EP1171606A2 - Vaccins a base d'acide nucleique contre les rickettsioses et utilisations - Google Patents

Vaccins a base d'acide nucleique contre les rickettsioses et utilisations

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Publication number
EP1171606A2
EP1171606A2 EP00930134A EP00930134A EP1171606A2 EP 1171606 A2 EP1171606 A2 EP 1171606A2 EP 00930134 A EP00930134 A EP 00930134A EP 00930134 A EP00930134 A EP 00930134A EP 1171606 A2 EP1171606 A2 EP 1171606A2
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EP
European Patent Office
Prior art keywords
seq
nucleic acid
nos
polynucleotide
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP00930134A
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German (de)
English (en)
Inventor
Anthony F. Barbet
Michael V. Bowie
Roman Reddy Ganta
Michael J. Burridge
Suman M. Mahan
Travis C. Mcguire
Fred R. Rurangirwa
Annie L. Moreland
Bigboy H. Simbi
William W. Whitmire
Arthur R. Alleman
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University of Florida
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University of Florida
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Publication of EP1171606A2 publication Critical patent/EP1171606A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/29—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Richettsiales (O)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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

Definitions

  • This invention relates to nucleic acid vaccines for nckettsial diseases of animals, including humans
  • the ⁇ ckettsiasare a group of small bacteriacommonlytransmittedby arthropod vectoi s to man and animals, in which they may cause serious disease
  • the pathogens causing human nckettsial diseases include the agent of epidemic typhus, Rickettsia prowazeku, which has resulted in the deaths of millions of people during wartime and natural disasters
  • the disease occurs throughout most of Africa and has an estimated endemic area of about 5 million square miles In endemic areas, heartwater is a latent infection in indigenous breeds of cattle that hav e been subjected to centuries of natural selection The problems occur where the disease contacts susceptible or naive cattle and other ruminants Heartwater has been confirmed to be on the island of Guadeloupe in the Caribbean and is spreading through the Caribbean Islands The tick vectors responsible for spreading this disease are already present on the American mainland and threaten the livestock industry in North and South America In acute cases of heartwater, animals exhibit a sudden rise in temperature, signs of anorexia, cessation of rumination, and nervous symptoms including staggering, muscle twitching, and convulsions Death usually occurs during these convulsions Peracute cases of the disease occur where the animal collapses and dies in convulsions having shown no preliminary symptoms Mortality is high in susceptible animals Angora sheep infected with the
  • Vaccines based on inactivated or attenuated ⁇ ckettsiae have been developed against certain nckettsial diseases, for example against R piowazeku and R rickettsn
  • these vaccines have major problems or disadvantages, including undesirable toxic reactions, difficult) in standardization. and expense (Woodward. T [1981 ] ' Rickettsial diseases certain unsettled problems tn their historical perspectiveJIn Rickettsia and Rickettsial Diseases. W Burgdorfer and R Anacker.
  • a vaccine currently used in the control of heartwater is composed of live infected sheep blood
  • This vaccine also has several disadvantages
  • First expertise is required for the intravenous inoculation techniques required to administer this vaccine
  • Second, vaccinated animals may experience shock and so require dailv monitoring for a period after vaccination There is a possibility of death due to shock throughout this monitoring period, and the drugs needed to treat any shock induced by vaccination are costly
  • Third, blood-borne parasites may be present in the blood vaccine and be transmittedto the vaccinates
  • the blood vaccine requires a cold chain to preserve the vaccine
  • nucleic acid vaccination has been shown to induce protective immune responses in non- v iral systems and in diverse animal species (Special Conference Issue, WHO meeting on nucleic acid vaccines [1994] Vaccine 12 1491 ) Nucleic acid vaccination has induced cytotoxic ly mphocyte (CTL). T-helper 1. and antibody responses, and has been shown to be protective against disease (UlmerJ .
  • mice with DNA encoding the influenza nucleoprotein caused the production ofhigh titer antibodies, nucleoprotein-specificCTLs. and protection against viral challenge
  • mice with plasmid DNA encoding the Plasmod m voelu circumsporozoite protein induced high antibody titers against malaria sporozoitesand CTLs. and protection against challenge infection (Sedegah M . R Hedstrom. P Hobart.
  • nucleic acid vaccines may be successful to protect against nckettsial disease or that a major surface protein conserved in rickettsia was protective against disease
  • novel vaccines for conferring immunity to rickettsia infection including Cowdriaruminantium causing heartwater
  • novel nucleic acid compositions and methods of using those compositions including to confer immunity in a susceptible host
  • nucleic acid vaccines can be driven by the human cytomegalov ⁇ rus(HCMV) enhancer-promoter
  • HCMV human cytomegalov ⁇ rus
  • the subject invention further concerns the genes designated C O dria riimmantium map 2, Cowdna ru nnantium lhwor ⁇ , Cowdt ia riimwantiim Cowdria riimmantium and Cowdria runnnantium igdoi ⁇ and the use of these genes in diagnostic and therapeutic applications
  • the subject invention further concerns the proteins encoded by the exemplified genes, antibodies to these proteins, and the use of such antibodies and proteins in diagnostic and therapeutic applications
  • the polynucleotide vaccines are administered in conjunction with an antigen
  • the antigen is the polypeptide which is encoded by the polynucleotideadministeredas the polynucleotidevaccine
  • the antigen is administered as a booster subsequent to the initial administration of the polynucleotide vaccine
  • Figures 1A-1C show a comparison of the amino acid sequences from alignment of the three nckettsial proteins, namely, Cowdria ruminantium (C r ), Ehrlichia chaffeensis (E c ).
  • FIG. 2A-2C shows the DNA sequence of the 28 kDa gene locus cloned from E chaffeensis (Fig 2A-2B) and E cams (Fig 2C)
  • Fig 2A-2B shows the DNA sequence of the 28 kDa gene locus cloned from E chaffeensis (Fig 2A-2B) and E cams (Fig 2C)
  • One letter amino acid codes for the deduced protein sequences are presented below the nucleotide sequence
  • the proposed s ⁇ gma-70-l ⁇ ke promoter sequences (38) are presented in bold and underlined text as -10 and -35 (consensus -35 and - 10 sequences are TTGAC A and TATAAT.
  • Figure 3A shows the complete sequence of the MAP2 homolog of Ehrlichia cams
  • the arrow (-*) represents the predicted start of the mature protein
  • the asterisk (*) represents the stop codon
  • Underlined nucleotides 5' to the open reading frame with -35 and -10 below represent predicted promoter sequences
  • Double underlined nucleotides represent the predicted nbosomal binding site Underlinednucleotides 3' to the open reading frame represent possible transcription termination sequences
  • Figure 3B shows the complete sequence of the M AP2 homolog of Ehrlichia chaffeensis
  • the arrow (- ⁇ ) represents the predicted start of the mature protein
  • the asterisk (*) represents the stop codon
  • Underlined nucleotides 5' to the open reading frame with -35 and - 10 below represent predicted promoter sequences
  • SEQ ID NO. 1 is the coding sequence of the MAPI gene from Cowdria ruminantium (Highway isolate)
  • SEQ ID NO. 2 is the polypeptide encoded by the polynucleotide of SEQ ID NO 1
  • SEQ ID NO. 3 is the coding sequence of the MAPI gene from Ehrlichia chaffeensis
  • SEQ ID NO. 4 is the polypeptide encoded bv the polynucleotide of SEQ ID NO 3
  • SEQ ID NO. 5 is the Anaplasma marginale MSP4 gene coding sequence
  • SEQ ID NO. 6 is the polypeptide encoded by the polvnucleotide of SEQ ID NO 5
  • SEQ ID NO. 7 is a partial coding sequence of the VSAl gene from Ehrlichia chaffeensis. also shown in Figures 2A-2B
  • SEQ ID NO. 8 is the coding sequence of the VSA2 gene from Ehrlichia chaffeensis. also shown in Figures 2A-2B
  • SEQ ID NO. 9 is the coding sequence of the VSA3 gene from Ehrlichia chaffeensis. also shown in Figures 2A-2B
  • SEQ ID NO. 10 is the coding sequence of the VSA4 gene from Ehrlichia chaffeensis. also shown in Figures 2A-2B
  • SEQ ID NO. 11 is a partial coding sequence of the VSA5 gene from Ehrlichia chaffeensis. also shown in Figures 2A-2B
  • SEQ ID NO. 12 is the coding sequence of the VSAl gene from Ehrlichia cams, also shown in Figure 2C
  • SEQ ID NO. 13 is a partial coding sequence of the VSA2 gene from Ehrlichia cams, also shown in Figure 2C
  • SEQ ID NO. 14 is the polypeptide encoded by the polynucleotide of SEQ ID NO 7, also shown in Figures 2A-2B
  • SEQ ID NO. 15 is the polypeptide encoded by the polynucleotide of SEQ ID NO 8, also shown in Figures 2A-2B
  • SEQ ID NO. 16 is the polypeptide encoded by the polynucleotide of SEQ ID NO 9, also shown in Figures 2A-2B
  • SEQ ID NO. 17 is the polypeptide encoded by the polynucleotide of SEQ ID NO 10, also shown in Figures 2A-2B
  • SEQ ID NO. 18 is the polypeptide encoded by the polynucleotide of SEQ ID NO 1 1. also shown in Figures 2A-2B SEQ ID NO. 19 is the polypeptide encoded by the polynucleotide of SEQ ID NO 12. also shown in Figure 2C
  • SEQ ID NO. 20 is the polypeptide encoded by the polynucleotide of SEQ ID NO 13. also shown in Figure 2C
  • SEQ ID NO. 21 is the coding sequence of the MAP2 gene from Ehrlichia cams, also shown in Figure 3A
  • SEQ ID NO.22 is the coding sequence of the MAP2 gene from Ehrlichia chaffeensis. also shown in Figure 3B
  • SEQ ID NO. 23 is the polypeptide encoded by the polynucleotide of SEQ ID NO 21. also shown in Figure 3A
  • SEQ ID NO. 24 is the polypeptide encoded by the polynucleotide of SEQ ID NO 22. also shown in Figure 3B
  • SEQ ID NO.25 is the coding sequence of the map! gene from Cowdria ruminantium
  • SEQ ID NO. 26 is the polypeptide encoded by the polynucleotide of SEQ ID NO 25
  • SEQ ID NO.27 is the coding sequence of the ihwor ⁇ gene from Cowdria ruminantium
  • SEQ ID NO.28 is the polypeptide encoded by the polynucleotide of SEQ ID NO 27
  • SEQ ID NO. 29 is the coding sequence of the 4hworfl gene from Cowdria ru inantium
  • SEQ ID NO. 30 is the polypeptide encoded by the polynucleotide of SEQ ID NO 29
  • SEQ ID NO. 31 is the coding sequence of the 18hworfl gene from Cowdria runnnantium
  • SEQ ID NO.32 is the polypeptide encoded by the polynucleotide of SEQ ID NO 31
  • SEQ ID NO.33 is the coding sequence of the 3gdor ⁇ gene from Cowdria ruminantium
  • SEQ ID NO.34 is the polypeptide encoded by the polynucleotide of SEQ ID NO 33
  • the subject invention concerns a novel strategy, termed nucleic acid vaccination, for eliciting an immune response protective against nckettsial disease
  • nucleic acid vaccination for eliciting an immune response protective against nckettsial disease
  • novel compositions that can be employed according to this novel strategy for eliciting a protective immune response
  • recombinant DNA or mRNA encoding an antigen of interest is inoculated directly into the human or animal host where an immune response is induced
  • Prokaryotic signal sequences may be deleted from the nucleic acid encoding an antigen of interest
  • problems of protein purification as can be encountered with antigen delivery using live vectors, can be virtually eliminated by employing the compositions or methods accordingto the subject invention Unlike live vector delivery, the subject invention can provide a further advantage in that the DNA or RNA does not replicate in the host, but remains episomal See. for example. Wolft. J A . J J Ludike. G Acsadi. P Williams. A Ja ( ⁇ 992) Hum Mol Genet 1 363
  • a complete immune response can be obtained as recombinant antigen is synthesized intracellularlyand presented to the host immune system in the context of autologous class I and class II MHC molecules
  • the subject inv ention concerns nucleic acids and compositions comprising those nucleic acids that can be effectiv e in protecting an animal from disease or death caused by rickettsia
  • a nucleic acid vaccine of the subject invention has been shown to be protectiv e against Cowdria ruminantium, the causative agent of heartwater in domestic rum inants
  • nucleotide sequences of nckettsial genes, as described herein can be used as nucleic acid vaccines against human and animal nckettsial diseases
  • the polynucleotide vaccines are administered in conjunction with an antigen
  • the antigen is the polypeptide which is encoded by the polynucleotideadministeredas the polynucleotide vaccine
  • the antigen is administered as a booster subsequent to the initial administration of the polynucleotidevaccine
  • the polynucleotidevaccine is administered in the form of a * cocktail" which contains at least two of the nucleic acid vaccines of the subject invention
  • the "cocktail" may be administered in conjunction with an antigen or an antigen booster as described above
  • the MAPI gene which can be used to obtain this protection, is also present in other r ⁇ cketts ⁇ ae ⁇ nclud ⁇ ngJ7?tf/?/ ⁇ sw ⁇ /w ⁇ * rg.77t7/e, Ehrlichia cams, and in a causative agent of human ehrhchiosis, Ehrlichia chaffeensis (van Vliet, A , F Jongejan.
  • VSA Variable Surface Antigen
  • the present invention also concerns polynucleotides encoding MAP2 or MAP2 homologs from Ehrlichia cams and Ehrlichia chaffeensis MAP2 polynucleotide sequences of the invention can be used as vaccine compositions and in diagnostic assays
  • the polynucleotides can also be used to produce the MAP2 polypeptides encoded thereby
  • the subject invention further concerns the genes designated Cow dria ruminantium map 2 Cowdria ruminantium Ihwor ⁇ , Cowdria ruminantium 4hw orfl. Cowdria ruminantium 18hworfl, and Cowdria ruminantium 3gdor ⁇ and the use of these genes in diagnostic and therapeutic applications.
  • the subject invention further concerns the proteins encoded by the exemplified genes, antibodies to these proteins, and the use of such antibodies and proteins m diagnostic and therapeutic applications
  • compositions comprising the subject polynucleotides can include appropriate nucleic acid vaccine vectors (plasmids), which are commercially available (e g , Vical. San Diego, CA)
  • compositionscan include a pharmaceutically acceptable carrier, e g . saline
  • a pharmaceutically acceptable carrier e g . saline
  • the pharmaceutically acceptable carriers are well known in the art and also are commercially available For example, such acceptable carriers are described in E W Martin's Remington's Pharmaceutical Science. Mack Publishing Company . Easton.
  • PA The subject invention also concerns polypeptides encoded by the subject polynucleotides Specificallyexemplifiedare the polypeptidesencoded by the MAP-1 and VSA genes of C rumimontium E chaffeensis E cams and the MP4 gene of Anaplasma marginale Polypeptides uncoded by E chaffeensis and E cams MAP2 genes are also exemplified herein Also encompassed within the scope of the present invention are fragments and variants of the exemplified polynucleotides and polypeptides Fragments would include, for example, portions of the exemplified sequences wherein procaryotic signal sequences have been removed Examples of the removal of such sequences are given in Example 3 Variants include polynucleotides and/or polypeptides having base or amino acid additions, deletions and substitutions in the sequence of the subject molecule so long as those variants hav e substantially the same activity or serologic reactivity as the native molecules Also included are allelic variant
  • Antibodies can be monoclonal or polyclonal and can be produced using standard techniques known in the art Antibodies of the invention can be used in diagnostic and therapeutic applications
  • the subject invention concerns a DNA vaccine ⁇ e g , VCL1010/M API ) containing the major antigenic protein 1 gene (MAPI) driven by the human cytomegalov ⁇ rus(HCMV) enhancer-promoter
  • MAPI major antigenic protein 1 gene driven by the human cytomegalov ⁇ rus(HCMV) enhancer-promoter
  • this vaccine was injected intramuscularly into 8- 10 week-old female DBA/2 mice after treating them with 50 ⁇ l/muscle of 0 5% bupivacaine 3 days previously Up to 75% of the VCL1010/MAP1 -immunized mice seroconverted and reacted with MAPI in antigen blots Splenocytesfiom immunized mice, but not from control mice immunized with VCL1010 DNA (plasmid vector.
  • Vical. San Diego proliferated in response to recombinant MAPI and C ruminantium antigens in in vitro lymphocyte proliferation tests These proliferating cells from mice immunized with VCL1010/MAP1 DNA secreted I FN-gamma and I L-2 at concentrations ranging from 610 pg/ml and 152 pg/ml to 1290 pg/ml and 310 pg/ml, respectively In experiments testing different VCLIOIO-NIAPI DNA vaccine dose regimens (25- 100 ⁇ g/dose.2 or 4 immunizations).
  • the subject invention concerns the discovery that the gene encoding the MAPI protein induces protective immunity as a DNA vaccine against rickettsial disease.
  • nucleic acid sequences described herein have other uses as well.
  • the nucleic acids of the subject invention can be useful as probes to identify complementary sequences within other nucleic acid molecules or genomes.
  • Such use of probes can be applied to identify or distinguish infectious strains of organisms in diagnostic procedures or in rickettsial research where identification of particular organisms or strains is needed.
  • probes can be made by labeling the nucleic acid sequences of interest according to accepted nucleic acid labeling procedures and techniques.
  • a person of ordinary skill in the art would recognize that variations or fragments of the disclosed sequences which can specifically and selectively hybridize to the DNA of rickettsia can also function as a probe.
  • Hybridization of immobilized DNA on Southern blots with 32P-labeled gene-specific probes can be performed by standard methods ( Maniatis et al.
  • hybridization and subsequent washes can be carried out under moderate to high stringency conditions that allow for detection of target sequences with homology to the exemplified polynucleotide sequence.
  • DNA gene probes hybridization can be carried out overnight at 20-25° C below the melting temperature (Tm) ofthe DNA hybrid in 6X SSPE.5X Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA.
  • Tm melting temperature
  • the melting temperature is described by the following formula (Beltz et al. et al [1983] Methods of Enzymolog . R Wu. L Grossman and K Moldave [eds ] Academic Press, New York 100 266-285)
  • Tm 81 5 °C+16 6 Log[Na+]+0 41 (%G+C)-0 61(%formam ⁇ de)-600/length of duplex in base pairs Washes are t pically carried out as follows
  • oligonucleotide probes hybridization can be carried out overnight at 10-20°C below the melting temperature (Tm) of the hybrid in 6X SSPE. 5X Denhardt's solution. 0 1 % SDS. 0 1 mg/ml denatured DNA Tm for oligonucleotide probes can be determined by the following formula
  • Tm (°C) 2(number T/A base pairs) +4(number G/C base pairs) (Suggs et al [1981 ] ICN-UCLA Svmp Dev Biol Using Purified Genes, O O Brown [ed ], Academic Press. New
  • Washes can be carried out as follows
  • salt and/or temperature can be altered to change stringency With a labeled DNA fragment >70 or so bases in length, the follow ing conditions can be used Low 1 or 2X SSPE room temperature Low 1 or 2X SSPE. 42 °C
  • the probe sequences of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest Mutations, insertions and deletions can be produced in a given polynucleotide sequence in many ways, and these methods are known to an ordinarily skilled artisan. Other methods may become known in the future.
  • restriction enzymes can be used to obtain functional fragments of the subject DNA sequences.
  • Ba 1 exonuclease can be conveniently used for time-controlled limited digestion of DNA (commonly referred to as "erase-a-base " procedures). See, for example, Maniatiset /. (1982) Molecular Cloning: A Laboratory Manual . Cold Spring Harbor Laboratory, New York; Wei et al. (1983)7. Biol. Chem. 258: 13006-13512.
  • nucleic acid sequences of the subject invention can be used as molecular weight markers in nucleic acid analysis procedures.
  • a nucleic acid vaccine construct was tested in animals for its ability to protect against death caused by infection with the rickettsia Co ri ⁇ ruminantium.
  • the vaccine constructtested was the MAPI gene of C. ruminantium inserted into plasmid VCL1010 (Vical. San Diego) under control of the human cytomegalovirus promoter-enhancer and intron A.
  • seven groups containing 10 mice each were injected twice at 2-week intervals with either 100, 75, 50, or 25 ⁇ g VCL1010/MAPl DNA (V/M in Table 1 below), or 100, 50 ⁇ g VCL1010 DNA (V in Table 1) or saline (Sal.), respectively .
  • mice/group were challenged with 30LD50 of C. ruminantium and clinical symptoms and survival monitored. The remaining 2 mice/group were not challenged and were used for lymphocyte proliferation tests and cytokine measurements. The results of the study are summarized in Table 1. below:
  • the VCLI OI O/MAPI nucleic acid vaccine increased survival on challenge in all groups, with a total of 20/30 mice surviving compared to 0/24 in the control groups
  • mice/group were challenged with 30LD50 of C ruminantium and 3 mice/group were sacrificed for lymphocyte proliferation tests and cvtokine measurements The results of this studv are summarized in Table 2, below
  • VCL1010/MAP1 DNA vaccine increased the numbers of mice surviving in both immunized groups, although there was no apparent benefit of 2 additional injections In these two experiments, there were a cumulative total of 35/92 (38%) survivingmice in groups receivingthe VCL1010/MAP1 DNA vaccine compared to 1/144
  • a G- ⁇ ch region -200 bases upstream from the initiation codon, s ⁇ gma-70-l ⁇ ke promoter sequences, putative ⁇ bosome binding sites (RBS), termination codons. and palindromic sequences near the termination codons are found in each of the E chaffeensis noncoding sequences
  • the £ cams noncoding sequence has the same feature except for the G- ⁇ ch region ( Figure 2C. SEQ ID NOs 12-13 and 19-20)
  • a further aspect of the subject invention are five additional genes which give protection when formatted as DNA vaccines
  • These genes are Cowdria ruminantium map 2, Cowdria ruminantium lhwor ⁇ . Cowdria ruminantium 4hworfl. Cowdria ruminantium 18hworfl. and Cowdria ruminantium 3gdorf3
  • the DNA and translated amino acid sequences of these five genes are shown in SEQ ID NOS 25-34
  • a homolog of map2 is present in Anaplasma marginale
  • a homolog of lhwor ⁇ is present in Brucella abortus
  • homologs of 4hworfl are present in Pseudomonas aerugmosa and Coxiella bur etii.
  • homologs of 18hworfl are present in
  • Coxiella burnetii and Rickettsia prow azekn This can be revealed by a search of DNA and protein databases with standard search algorithms such as "Blast" Based on the protective ability of these genes against Cowdria ruminantium and their presence in other bacterial pathogens, the subject invention further concerns the use of these genes, their gene products, and the genes and gene products of the homologs as vaccines against bacteria
  • vaccinia or other liv e viruses
  • Salmonella or other live bacteria
  • Table 3 shows the protective ability of the 5 genes against death from C owdria ruminantium challenge in mice
  • Genes were inserted into VR1012 according to the manufacturers ⁇ nstruct ⁇ ons(V ical. San Diego) and chal lenge studies were conducted as described in Example 1 N-terminal sequences which putatively encoded prokaryotic signal peptides were deleted because of the potential for their affects on expression and and immune responses in eukaryotic expression systems or challenged animals
  • the inserts were as follows map2. SEQ ID NO 25. beginning at base 46. 18hworfl . SEQ ID NO 31. beginning at base 67. 3gdorf3. SEQ ID NO 33. beginning at base 79. lhwor ⁇ . SEQ ID NO 27. beginning at base 76. and 4hworfl . SEQ ID NO 29. beginning at base 58 Table ⁇ 3

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Abstract

L'invention concerne des vaccins à base d'acide nucléique contenant des gènes destinés à protéger les animaux ou les humains contre les rickettsioses. L'invention concerne également des polypeptides, et des méthodes consistant à utiliser ces derniers afin de détecter des anticorps dirigés contre certains pathogènes.
EP00930134A 1999-04-22 2000-04-21 Vaccins a base d'acide nucleique contre les rickettsioses et utilisations Withdrawn EP1171606A2 (fr)

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US20030044422A1 (en) * 2001-02-20 2003-03-06 Barbet Anthony F. Ehrlichia ruminantium polypeptides, antigens, polynucleotides, and methods of use
NZ532634A (en) 2001-11-01 2006-09-29 Res Dev Foundation DNA encoding disulfide bond formation (Dsb) proteins from bacterial species of the genus Ehrlichia, such as Ehrlichia chaffeensis and Ehrlichia canis disulfide bond formation proteins.
US7304139B2 (en) 2003-10-28 2007-12-04 University Of Florida Research Foundation, Inc. Polynucleotides and polypeptides of Anaplasma phagocytophilum and methods of using the same
RU2595427C2 (ru) * 2015-09-11 2016-08-27 Федеральное государственное бюджетное научное учреждение "Центр экспериментальной эмбриологии и репродуктивных биотехнологий" (ФГБНУ ЦЭЭРБ) НАБОР СИНТЕТИЧЕСКИХ ОЛИГОДЕЗОКСИРИБОНУКЛЕОТИДОВ ДЛЯ ДЕТЕКЦИИ ГЕНА msp4 РИККЕТСИИ Anaplasma marginale МЕТОДОМ ПОЛИМЕРАЗНОЙ ЦЕПНОЙ РЕАКЦИИ В РЕЖИМЕ "РЕАЛЬНОГО ВРЕМЕНИ"
AU2023224276A1 (en) * 2022-02-24 2024-08-22 Kansas State University Research Foundation Anaplasma vaccines and methods of use thereof

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WO2000065063A9 (fr) 2002-02-14
AU4801000A (en) 2000-11-10
WO2000065063A3 (fr) 2001-04-12
WO2000065063A2 (fr) 2000-11-02

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