequence of OspC system type A bacterial strain B31 and other OspC system type.
Detailed description of the invention
In order to understand subject application better, provide following non-limiting definition:
Term " at least 95% is consistent " comprises all concordance percentage ratio between 95% and 100% and comprises 95% and 100%, and such as 96%, 97%, 98%, 99% etc.
Term " α spiral 5 district " or " spiral 5 district " refer to the aminoacid sequence between the residue 160 and 200 of OspC system type A bacterial strain B31, and containing Secondary structural elements, comprise part people such as (, 2001) storehouse Herba Kalimeridis (Kumaran) that ring 6, α spiral 5 and destructuring C hold territory.
Term " conservative replacement " represents that amino acid residue is by the biologically similar residue substitutions of another kind, or the displacement of nucleotide sequence nucleotide, such that encoded amino acid residue does not change or the residue that another kind is biologically similar.The example that conservative replaces comprises a kind of hydrophobic residue (as isoleucine, valine, leucine or methionine) and is replaced by another kind of hydrophobic residue, or a kind of polar residues is replaced by another kind of polar residues, as arginine is replaced by aspartic acid by from propylhomoserin replacement, glutamic acid, or glutamine is by N replacement etc.Term " conservative replacement " also comprises the parent amino acid using the amino acid replacement be substituted to be unsubstituted, and its condition is that the antibody cultivated for the polypeptide be substituted also carries out immunoreation with the polypeptide be unsubstituted.
" the conservative change " of term reference protein or reference nucleic acid refers to the protein different from reference molecule or nucleic acid by means of only conservative replaces respectively.
Term " construct " (such as N-construct or I-construct) before system type title refers to the aminoacid sequence comprising cyclic peptide and helical peptides.
" helical peptides " or " alpha helical peptides " of a certain system type of term OspC refers to and determines with the immunodominant antigen in α spiral 5 district of the OspC protein from described system type the peptide that base at least 95% is consistent.Therefore, for example, helical peptides N refers to and determines with the immunodominant antigen in α spiral 5 district from OspC system type N the peptide that base at least 95% is consistent.
Term " immunodominant antigen decision base " refers to the epitope on molecule, and it induces dominant or intensive immunoreation compared with other epitope, comprises one or both B cell and t cell responses.
Term " Linear antigenic decision base " refers to the epitope comprising amino acid whose single, non-interrupted, the continuous chain that are bonded together to be formed peptide or polypeptide by peptide bond.This kind of epitope can by its primary structure, and namely in chain, amino acid whose linear order describes.When expressing in the recombinant type protein subunit at OspC, this kind of epitope keep with wild-type protein in conjunction with the ability of similar mode in conjunction with infection induced antibody.
" cyclic peptide " of a certain system type of term OspC refers to and determines with the immunodominant antigen in ring 5 district of the OspC protein from described system type the peptide that base at least 95% is consistent.Therefore, for example, cyclic peptide N refers to and determines with the immunodominant antigen in ring 5 district from OspC system type N the peptide that base at least 95% is consistent.
Term " ring 5 district " refers to aminoacid sequence, containing Secondary structural elements, comprises a part for α spiral 3, ring 5 and α spiral 4 between its residue 131 and 159 being usually located at OspC system type A bacterial strain B31.See people such as storehouse Herba Kalimeridis, 2001.The sequence of OspC system type A bacterial strain B31 is provided in SEQ ID NO:35 and Fig. 4.
As used herein term " treatment effective dose " means the amount being enough to cause in person under inspection immunoreactive microorganism or subunit antigen or polypeptide or polynucleotide molecule and its combination when casting.Immunoreation can including (but not limited to) the induction of cell and/or humoral immunity.
As used herein term " vaccine " and " vaccine combination " mean a kind of compositions, and its prevention or minimizing are infected, or it prevents or reduces one or more symptom or symptom of infecting.Protective effect for the vaccine combination of pathogen is realized by the reaction of induction of immunity in person under inspection (cell-mediated or humoral immune reaction or both combinations) usually.In general, eliminate or reduce to infect and occur, improve symptom or symptom, or accelerate to eliminate from infected person under inspection the protective effect that microorganism represents vaccine combination.
In extensive, the invention provides the immunogenic composition of the antibody of OspA and the OspC protein can induced for Borrelia burgdoyferi.Therefore, compositions will comprise two kinds of protein: the first protein comprising OspA or its fragment, and the second protein comprising OspC protein or its fragment.In certain embodiments, the second protein is the chimeric protein of multiple fragments of the OspC protein comprising different system type.
In certain embodiments, first protein comprises the fragment (SEQ IDNO:2) (MGKQNVSSLDEKNSVSVDLPGEMNVLVSKEKNKDGKYDLIATVDKLELKGTSDKNN GSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFN EKGEVSEKIITRADGTRLEYTEIKSDGSGKAKEVLKSYVLEGTLTAETTLVVKEGT VTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKE NTITVQQYDSNGTKLEGSAVEITKLDEIKNALK) of OspA protein, it is virus protein (the such as fragment of influenza virus NS 1 protein matter immediately, it is SEQ IDNO:3 (MDPNTVSSFQVDSFLWHVRKRVADQELGDAPFLDRLRRDQKSLRGRGSTLGLDIET ATRAGKQIVERILKEESDEALKMT)) downstream.That it can produce anti-OspA antibody in vaccinated animal to an important requirement of the first protein.Therefore, without the need to the full length sequence of OspA fragment, and also without the need to consistent with SEQ IDNO:2100%.
As in subject application pointed by other place, 95% sequence identity may be enough to provide suitable antibody production.Different aminoacids can be that conservative replaces, and/or the immunodominant antigen being positioned at OspA fragment determines the outside of base.
In other embodiments, shorter OspA fragment can be used.By knowing, how those of ordinary skill in the art determines which OspA fragment contains the immunodominant antigen that can produce spirillum acid antibody and determines base.
The present inventor surprisingly finds, comprise the fragment of (holding C to hold from N) influenza virus NS-1 protein, the first protein then removing the OspA protein of signal sequence is particularly useful for immunogenic composition of the present invention.
Popular in the aggressive Lyme disease of which kind of system type in Canis familiaris L. that prior art studies undeclared OspC.Major part research is carried out to human sample.It is K and A that the people such as Jones (Jones) are reported in the most popular system type found in the joint fluid of the scorching patient of human joint, and typically, and find system type A, B, C, D, H, K, N.Arthritis and Rheumatism (ArthritisRheum) 200960 (7) 2174.The people such as her grace Hart (Earnhart) find system type A, B, I, K, C, D, N in blood and/or CSF sample.Immunology of infection (Infect Immun.) 200573 (12): 7869.System type A, B, I and K are typically associated with the aggressive form of Lyme disease in the mankind by other research.
But unexpected discovery is in Canis familiaris L., and most popular system type is OspC F, and it is all not relevant to the aggressive form of Lyme disease in the mankind or Canis familiaris L. before this.System type N, it is relevant to the aggressive Lyme disease in the mankind, also relevant to the aggressive Lyme disease in Canis familiaris L..In addition, the present inventor finds system type T and U, not relevant to the aggressive Lyme disease in the mankind before this, may cause the aggressive Lyme disease in Canis familiaris L..
According to some embodiments, the second protein contains the immunoreactive immunodominant antigen that can produce for different OspC protein system type and determines base.More particularly, the second protein of immunogenic composition required in the present invention is chimeric protein, and its immunodominant antigen comprising OspC system type F and N determines base.Immunodominant antigen determines that base can in ring as discussed below and/or helical peptides form, or it can be present in the comparatively large fragment of target OspC protein.The suitable limiting examples of this kind of fragment is SEQ IDNO:32 (NNSGKDGNTSANSADESVKGPNLTEISKKITESNAVVLAVKEIETLLSSIDELATK AIGQKIDANGLGVQANQNGSLLAGAYAISTLITQKLSALNSEDLKEKVAKVKKCSE DFTNKLKNGNAQLGLAAATDDNAKAAILKTNGTNDKGAKELKDLSDSVESLVKAAQ VMLTNSVKELTSPVVAESPKKP), and it is the fragment of OspC system type F protein matter.
Previous research shows, the conserved domains (namely in different system type conservative) comprising OspC protein is for mice and the mankind but not to produce in Canis familiaris L. for anti-burgdorferi antibody be important.See people such as Loverich (Lovrich), clinical and vaccine immunity (Clin.and VaccineImmunol.) in May, 2007,635-637 page.But the more long segment that the present inventor surprisingly finds to add a kind of OspC system type (such as system type F) is favourable and therefore can make more effectively to produce the second protein to expression.
The people such as Bu Kesi (Buckles) prove the surface of the ring 5 of OspC protein expose to the open air and can be produce spirillum acid antibody suitable targets.Clinical vaccine immunology (Clin VaccineImmunol.) in October, 2006; 13 (10): 1162-5.Also see WO09135118.But, consider at least 21 kinds of system type (people such as Sai Nuosite (Seinost), immunology of infection (Infect Immun.) in the July, 1999 that describe OspC; 67 (7): 3518-241999), still need to determine which kind of combination provides the appropriate protection for Lyme disease.
Therefore, in certain embodiments, the Linear antigenic that the second protein comprises from ring 5 district (cyclic peptide) of the OspC protein of different system type and spiral 5 district (spiral 5 peptide) determines base.The system type of current consideration is T, U, E, A, B, K, I, H, N, C, M, D and F.Therefore the second protein can comprise ring from 2-13 the system type (such as 2,3,4,5,6,7,8,9,10,11,12,13 system type) of OspC and helical peptides.The order of peptide is unimportant.In certain embodiments, cyclic peptide is by helical peptides interval, and vice versa.In other words, ring and helical peptides are continuous arrangements: in this kind of embodiment, should there is not the situation that there is not helical peptides between two cyclic peptide in the second protein, and should not there is cyclic peptide between two helical peptides.
How those of ordinary skill in the art determines to determine base from the ring region of different OspC system type and the immunodominant antigen of coil region by knowing.For example, serum from the person under inspection of the B. barrgdorferi infection by different system type can react with from the ring region of corresponding system type and the specific peptide of coil region, and can carry out quantitatively (such as by ELISA, immunoblotting etc.) the combination of the antibody existed in serum and cyclic peptide and/or helical peptides, the clue which peptide to contain the immundominance Linear antigenic decision base from set OspC system type about is provided thus.
Similarly, the spirillum acid activity of antibody can be measured by method well known in the art, such as usual by will the Borrelia burgdoyferi of cultivation be passed through and carry out the serum incubation that personal immundominance Linear antigenic as described above determines the person under inspection that base is attacked, and to quantitatively carrying out with dead burgdorferi of living.
In certain embodiments, the sequence of cyclic peptide and helical peptides is as follows:
Cyclic peptide I is consistent with SEQ ID NO:4 (AKLKGEHTDLGKEGVT) at least 95%;
Helical peptides I is consistent with SEQ ID NO:5 (KGADELEKLFESVKNLSKAAKEMLTNSVKE) at least 95%;
Cyclic peptide H is consistent with SEQ ID NO:6 (SEKFAGKLKNEHASLGKKDAT) at least 95%;
Helical peptides H is consistent with SEQ ID NO:7 (KGAKELKDLSDSVESLVKA) at least 95%;
Cyclic peptide N is consistent with SEQ ID NO:8 (SDDFTKKLQSSHAQLGVAGGATT) at least 95%;
Helical peptides N is consistent with SEQ ID NO:9 (ADELEKLFKSVESLAKAAQDALANSVNELTS) at least 95%;
Cyclic peptide C is consistent with SEQ ID NO:10 (KKLKEKHTDLGKKDAT) at least 95%;
Helical peptides C is consistent with SEQ ID NO:11 (AAELEKLFESVENLAKAAKEMLSNS) at least 95%;
Cyclic peptide M is consistent with SEQ ID NO:12 (NKAFTDKLKSSHAELGIANGAAT) at least 95%;
Helical peptides M is consistent with SEQ ID NO:13 (KGAQELEKLFESVKNLSKAAQETLNNSVKE) at least 95%;
Cyclic peptide D is consistent with SEQ ID NO:14 (SESFTKKLSDNQAELGIENAT) at least 95%;
Helical peptides D is consistent with SEQ ID NO:15 (KGAEELVKLSESVAGLLKAAQAILANSVKELTSPVVAESPKKP) at least 95%;
Cyclic peptide F is consistent with SEQ ID NO:16 (SEDFTNKLKNGNAQLGLAAAT) at least 95%;
Helical peptides F is consistent with SEQ ID NO:17 (KGAKELKDLSDSVESLVKAAQVMLTNS) at least 95%;
Cyclic peptide T is consistent with SEQ ID NO:18 (STGFTNKLKSGHAELGPVGGNAT) at least 95%;
Helical peptides T is consistent with SEQ ID NO:19 (KGAKELKDLSESVEALAKAAQAMLTNS) at least 95%;
Cyclic peptide U is consistent with SEQ ID NO:20 (SEKFTKKLSESHADIGIQAAT) at least 95%;
Helical peptides U is consistent with SEQ ID NO:21 (KGAEELDKLFKAVENLSK) at least 95%;
Cyclic peptide E is consistent with SEQ ID NO:22 (STEFTNKLKSEHAVLGLDNLT) at least 95%;
Helical peptides E is consistent with SEQ ID NO:23 (KGAAELEKLKAVENLSKAAQDTLKNAVKELTSPIVAE SPKKP) at least 95%;
Cyclic peptide A is consistent with SEQ ID NO:24 (SETFTNKLKEKHTDLGKEGVT) at least 95%;
Helical peptides A is consistent with SEQ ID NO:25 (KGAEELGKLFESVEVLSKAAKEMLANSVKELTS) at least 95%;
Cyclic peptides B is consistent with SEQ ID NO:26 (SEEFSTKLKDNHAQLGIQGVT) at least 95%;
Helical peptides is consistent with SEQ ID NO:27 (KGVEELEKLSGSLESLS) at least 95%;
Cyclic peptide K is consistent with SEQ ID NO:28 (SEDFTKKLEGEHAQLGIENVT) at least 95%; With
Helical peptides K is consistent with SEQ ID NO:29 (AAELEKLFKAVENLAKAAKEM) at least 95%.
In certain embodiments, be placed together from the ring of identical systems type and helical peptides, namely adjacent one another are.For example, should by from the ring of other OspC system type any or helical peptides not separately from the cyclic peptide of OspC system type A and the helical peptides from OspC system type A.
In addition, although in certain embodiments, be closely adjacent to each other from the ring of identical OspC system type and helical peptides, in other embodiments, cyclic peptide and helical peptides can by not affecting the connexon sequence of final protein structure separately.Aminoacids characteristic and its to protein structure act on well known in the art and one of ordinary skill in the art can identify which kind of aminoacid is applicable to connexon.
As illustrated in example, the present inventor surprisingly finds that F with N is the most popular OspC system type relevant to the Lyme disease in Canis familiaris L..The present inventor also finds to exist can provide the extremely good level of protection for Lyme disease from the ring of system type I, H, N, C, M, D and F and helical peptides in Canis familiaris L..Although from the ring of different system type and the order of helical peptides inessential, but in certain embodiments, second protein comprises (directed by N to C) I-construct, H-construct, N-construct, C-construct, M-construct, D-construct is then the aminoacid sequence (such as SEQ ID NO:32) consistent with the fragment at least 95% of OspC system type F protein matter.Therefore, in certain embodiments, the second protein will comprise the aminoacid sequence consistent with SEQ ID NO:31 (A12CF) at least 95% (such as 96%, 97%, 98%, 99% and preferably 100%).
In other embodiments, be included in the second protein from the ring of system type F, T, U, E, A, B, K and helical peptides.In certain embodiments, therefore the second protein will comprise following (directed by N to C): T-construct, U-construct, E-construct, A-construct, B-construct, K-construct, I-construct, H-construct, N-construct, C-construct, M-construct and D-construct.Second protein can also optionally comprise F-construct, and it is the upstream of T-construct in certain embodiments.Or or in addition, the second protein can containing the aminoacid sequence (SEQ ID NO:32) consistent with the fragment at least 95% (such as 96%, 97%, 98%, 99%) of OspC system type F protein matter.
Other suitable example of second protein with and production and preparation method thereof be provided in application case PCT/US2011/056854 (on October 19th, 2011 submits to, inventor R. Marconi (R.Marconi) and C. she grace Hart (C.Earnhart)).
In certain embodiments, immunogenic composition will comprise SEQ ID NO:1; With any one in SEQ ID NO:30 or SEQ ID NO:31.
Sequence described herein can by the method manufacture be well known in the art.Polypeptide can use solid phase technique to be produced (see people (1969) Solid phase peptide synthesis (Solid-Phase Peptide Synthesis) such as such as Stewarts (Stewart) by direct peptide synthesis, San Francisco WH freeman company (WH Freeman Co, San Francisco); Merifield J. (Merrifield J.) (1963) JACS (J Am Chem Soc) 85:2149-2154).Peptide symthesis can be used manual technique or be undertaken by automatization.Automatic synthesis (such as) can use Applied Biosystems, Inc. 431A peptide synthesizer (Applied Biosystems 431A Peptide Synthesizer) (PerkinElmer (Perkin Elmer), California Foster City (Foster City, Calif)) explanation that provides according to manufacturer realizes.For example, subsequence chemically can be synthesized separately and be made chemically to combine, thus provides full-length polypeptide or its fragment.Or this kind of sequence can be ordered from the company of many special production polypeptide.The most usually polypeptide can be produced by expressing code nucleic acid and reclaiming polypeptide, as described below.
For example, in an embodiment, when cyclic peptide and helical peptides are consistent with the fragment 100% of the OspC protein of goal systems type, the nucleotide sequence of this kind of ring and helical peptides is also known or be easy to obtain from openly available data base (such as Genbank).If institute's ring selection/helical peptides is slightly different from the fragment of naturally occurring OspC protein, so can easily use well-known gene code to design nucleic acid sequence encoding.
Multiple organism presents preference and uses concrete codon to encode to amino acid whose insertion concrete in growthing peptide chain.Codon preference or codon-bias, the codon use between organism is different, is fully confirmed in multiple organism.Codon-bias is usually relevant to the translation efficiency of messenger RNA (mRNA), and believes that translation efficiency especially depends on the characteristic of translated codon and the availability of concrete transfer RNA (tRNA) molecule.In cell, the leading position of selected tRNA reflects the codon the most often used in peptide symthesis usually.Therefore, because Most amino-acids is encoded (methionine exception), so can customize nucleotide sequence for the best gene expression in given organism based on codon optimized by multiple codon.
Also comprise the method producing recombinant type polypeptide.These class methods a kind of comprise to be introduced in cell colony by any nucleic acid as described above, these nucleic acid are operably connected to the adjustment sequence that effectively can produce encoded polypeptide, cultivate host cell (such as yeast, insecticide, mammalian cell, plant cell etc.) in the medium so that express polypeptide, and from cell or from culture medium isolated polypeptide.By any transmission method as known in the art, nucleic acid is introduced this kind of cell, comprise such as transition, transfection, injection, particle gun, Passive intake etc.As skilled in the art will recognize, nucleic acid can be a part for carrier, as regroup expression vector, comprises any carrier known in DNA plasmid vector or affiliated field.
Or, can use based on acellular prokaryote or Eukaryotic expression system.
In certain embodiments, coding the first and/or second nucleic acid sequences to proteins more can comprise the sequence (" merging collocation thing ") of coded polypeptide, and itself and the first and/or second protein merge, thus promote the purification of fused protein.In some embodiment of this one side of the present invention, merging collocation thing is six histidine peptides (SEQ ID NO:47, HHHHHH), as pQE carrier (Kai Jie company (Qiagen, Inc.) provided), with people such as Jie Ensi (Gentz), described in institute of NAS periodical (Proc Natl Acad Sci USA) 86:821-824 (1989), or it can be HA labelling, it is corresponding to the epitope (Wilson's deriving from influenza hemagglutinin protein matter, I (Wilson, the people such as I), cell (Cell) 37:767, 1984).Polynucleotide can also contain non-coding 5' and 3' sequence, as transcribed, non-translated sequence; Montage and polyadenylation signal; Ribosome binding site and the sequence making mRNA stable.
Immunogenic composition described herein is specially adapted to the seriousness of preventing the Lyme disease in Canis familiaris L. or reducing its symptom.Therefore, on the other hand, the invention provides a kind of vaccine, it comprises immunogenic composition any one of above-described embodiment and suitable adjuvant.
The first and second protein in immunogenic composition of the present invention should exist with immune effective dose (being namely enough to cause immunoreactive amount in Canis familiaris L.).In certain embodiments, the concentration of the first protein between 1 mcg/ml and 100 mcg/ml (such as 5,10,20,30,40,50,60,70,80,90 mcg/ml), and the concentration of the second protein between 1 mcg/ml and 200 mcg/ml (such as 5,10,20,30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190 mcg/ml).In certain embodiments, the amount of the first protein between about 10 mcg/ml and 50 mcg/ml and the amount of the second protein between 20 mcg/ml and 100 mcg/ml.
Be applicable to adjuvant used according to the invention and include, but is not limited to some adjuvant types, as: mineral salt, such as vitriol, aluminium hydroxide, gel aluminum hydroxide are (such as
), aluminum phosphate and calcium phosphate; Surfactant and micron particle, such as non-ionic block polymers surfactant, cholesterol, virion, saponin (such as Quil A, QS-21 and GPI-0100), albuminous body, immunostimulating complex, snail like body, quaternary amine (DDA (DDA)), pyridine, VitAVitE; Bacterial product, as RIBI adjuvant system (Rui Bi company (Ribi Inc.)), Mycobacterium phlei (Mycobacterum phlei) cell wall skeleton (
), Romurtide (MDP) and tripeptides (MTP), monophosphoryl lipid A, bacillus calmette-guerin vaccine (Bacillus Calmete-Guerin), heat-labile enterotoxins of Escherichia coli, cholera toxin, trehalose dimycolate, CpG oligodeoxynucleotide; Cytokine and hormone, such as interleukin (IL-1, IL-2, IL-6, IL-12, IL-15, IL-18), granulocyte-macrophage colony stimulating factor, dehydroepiandrosterone, 1,25-dihydroxyvitamin D
3; Polyanion, such as glucosan; Polyacrylic acid (such as, polymethyl methacrylate, carbopol (Carbopol) 934P); Carrier, the saltant type heat labile enterotoxin (rmLT) of such as tetanus toxoid, diphtheria toxoid, choleratoxin B subunit, enterotoxigenic Escherichia coli, heat shock protein; O/w emulsion, such as
(Hai De Knicks company of the U.S. (Hydronics, USA)); And water-in-oil emulsion, such as not formula is completely and Freund's incomplete adjuvant (Freund's complete and incompleteadjuvant).In other embodiments, SP oil can also be used.As used herein, term " SP oil " represents fat liquor, and it comprises polyox-yethylene-polyoxypropylene block copolymer, squalane, Polysorbate 80 and buffer salt solution.Usually, SP fat liquor will comprise about 1% to 3% (volume/volume) block copolymer, about 2% to 6% (volume/volume) squalane, more particularly, about 3% to 6% squalane and about 0.1% to 0.5% (volume/volume) Polysorbate 80, all the other are buffer salt solution.
Vaccine described herein can be combination-vaccine, and it comprises above-mentioned immunogenic composition, combines with the antigen of at least one from other canine pathogen, can induce the protective immunological reaction for the disease caused by other pathogen this kind of in Canis familiaris L..
Other pathogen this kind of includes, but is not limited to canine distemper (CD) virus, hepatitis infectiosa canis virus 2 type (CAV-2), dog parainfluenza (CPI) virus, dog small virus (CPV), canine coronavirus (CCV), canine alphaherpesvirus and rabies virus.The antigen from these pathogen for vaccine combination of the present invention can in modified live virus preparation, nonactive virus formulation or subunit protein formulation form.In other embodiments, recombinant type CDV (canine distemper virus) can also be used.The method of the virulent virus strain of these viruses being carried out to attenuation is known in affiliated field with the method for the nonactive virus formulation of preparation, and is described in such as United States Patent (USP) the 4th, 567, No. 042 and the 4th, 567, No. 043.
Other pathogen also comprises leptospira bataviae, leptospira biflexa, leptospira grippotyphosa, icterohemorrhagic form leptospira, leptospira pomona, Ha Boweisi leptospira, Porphyromonas Pseudomonas, Bacteroides, leishmaniasis, Ai Lixi body genus, mycoplasm hyopneumoniae genus, Anaplasma (Anaplasma spp.) and Sabouraudites lanosus.Use method well known in the art, the antigen from these pathogen for vaccine combination of the present invention can be the nonactive form of cell preparation wholly or in part.For example, the method preparing nonactive Leptospira cell preparation is wholly or in part known in affiliated field and is described in such as with in Publication about Document: face (Yan), K-T, " the immunity aspect (Aspects of Immunity to Leptospira borgpeterseniiserovar hardjo) for Bo Shi leptospira hardjo serovar ", thesis for the doctorate, annex I, 1996, agriculture and food science institute (Facultyof Agriculture and Food Science), The Queen's Univ. of Belfast (The Queen'sUniversity of Belfast), the people such as Macintosh (Mackintosh), " Ha Erqiao-Bo Mengna type vaccine prevents the purposes (The use of a hardjo-pomona vaccine toprevent leptospiruria in cattle exposed to natural challenge with Leptospiainterrogans serovarhardjo) of leptospiruria in the cattle naturally attacked carried out with question mark shape leptospira hardjo serovar being exposed to ", veterinary of New Zealand magazine (New Zealand Vet.J.) 28:174-177,1980, the people such as Bo Lin (Bolin), " vaccination of pentavalent Lai Shi vaccine is to the effect (Effect ofvaccination with a pentavalent leptopsiral vaccine on Leptospirainterrogans serovar hardjo type hardjo-boivs infection of pregnant cattle) of the hardjo cattle infected of leptospira hardjo serovar cattle period of pregnancy ", American Veterinary research magazine (Am.J.Vet.Res.) 50:161-165,1989.
According to the present invention, can at least 6 ages in week, or at least 7 ages in week, or the Canis familiaris L. at least 8 or 9 week age casts vaccine.Cast and undertaken by any known approach, comprise per os, intranasal, through surface mucosa, percutaneous and parenteral (such as intravenous, intraperitoneal, Intradermal, subcutaneous or intramuscular).Cast to use and realize without needle-like delivery apparatus.Cast and approach can also be used to combine realization, such as, first use
approach casts, and then uses through mucous membrane approach to cast.In certain embodiments, the approach that casts comprises subcutaneous and intramuscular and casts.
The all publication (patent publication and non-patent publication) quoted in this description represent the level of skill of those skilled in the art in the invention.The mode that all these publication are quoted in this article all is in full incorporated to, and its degree as the indivedual publication of each section specifically and be illustrated as individually and be incorporated to way of reference.
In order to more clearly understand the present invention, hereafter set forth following instance.These examples are only illustrative, and are not understood to limit the scope of the invention by any way or ultimate principle.In fact, except herein and except describing, those skilled in the art easily knows various amendment of the present invention by being become aobvious by the example of hereinafter setting forth and aforementioned explanation.This kind of amendment is also intended to belong in the scope of appended claims.
Example
Example 1. determines the Borrelia burgdoyferi OspC system type relevant to the Lyme disease in Canis familiaris L.
Ixodes scapularis (Ixodes scapularis tick) of growing up is collected by being marked at southern Rhode Island State (Rhode Island).Use the anti-Borrelia burgdoyferi antibody of standard method and labelling, measured the Ticks worm percentage ratio infecting Borrelia burgdoyferi by direct fluorescence microscopy.
All programs are all carried out according to the regulation of Animal Welfare Law (Animal Welfare Act) and are supported Canis familiaris L. according to farm dog breeders S.O.P. (Farm Canine Husbandry StandardOperating Procedures).(7 male, and 8 female for the Canis familiaris L. of specifying 15 objects of two kinds of sexes to breed; 9 to 10 ages in week; Marshall living resources company (MarshallBioresources)) identiflication number and be divided into four seminar, be expressed as T01 (n=4), T02 (n=4), T03 (n=4) and T04 (n=3).Canis familiaris L. is equipped with Elizabethan to lead (Elizabethancollars) and stable breeding in apartment-type cage one by one.In the previous day of Ticks insect infection, collect serum from each Canis familiaris L..Use is placed in the every fixing infection room on one side of mesothorax (midthorax), uses the Canis familiaris L. in 0,25,50 or 75 adult ixodes scapularis infection research group T01, T02, T03 and T04 respectively.Ticks worm is supported full, removes and collect blood serum sample and skin biopsy body the 49th day and the 90th day (relative to starting to infect).Test (IDEXX) by SNAP 4DX and assess seroconversion.In order to cultivate spirillum, a part for every a skin biopsy body is placed in BSK-H culture medium (6% rabbit anteserum; 37EC, 5%CO
2) in.As described previously, clonal population is obtained by subsurface coating from culture.Excise colony from plate and be placed in BSK-H culture medium and cultivate.
Use triumphant outstanding DNeasy test kit (Qiagen DNeasy Kit), illustrate from skin biopsy body according to supplier and extract DNA.In addition, as described previously, DNA is extracted from the culture of the clonal population of Borrelia burgdoyferi.Use from the DNA organizing (100 nanogram) to extract and Borrelia burgdoyferi cytolysis thing (the 1:1 supernatant from boiling; GoTaq polymerase) DNA that obtains carries out pcr amplification to ospC gene.All PCR all use standard conditions to carry out.A part for each reactant is assessed by agarose gel electrophoresis and ethidium bromide staining.From gel (triumphant outstanding gel extraction kit (Qiagen Gel Extraction Kit); Kai Jie company (QIAGEN)) remove remaining PCR primer and bonding with pET46Kk/LIC carrier (Nuo Jie company (Novagen)).Plasmid is breeding in escherichia coli NovaBlue cell (Nuo Jie company).By the ospC gene of PCR screening colony.Seethe with excitement by making a part for every a ospC Positive E. coli colony and produce the template of PCR.Also part colony is inoculated in LB culture medium (2 milliliters), grow overnight, uses triumphant outstanding MiniPrep test kit (Kai Jie company) to extract plasmid by collected by centrifugation.Primer following (5' to 3') for PCR:
OspC-F1
GACGACGACAAGATTGAATACATTAAGTGCAATATTAATGAC(SEQ ID NO:33)
OspC-R1
GAGGAGAAGCCCGGTTTACAAATTAATCTTATAATATTGATCTTAATTAAGG(SEQ ID NO:34)
DNA sequencing is carried out by Eurofins MWG Operon.Use ClustalX 2.0.10 software, under multisequencing alignment pattern, produce adjacent bond tree by default configuration and Gonnet matrix, and use N-J Plot (2.2 editions) to observe.
Result
The analysis of Borrelia burgdoyferi sickness rate from the Ticks worm that Rhode Island is collected. use direct fluorescence microscopy, the ixodes scapularis from southern Rhode Island on-site collection measuring about 50% infects Borrelia burgdoyferi.This is consistent with the Ticks insect infection rate previously reported in this region.
B. barrgdorferi infection in the Canis familiaris L. occurred by Ticks insect infection. when this research starts, using SNAP 4DX analytic process (IDEXX), by immunoblotting assay with by using Borrelia burgdoyferi, confirming that all Canis familiaris L.s are seronegativity.In order to infect Canis familiaris L. with Borrelia burgdoyferi by natural propagation approach, Canis familiaris L. health is supported the Ticks worm of on-site collection.Because the infection rate in Ticks worm is about 50%, the Ticks worm (0,25,50 or 75) increasing number is placed on Canis familiaris L. health.Within the 49th day after Ticks insect infection, collect blood serum sample and assess immune state.In the Canis familiaris L. being subject to Ticks insect infection, 10 in 11 Canis familiaris L.s is seropositivity to Borrelia burgdoyferi.All negative control Canis familiaris L.s (not by Ticks insect infection) are seronegativity.The skin biopsy body collected from every Canis familiaris L. is extracted STb gene and with ospC and flaB primer collection, is tested by PCR for Borrelia burgdoyferi.All seropositivity Canis familiaris L.s produce ospC and the flaB amplicon estimating size.All seronegativity Canis familiaris L.s are all negative in PCR to two kinds of genes.
The multifarious analysis of OspC in the bacterial strain found in infected Canis familiaris L. tissue. in order to measure the ospC genotype being deposited in the bacterial strain be exposed in the dog skin skin of Ticks worm, by the DNA extracted from skin biopsy body, pcr amplification is carried out to ospC.Plasmid will be bred in gained amplicons cloned to pET46Ek/LIC in escherichia coli.Then from being no less than 5 independently escherichia coli colony separation quality grains and measure ospC sequence.Sequence is aimed at and dendrogram construction (Fig. 1) shows that in 10 Canis familiaris L.s 6 merely hit, and produces the persistency (table 1) of the bacterial strain of some different ospC types.Because use multiple Ticks insect infections every Canis familiaris L., so this observed result is not unexpected.Differentiate OspC type A, B, F, I and N, wherein type F and N is most popular (being respectively 7 in 10 Canis familiaris L.s 5 and 10 Canis familiaris L.s).
In order to define the genotypic scope of the ospC existed in infected Canis familiaris L. further, be coated with culture from skin biopsy body to produce clonal population.By this method, the bacterial strain of the expression ospC type do not detected by the PCR of biopsy body sample can be differentiated.Then indivedual Borrelia burgdoyferi colony (Fig. 2) is tested by PCR for ospC.In 6 Canis familiaris L.s 3 merely hit differentiates other ospC type.Previously do not differentiated two kinds of OspC types differentiated, it all derives from same Canis familiaris L..These system type are expressed as DRI85a and DRI85e.Other ospC type differentiated by this method comprises type E, F, H, I, N, U and T (table 1).In general, in these are analyzed, 11 kinds of different OspC types are altogether detected.
From the OspC type of sequencing biopsy body and clone and separate thing in each group, table 1. and every only indivedual Canis familiaris L..
In this research, the present inventor is determined in Canis familiaris L. successfully to cause and infects and the ospC genotype of the Lyme disease spirillum bacterial strain retained.The health of laboratory Canis familiaris L. is supported the ixodes scapularis from Rhode Island on-site collection and the ospC genotype of the bacterial strain existed mensuration skin after 49 days.The OspC type that discriminating 11 kinds is different altogether.OspC type F (previously not detecting in the mankind) is the OspC type (50% infected Canis familiaris L.) the most frequently detected.Few type B, N and U occurred in the mankind also detected.Two kinds of ospC types (DRI85a and DRI85e) previously do not defined also are reclaimed.The multiformity observed in this research is consistent with research comparatively early, and described research shows to keep some ospC system type in endemicity Borrelia burgdoyferi colony.Because the Ticks worm for this research is collected from single geographic area, so the bacterial strain of probably expressing other OspC type well do not presented in Rhode Island also can infect Canis familiaris L..Do not consider this supplemental instruction, first this research prove that previous not relevant to human infection OspC type can infect Canis familiaris L. effectively, therefore contributes to the appropriate design of canine lyme disease vaccine of new generation.
In example 2. Canis familiaris L., recombinant type is fitted together to effect of Borrelia burgdoyferi OspC/OspA vaccine.
Select 30 Canis familiaris L.s being all in good general health situation for research.Blood sample was collected before initial vaccination.Canis familiaris L. accepts the one in following vaccine, described by middle table 2: T01:PBS (reference product); T02:20 mcg/ml OspA+30 mcg/ml A12CF (SEQ ID NO:31); T03:20 mcg/ml OspA+30 mcg/ml A10CF (SEQ ID NO:30).(A12CF is made up of the epitope from multiple OspC system type, and it is joined together to form single polypeptide.A10CF is also made up of the epitope from multiple OspC system type; The design class of its design and A12CF seemingly.) Canis familiaris L. vaccination twice when 8 week age and 11 week age, and then accepted attack when 14 week age.After vaccination, observe the reaction in Canis familiaris L. or extremely last 20 minutes.Observed swelling, pain, heating, the abscess of injection site at the 1st, 2,3 and 22,23,24 day, suppurate.Be that every Canis familiaris L. is equipped with Elizabethan [E] neck in storing Ticks worm the previous day, and the action of monitoring Canis familiaris L. when Elizabethan's neck is in correct position, feed and drinking-water ability.The rear dorsal line of 20 to the 40 adult ixodes scapularises collected from Northeastern United States (male, female) along every bar Canis familiaris L. is put, and lasts 7 to 10 days and carry out feeding until full.Collect blood serum sample and skin biopsy body in the interval of regulation, and carry out analyzing to monitor infection.Collect full or that do not adhere to, non-viable Ticks worm and store at 4 DEG C.At the end of attack, remove and store remaining Ticks worm, and according to labelling with surperficial acaricide process Canis familiaris L., then carrying out second time after 30 days and use.Observe overall physical appearance and the state of Canis familiaris L. every day.Carry out clinical observation (walking lamely and ataxia); If observe any one, so measure/record the body temperature (tympanic temperature) of Canis familiaris L. every day until clinical symptom disappears.Receive by Ticks worm and based on Subsequent infection, carry out blood collecting and skin biopsy (except the difference of schedule/sequential) according to agreement.Back cervical vertebra district gathers perforated skin biopsy body near general Ticks worm attachment area, and timing is with consistent with blood collecting.Final biopsy body is gathered immediately after euthanasia and before postmortem.
Table 2. research design
Result
After vaccination, in any Canis familiaris L., do not observe reaction or abnormal, and there is not any exception (swelling, pain, heating, abscess, suppurate) in injection site place yet.The body temperature of tympanum probe measurement is used not show remarkable or lasting rising.In two Canis familiaris L.s carrying out vaccination with A12CF+OspA (T02), occur that discontinuity is walked lamely, and a Canis familiaris L. in non-vaccination group (T01) walked lamely at 192-193 days.During studying, in any Canis familiaris L., do not observe ataxia.During studying, in some Canis familiaris L.s, observe abnormal health event, comprise pyoderma, bite, abrade, loose stool, external otitis etc., but all not owing to vaccine or vaccination.
At the 146th day, observe serological reaction in a Canis familiaris L. in 8 in 10 contrasts Canis familiaris L. (T01) and T02, represent active B. barrgdorferi infection.9 contrasts Canis familiaris L. (T01) were positive serology at the 177th day, and all Canis familiaris L.s in T01 are positive at the end of research.By contrast, terminate from the 177th day to research, in each vaccination group, only a Canis familiaris L. is positive.
For the Ticks worm studied through Borrelia burgdoyferi and limit worm double infection.The result of serological analysis shows that limit worm is successfully propagated to Canis familiaris L. by Ticks worm.This supports that vaccine constructs (T02, T03) is only for the specificity of Borrelia burgdoyferi.
In all skies, except except the OspC that the 146th day compares T01 and T02, when comparing T01 and T02 and comparing T01 and T03, ELISA value (being expressed as the geometric mean titer for each in OspA and OspC) significantly different (table 3).
The ELISA geometric average OspA in each group, table 3. and each stage, OspC
Borrelia burgdoyferi organism of living is had in specific ELISA to the blood serum sample analyzed and collect from the Canis familiaris L. (A12CF+OspA) contrasted Canis familiaris L. (T01) and T02 during attack after-stage.The geometric mean titer that T01 contrasts T02 is: the 146th day, 90 to 6; 177th day, 116 to 7; With the 198th day, 87 to 7.Therefore, these results support that vaccine is for the protective effect of Borrelia burgdoyferi.
For living, spirillum cultivates skin punctures biopsy samples.In T01 group, within the 146th day, there are 4 Canis familiaris L.s and within the 177th day, have 5 Canis familiaris L.s to be that culture is positive.A Canis familiaris L. is had to have spirillum positive skin culture at the 177th day in each in T02 and T03.At the end of research, do not obtain positive culture from any group.At the 146th day, also use flaB and ospC Auele Specific Primer for whether there is Borrelia burgdoyferi to assess skin punctures biopsy body.There are 5 Canis familiaris L.s in T01 to flaB for positive, and have 3 Canis familiaris L.s to ospC for positive.Canis familiaris L. is all there is not to arbitrary PCR reaction for positive in T02 or T03.
Joint and skin biopsy microscopy show that the vaccination of T02 or T03 vaccine can be protected from infection (data do not show).The joint of vaccinated Canis familiaris L. and the change of skin less, joint and change of skin can be used as the feature of Lyme disease.If there is this kind of change, so when with compared with the not vaccinated tissue contrasting Canis familiaris L. time, its seriousness in vaccinated Canis familiaris L. is lower.Based on the number (in T02 6 of Canis familiaris L. that there is focus in joint; In T03 7), two kinds of vaccine (T02; T03) exist slightly different between.But, the decisive conclusion that better protection about which kind of construct is provided cannot be drawn.
In a word, A12CF+OspA (T02) and A10CF+OspA (T03) all can available protecting Canis familiaris L. from the B. barrgdorferi infection propagated by ixodes scapularis.
Claims (26)
1. an immunogenic composition, it comprises:
A) the first protein, it comprises the aminoacid sequence consistent with SEQ ID NO:1 at least 95%; With
B) the second protein, its immunodominant antigen comprising OspC system type F and N determines base.
2. immunogenic composition according to claim 1, wherein said first protein comprises SEQ ID NO:1.
3. immunogenic composition according to claim 1 and 2, wherein said second protein comprises:
I) with from ring 5 district (cyclic peptide) of OspC system type I, H, C, M and D and the immunodominant antigen in α spiral 5 district (helical peptides) multiple peptides that base at least 95% is consistent are determined; With
Ii) at least one in following each:
The cyclic peptide of OspC system type F a) adjacent one another are and the helical peptides of OspC system type F, or
B) consistent with SEQ ID NO:3295% aminoacid sequence.
4. according to the immunogenic composition in claim 1 and 2 described in arbitrary claim, wherein:
Cyclic peptide I is SEQ ID NO:4;
Helical peptides I is SEQ ID NO:5;
Cyclic peptide H is SEQ ID NO:6;
Helical peptides H is SEQ ID NO:7;
Cyclic peptide N is SEQ ID NO:8;
Helical peptides N is SEQ ID NO:9;
Cyclic peptide C is SEQ ID NO:10;
Helical peptides C is SEQ ID NO:11;
Cyclic peptide M is SEQ ID NO:12;
Helical peptides M is SEQ ID NO:13;
Cyclic peptide D is SEQ ID NO:14;
Helical peptides D is SEQ ID NO:15.
5. the immunogenic composition according to claim arbitrary in Claim 1-3, wherein said second protein comprises the aminoacid sequence consistent with SEQ ID NO:3295% at the c-terminus of described second protein.
6. the immunogenic composition according to claim arbitrary in claim 1 to 5, wherein press N to C directed, described second protein comprises I-construct, H-construct, N-construct, C-construct, M-construct, D-construct.
7. the immunogenic composition according to claim arbitrary in claim 1 to 6, wherein said second protein more comprises and determines with from ring 5 district (cyclic peptide) of OspC system type T, U, E and the immunodominant antigen in α spiral 5 district (helical peptides) multiple peptides that base 95% is consistent.
8. immunogenic composition according to claim 7, wherein press N to C directed, described second protein comprises T-construct, U-construct, E-construct.
9. the immunogenic composition according to claim 7 or 8, wherein
Cyclic peptide T is SEQ ID NO:18;
Helical peptides T is SEQ ID NO:19;
Cyclic peptide U is SEQ ID NO:20;
Helical peptides U is SEQ ID NO:21;
Cyclic peptide E is SEQ ID NO:22;
Helical peptides E is SEQ ID NO:23.
10. the immunogenic composition according to claim arbitrary in claim 1 to 9, wherein said second protein more comprises and determines with from ring 5 district (cyclic peptide) of OspC system type A, B, K and the immunodominant antigen in α spiral 5 district (helical peptides) multiple peptides that base 95% is consistent.
11. immunogenic compositions according to claim 10, wherein press N to C directed, described second protein comprises A-construct, B-construct, K-construct.
12. immunogenic compositions according to claim 11, wherein
Cyclic peptide A is SEQ ID NO:24;
Helical peptides A is SEQ ID NO:25;
Cyclic peptides B is SEQ ID NO:26;
Helical peptides B is SEQ ID NO:27;
Cyclic peptide K is SEQ ID NO:28;
Helical peptides K is SEQ ID NO:29.
13. immunogenic compositions according to claim arbitrary in claim 10 to 12, wherein press N to C directed, described second protein comprises T-construct, U-construct, E-construct, A-construct, B-construct, K-construct.
14. immunogenic compositions according to claim 13, wherein press N to C directed, described second protein comprises T-construct, U-construct, E-construct, A-construct, B-construct, K-construct, I-construct, H-construct, N-construct, C-construct, M-construct, D-construct.
15. immunogenic compositions according to claim 12, wherein said F-construct is the upstream of described T-construct, and the wherein said aminoacid sequence consistent with SEQ ID NO:3295% is the downstream of described D-construct.
16. immunogenic compositions according to claim 15, wherein
Cyclic peptide F is SEQ ID NO:16;
Helical peptides F is SEQ ID NO:17.
17. immunogenic compositions according to claim arbitrary in claim 1 to 16, wherein from the described cyclic peptide of each system type and described helical peptides adjacent one another are.
18. immunogenic compositions according to claim arbitrary in claim 1 to 17, wherein the described cyclic peptide of each system type and described helical peptides are continuously arranged.
19. immunogenic compositions according to claim arbitrary in claim 1 to 18, wherein the described cyclic peptide of each system type is the upstream of the corresponding helical peptides of described system type.
20. immunogenic compositions according to claim arbitrary in claim 1 to 19, wherein
A. described first protein is SEQ ID NO:1; And
B. described second protein is SEQ ID NO:30.
21. immunogenic compositions according to claim arbitrary in claim 1 to 19, wherein
A. described first protein is SEQ ID NO:1; And
B. described second protein is SEQ ID NO:31.
22. immunogenic compositions according to claim arbitrary in claim 1 to 21, it more comprises at least one additional antigens, and described at least one additional antigens can prevent or reduce the pathology effect that can cause the infected by microbes of disease in Canis familiaris L..
23. immunogenic compositions according to claim 22, wherein said microorganism is selected from by the following group formed: canine distemper (CD) virus, hepatitis infectiosa canis virus 2 type (CAV-2), dog parainfluenza (CPI) virus, dog small virus (CPV), canine coronavirus (CCV), canine alphaherpesvirus and rabies virus.
24. 1 kinds of vaccine combinations, it comprises immunogenic composition according to claim arbitrary in claim 1 to 23 and adjuvant.
25. vaccine combinations according to claim 24, wherein said adjuvant is selected from by the following group formed: mineral salt, surfactant and micron particle, bacterial product, cytokine and hormone, supporting agent, O/w emulsion and water-in-oil emulsion.
26. 1 kinds of preventions or reduce the methods of the pathology effect that Borrelia burgdoyferi (Borrelia burgdorferi) infects in dog, its comprise cast described to its dog immunity effective dose in need according to the vaccine combination in claim 24 and 25 described in arbitrary claim.
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| US201261635031P | 2012-04-18 | 2012-04-18 | |
| US61/635,031 | 2012-04-18 | ||
| PCT/US2013/037063 WO2013158818A2 (en) | 2012-04-18 | 2013-04-18 | Vaccines and methods to treat lyme disease in dogs |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110483624A (en) * | 2019-08-22 | 2019-11-22 | 中国疾病预防控制中心传染病预防控制所 | Borrelia garinii OspA PROTEIN C end peptide fragment and its application |
| CN111432835A (en) * | 2017-12-04 | 2020-07-17 | 英特维特国际股份有限公司 | Canine Lyme disease vaccine |
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| EP3204404A4 (en) | 2014-10-08 | 2018-06-06 | Virginia Commonwealth University | Stage specific diagnostic antigens, assay and vaccine for lyme disease |
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| US8808705B2 (en) * | 2010-10-20 | 2014-08-19 | Virginia Commonwealth University | Polyvalent chimeric OspC vaccinogen and diagnostic antigen |
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2013
- 2013-04-16 UY UY0001034745A patent/UY34745A/en not_active Application Discontinuation
- 2013-04-17 AR ARP130101267A patent/AR090726A1/en unknown
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- 2013-04-18 WO PCT/US2013/037063 patent/WO2013158818A2/en active Application Filing
- 2013-04-18 JP JP2015507162A patent/JP2015514776A/en active Pending
- 2013-04-18 EP EP13719257.1A patent/EP2838555A2/en not_active Withdrawn
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Cited By (3)
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| CN111432835A (en) * | 2017-12-04 | 2020-07-17 | 英特维特国际股份有限公司 | Canine Lyme disease vaccine |
| US11883476B2 (en) | 2017-12-04 | 2024-01-30 | Intervet Inc. | Canine lyme disease vaccine |
| CN110483624A (en) * | 2019-08-22 | 2019-11-22 | 中国疾病预防控制中心传染病预防控制所 | Borrelia garinii OspA PROTEIN C end peptide fragment and its application |
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| EP2838555A2 (en) | 2015-02-25 |
| EA201491668A1 (en) | 2015-08-31 |
| JP2015514776A (en) | 2015-05-21 |
| AR090726A1 (en) | 2014-12-03 |
| BR112014025740A2 (en) | 2017-07-04 |
| WO2013158818A2 (en) | 2013-10-24 |
| AU2013249229A1 (en) | 2014-10-23 |
| CA2870179A1 (en) | 2013-10-24 |
| WO2013158818A3 (en) | 2014-04-17 |
| CA2870179C (en) | 2018-09-04 |
| UY34745A (en) | 2013-11-29 |
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