EP1171606A2

EP1171606A2 - Nucleic acid vaccines against rickettsial diseases and methods of use

Info

Publication number: EP1171606A2
Application number: EP00930134A
Authority: EP
Inventors: 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
Original assignee: University of Florida
Current assignee: University of Florida
Priority date: 1999-04-22
Filing date: 2000-04-21
Publication date: 2002-01-16
Also published as: WO2000065063A9; WO2000065063A3; WO2000065063A2; AU4801000A

Abstract

Described are nucleic acid vaccines containing genes to protect animals or humans against rickettsial diseases. Also described are polypeptides and methods of using these polypeptides to detect antibodies to pathogens.

Description

DESCRIPTION

NUCLEIC ACID VACCINES AGAINST RICKETTSIAL DISEASES AND METHODS OF USE

This invention was made with government support under USAID Grant No LAG- 1328- G-00-3030-00 The government has certain rights in this invention

Technical Field This invention relates to nucleic acid vaccines for nckettsial diseases of animals, including humans

Background of the Invention The πckettsiasare 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 causative agents of spotted fever, e g . Rickettsia rickettsu and Rickettsia conoru, are also included within this group Recently, new types of human nckettsial disease caused by members of the tribe Ehrhchiae have been described Ehrhchiae infect leukocytes and endothelial cells of mam different mammalian species, some of them causing serious human and veterinary diseases Over 400 cases of human ehrhchiosis, including some fatalities, caused b\ Ehrhchia chaff ensi have now been reported Clinical signs of human ehrhchiosis are similar to those of Rock\ Mountain spotted fever, including fever, nausea, vomiting, headache, and rash Heartwater is another infectious disease caused by a nckettsial pathogen. nameK

Cowdria r minantium. and is ticks of the genus Amblvomma 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 disease have a 90% mortality rate while susceptible cattle strains have up to a 60% mortality rate

If detected earl>, tetracycline or chloramphenicol treatment are effective against nckettsial infections, but symptoms are similar to numerous other infections and there are no satisfactory diagnostic tests (Helmick C , K Bernard, L D'Angelo [1984] J Infect Dis

150 480)

Animals which have recovered from heart ater are resistant to further homologous, and in some cases heterologous, strain challenge It has similarly been found that persons recovering from a nckettsial infection may develop a solid and lasting immunity Individuals recovered from natural infections are often immune to multiple isolates and even species For example, guinea pigs immunized with a recombinant R conorii protein were partially protected even againstR πc/ce/tszz Vishwanath, S , G McDonald, Ν Watkins [1990] Infect Immun 58 646) It is known that there is structural variation in nckettsial antigens between different geographical isolates Thus, a functional recombinant vaccine against multiple isolates would need to contain multiple epitopes. e g , protective T and B cell epitopes, shared between isolates It is believed that serum antibodies do not play a significant role in the mechanism of immunity against πckettsιa(Uιlenberg, G [1983] Advances in Vet Sci and Co p Med 27 427-480, Du Plessis Plessis. J L [1970] Onderstepoort J Vet Res 37(3) 147-150)

Vaccines based on inactivated or attenuated πckettsiae have been developed against certain nckettsial diseases, for example against R piowazeku and R rickettsn However, 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. eds , Academic Press New York pp 17-40) 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 Finally, the blood vaccine requires a cold chain to preserve the vaccine

Clearly, a safer, more effective vaccine that is easily administered would be particularly advantageous For these reasons, and with the advent of new methods in biotechnology, investigators have concentrated recently on the development of new types of vaccines, including recombinant vaccines However, recombinant v accine antigens must be carefully selected and presented to the immune system such that shared epitopes are recognized These factors have contributed to the search for effective vaccines A protective vaccine against πckettsiae that elicits a complete immune response can be advantageous A tew antigens which potentially can be useful as vaccines have now been identified and sequenced for various pathogenic rickettsia The genes encoding the antigens and that can be employed to recombinantly produce those antigen have also been identified and sequenced Certain protective antigens identified for R rickettsi R conoru, and R prow azeku (e g , rOmpA and rOmpB) are large (> 100 kDa). dependent on retention of native conformation for protective efficacy, but are often degraded when produced in recombinant systems This presents technical and quality-control problems if purified recombinant proteins are to be included in a vaccine The mode of presentation of a recombinant antigen to the immune system can also be an important factor in the immune response 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 . j Donelly , S Parker et al [ 1993] Science 259 1745) For example direct intramuscular injection of mice with DNA encoding the influenza nucleoprotein caused the production ofhigh titer antibodies, nucleoprotein-specificCTLs. and protection against viral challenge Immunization of 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. S Hoffman [ 1994] Proc hall Acad Sci USA 91 9866) Cattle immunized with plasmids encoding bovine herpes irus 1 (BHV- 1 ) glycoprotein IV dev eloped neutralizing antibody and were partially protected (Cox, G , T Zamb, L Babiuk [ 1993] J Virol 67 5664) However, it has been a question in the field of immunization whether the recently discovered technology of nucleic acid vaccines can provide improved protection against an antigenic drift variant Moreover it has not heretofore been recognized or suggested that nucleic acid vaccines may be successful to protect against nckettsial disease or that a major surface protein conserved in rickettsia was protective against disease

Brief Summary of the Invention

Disclosed and claimed here are novel vaccines for conferring immunity to rickettsia infection, including Cowdriaruminantium causing heartwater Also disclosed are novel nucleic acid compositions and methods of using those compositions, including to confer immunity in a susceptible host Also disclosed are novel materials and methods for diagnosing infections by Ehrhchia in humans or animals

One aspect of the subject invention concerns a nucleic acid, e g , DNA or mRNA vaccine containing the major antigenic protein 1 gene (MAPI ) or the major antigenic protein 2 gene (MAP2) of rickettsial pathogens In one embodiment, the nucleic acid vaccines can be driven by the human cytomegalovιrus(HCMV) enhancer-promoter In studies immunizingmice by intramuscular injection of a DNA vaccine composition according to the subject invention, immunized mice seroconverted and reacted with MAPI in antigen blots Splenocytes from immunized mice, but not from control mice immunized with vector only, proliferated in response to recombinant MAPI and nckettsial antigens in m vitro lymphocyte prohferationtests In experiments testing different DNA vaccine dose regimens, increased survival rates as compared to controls were observed on challenge with rickettsia Accordingly , the subject invention concerns the discovery that DNA vaccines can induce protective immunity against rickettsial disease or death resulting therefrom

The subject invention further concerns the genes designated C O dria riimmantium map 2, Cowdna ru nnantium lhworβ, Cowdt ia riimwantiim Cowdria riimmantium and Cowdria runnnantium igdoiβ 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

In one embodiment of the subject invention the polynucleotide vaccines are administered in conjunction with an antigen In a preferred embodiment, the antigen is the polypeptide which is encoded by the polynucleotideadministeredas the polynucleotidevaccine As a particularly preferred embodiment, the antigen is administered as a booster subsequent to the initial administration of the polynucleotide vaccine Brief Description of the Drawings 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 ). and Anaplasma marginale (A m ) Figures 2A-2C 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 sιgma-70-lιke promoter sequences (38) are presented in bold and underlined text as -10 and -35 (consensus -35 and - 10 sequences are TTGAC A and TATAAT. respectively) Similarly, consensus nbosomal binding sites and transcription terminator sequences (bold letter sequence) are identified G-nch regions identified in the E chaffeensis sequence are underlined The conserved sequences from within the coding regions selected for RT-PCR assay are identified with italics and underlined text

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 Double underlined nucleotides represent the predicted nbosomal binding site Underlined nucleotides 3' to the open reading frame represent possible transcription termination sequences

Brief Description of the 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 ihworβ 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 3gdorβ gene from Cowdria ruminantium SEQ ID NO.34 is the polypeptide encoded by the polynucleotide of SEQ ID NO 33

Detailed Disclosure of the Invention In one embodiment, the subject invention concerns a novel strategy, termed nucleic acid vaccination, for eliciting an immune response protective against nckettsial disease The subject invention also concerns novel compositions that can be employed according to this novel strategy for eliciting a protective immune response

According to the subject invention, 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 Advantageously, 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

In one embodiment, 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 For example, 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 Accordingly, nucleotide sequences of nckettsial genes, as described herein can be used as nucleic acid vaccines against human and animal nckettsial diseases

In one embodiment of the subject invention, the polynucleotide vaccines are administered in conjunction with an antigen In a preferred embodiment, the antigen is the polypeptide which is encoded by the polynucleotideadministeredas the polynucleotide vaccine As a particularly preferred embodiment, the antigen is administered as a booster subsequent to the initial administration of the polynucleotidevaccine In another embodiment of the invention, 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 rιckettsιae ιncludιngJ7?tf/?/αswα/wα^*rg.77t7/e, Ehrlichia cams, and in a causative agent of human ehrhchiosis, Ehrlichia chaffeensis (van Vliet, A , F Jongejan. M van Kleef, B van der Zeijst [1994] Infect Immun 62 1451) The MAPI gene or a MAPI-like gene can also be found in certain Rickettsia spp MAPI -like genes from Ehrlichia chaffeensis and Ehrlichia cams have now been cloned and sequenced These MAP- 1 homologs are also referred to herein as Variable Surface Antigen (VSA) genes

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 Ihworβ, Cowdria ruminantium 4hw orfl. Cowdria ruminantium 18hworfl, and Cowdria ruminantium 3gdorβ 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)

In addition, the compositionscan include 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 variants of the subject polynucleotides The polypeptides of the present invention can be used to raise antibodies that are reactive with the polypeptidesdisclosed herein The polypeptidesand polynucleotides can also be used as molecular weight markers Another aspect of the subject invention concerns antibodies reactive with MAP- 1 and

MAP2 polypeptidesdisclosed herein 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

In a specific embodiment, the subject invention concerns a DNA vaccine {e g , VCL1010/M API ) containing the major antigenic protein 1 gene (MAPI) driven by the human cytomegalovιrus(HCMV) enhancer-promoter In a specific example, this vaccine was injected intramuscularly into 8- 10 week-old female DBA/2 mice after treating them with 50 μl/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 μg/dose.2 or 4 immunizations). survival rates of 23% to 88% (35/92 survivors/total in all VCL1010/M P1 immunized groups) were observed on challenge with 30LD50 of C ruminantium Survival rates of 0% to 3% (1/144 survivors/total in all contiol groups) were recorded for control mice immunized similarly with VCL1010 DNA or saline Accordingly, in a specific embodiment, the subject invention concerns the discovery that the gene encoding the MAPI protein induces protective immunity as a DNA vaccine against rickettsial disease.

The nucleic acid sequences described herein have other uses as well. For example, 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. As is well known in the art, 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. It is within the ordinary skill of persons in the art, and does not require undue experimentation in view of the description provided herein, to determine whether a segment of the claimed DNA sequences is a fragment or variant which has characteristicsof the full sequence, e.g.. whether it specifically and selectively hybridizes or can confer protection against rickettsial infection in accordance with the subject invention. In addition, with the benefit of the subject disclosure describing the specific sequences, it is within the ordinary skill of those persons in the art to label hybridizing sequences to produce a probe.

Various degrees of stringency of hybridization can be employed. The more severe the conditions, the greater the complementarity that is required for duplex formation. Severity of conditionscan be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybridization is conducted under moderate to high stringency conditions by techniques well known in the art. as described, for example, in Keller. G.H., M.M. Manak (1987) DNA Probes. Stockton Press. Ne York. NY., pp. 169- 170. Examples of various stringency conditions are provided herein. Hybridization of immobilized DNA on Southern blots with 32P-labeled gene-specific probes can be performed by standard methods ( Maniatis et al. ( 1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory. New York.). In general, 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. For double-stranded

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. 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(%formamιde)-600/length of duplex in base pairs Washes are t pically carried out as follows

(1 ) twice at room temperature for 15 minutes in I X SSPE. 0 1% SDS (low stringency wash).

(2) once at Tm-20°C for 15 minutes in 0 2X SSPE, 0 1 % SDS (moderate stringency wash) For 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

York, 23 683-693)

Washes can be carried out as follows

( 1 ) twice at room temperature for 15 minutes 1 X SSPE, 0 1 % SDS (low stringency wash (2) once at the hybridization temperature for 15 minutes in IX SSPE. 0 1% SDS

(moderate stringency wash) In general, 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

Moderate 0 2X or 1 X SSPE. 65 °C

Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid and. as noted above, a certain degree of mismatch can be tolerated Therefore. 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.

It is also well known in the art that restriction enzymes can be used to obtain functional fragments of the subject DNA sequences. For example. Ba 1 exonucleasecan 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.

In addition, the nucleic acid sequences of the subject invention can be used as molecular weight markers in nucleic acid analysis procedures.

Following are examples which illustrate procedures for practicingthe invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1

A nucleic acid vaccine construct was tested in animals for its ability to protect against death caused by infection with the rickettsia Co riα 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. In this study, seven groups containing 10 mice each were injected twice at 2-week intervals with either 100, 75, 50, or 25 μg VCL1010/MAPl DNA (V/M in Table 1 below), or 100, 50 μg VCL1010 DNA (V in Table 1) or saline (Sal.), respectively . Two weeks after the last injections, 8 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:

Table 1

100 μg 75 μg 50 μg 25 μg 100 μg 50 μg

V/M V/M V/M V/M^~ V v ^" Sal.

Survived 5 1 5 0 0 0 Died -> 1 J 5 8 8 8 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

This study was repeated with another 6 groups, each containing 33 mice (a total of 198 mice) Three groups received 75 μg VCL1010/MAP1 DNA or VCLI OIO DNA or saline (4 injections in all cases) Two weeks after the last injection.30 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

Table 2

V/M 2 inj V 2 inj Sal 2 inj V/M 4 inj V 4 inj Sal 4 ιnj Died* 23 30 30 22 30 29

*In mice that died in both V/M groups, there was an increase in mean survival time of approximately 4 days compared to the controls (p<0 05)

Again, as summarized in Table 2, the 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

(0 7%) surviving mice in the control groups In both immunization and challenge trials described above, splenocytes from VCL1010/MAP1 immunized mice, but not from control mice, specifically proliferated to recombinant MAPI protein and to C ruminantium in lymphocyte proliferation tests These proliferating splenocytes secreted IL-2 and gamma- interferon at concentrations up to 310 and 1290 pg/ml respectively These data show that protection against nckettsial infectionscan be achieved with a DNA vaccine In addition, these experiments show MAP l -i elated proteins as vaccine targets

Example 2 — Cloning and sequence analysis of MAPI homologue genes of £ chaffeensis and E cams

Genes homologous to the major surface protein of ruminantium MAPI were cloned from E chaffeensis and E cams by using PCR cloning strategies The cloned segments represent a 4 6 kb genomic locus of E chaffeensis and a 1 6 kb locus of is cams DNA sequence generated from these clones was assembled and is presented along with the deduced amino acid sequence in Figures 2A-2B (SEQ ID NOs 7- 1 1 and 14- 18) and Figure 2C (SEQ ID NOs 12-13 and 19-20) Significant features of the DNA include five very similar but nonidentical open reading frames (ORFs) for E chaffeensis and two very similar, nonidentical ORFs for the E cams cloned locus The ORFs for both Ehrlichia spp are separated by noncoding sequences ranging from 264 to 310 base pairs The noncoding sequences have a higher A+T content

(71 6% for E chaffeensis and 76 1 % for E cams) than do the coding sequences (63 5% for E chaffeensis and 68 0% for E cams) A G-πch region -200 bases upstream from the initiation codon, sιgma-70-lιke promoter sequences, putative πbosome 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-πch region (Figure 2C. SEQ ID NOs 12-13 and 19-20)

Sequence comparisons of the ORFs at the nucleotide and translated amino acid levels revealed a high degree of similarity between them The similarity spanned the entire coding sequences, except in three regions where notable sequence variations were observed including some deletιons/ιnsertιons(Vaπable Regions I, II and III) Despite the similanties.no two ORFs are identical The cloned ORF 2, 3 and 4 of £ chaffeensis have complete coding sequences The ORF1 is a partial gene having only 143 amino acids at the C-terminus whereas the ORF5 is nearly complete but lacks 5-7 amino acids and a termination codon The cloned ORF2 of £ cams also is a partial gene lacking a part of the C-terminal sequence The overall similarity between different ORFs at the amino acid level is 56 0% to 85 4% for £ chaffeensis, whereas for £ cams it is 53 3% The similarity of £ chaffeensis ORFs to the MAPI coding sequences reported for C ruminantium isolates ranged from 55 5% to 66 7% while for £ ams to C ruminantium it is 48 5% to 54 2% Due to their high degree of similarity to MAPI surface antigen genes of C ruminantium and since they are nonidenticalto each other, the £ chaffeensis and £ cams ORFs are referred to herein as putative Variable Surface Antigen (VSA) genes The apparent molecular masses of the predicted mature proteins of £ chaffeensis were 28 75 kDa for VSA2. 27 78 for VSA3, and 27 95 for VSA4, while £ cams VSA l was slightly higher at 29 03 kDa The first 25 amino acids in each VSA coding sequence were eliminated when calculating the protein size since they markedly resembled the signal sequence of C ruminantium MAPI and presumably would be absent from the mature protein

The amino acid sequence derived from the cloned £ chaffeensis MAPI-like gene and alignment with the corresponding genes of C ruminantium and 4 marginale is shown in Figure 1 Example 3

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 lhworβ. 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

There is published information showing that gene homologs of all five genes are present in other bacteria For example, a homolog of map2 is present in Anaplasma marginale, a homolog of lhworβ is present in Brucella abortus, homologs of 4hworfl are present in Pseudomonas aerugmosa and Coxiella bur etii. and 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 This includes their use as DNA or nucleic acid vaccines or when formulated in vaccines employing other methods of delivery, e g , recombinantproteins or synthetic peptides in adjuvants, recombinant live vector delivery systems such as vaccinia (or other liv e viruses) or Salmonella (or other live bacteria) These methods of delivery are standard to those familiar with the field This also includes vaccines against heartwater disease, vaccines against nckettsial diseases in general and vaccines against other bacteria containing homologs of these genes

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 ιnstructιons(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. lhworβ. SEQ ID NO 27. beginning at base 76. and 4hworfl . SEQ ID NO 29. beginning at base 58 Table Ϊ 3

DNA Construct MWT Survival Rate Size Vaccinated Contro 1 P value

TMMAP 2 21 kd 9/28* 32% 0/29 0% 0.004

MB18HWORF1 28 kd 10/30* 33% 1/27 4% 0.021

AM3GDORF3 16 kd 7/26 27% 1/27 4% 0.060

TM 1 HWORF3 36 kd 8/29 28% 2/30 7% 0.093

TM4HWORF1 19 kd 10/30* 33% 2/30 7% 0.054

Control - VR1012 DNA vector plasmid only ^Statistically significant difference (Fisher^'s Exact test)

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

Claims

1. A composition comprising a polynucleotide which encodes a polypeptide having the characteristic of eliciting an immune response protective against disease or death caused by a rickettsial pathogen.

2. The composition, according to claim 1 , wherein said rickettsial pathogen is selected from the group consisting of Rickettsia spp., Ehrlichia spp.. Anaplasma spp.. and Cowdria spp.

3. The composition, according to claim 1. wherein said polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4. SEQ ID NO. 6. SEQ ID NO. 14. SEQ ID NO. 15. SEQ ID NOS. 16-20. SEQ ID NO. 23, SEQ ID NO. 24. SEQ ID NO. 26, SEQ ID NO. 28. SEQ ID NO. 30. SEQ ID NO. 32. SEQ ID NO. 34. homologs thereof, and immunogenic fragments thereof.

4. The composition, according to claim 1 , wherein said polynucleotide has a nucleic acid sequence selected from the group consistingof SEQ ID NO. 1 , SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7. SEQ ID NO. 8, SEQ ID NOS. 9-13, SEQ ID NO. 21 , SEQ ID NO. 22, , SEQ ID NO. 25. SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31. SEQ ID NO. 33. homologs thereof, and fragments thereof which encode immunogenic polypeptides.

5. The composition, according to claim 4. wherein said polynucleotide has a nucleic acid sequence of SEQ ID NO. 3. or a fragment thereof.

6. The composition, according to claim 1. wherein said polynucleotide further comprises a nucleic acid vaccine vector.

7. The composition, according to claim 1. further comprising a pharmaceutically acceptable carrier.

8. A polynucleotide encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NOS. 14-20. SEQ ID NOS. 23-24, SEQ ID NO. 26. SEQ ID NO. 28. SEQ ID NO. 30. SEQ ID NO. 32. SEQ ID NO. 34, and fragments thereof.

9. The polynucleotide, according to claim 8. said polynucleotide having a nucleic acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NOS. 7-13. SEQ ID NOS. 21 -22, SEQ ID NOS. 25. SEQ ID NO. 27. SEQ I D NO. 29. SEQ ID NO. 31 , and SEQ ID NO. 33.

10. A method for protecting a susceptible host against disease or death caused by a rickettsial pathogen, said method comprising administering an effective amount of a polynucleotideencoding polypeptide having the characteristic of eliciting an immune response protective against said rickettsial pathogen.

1 1 . The method, according to claim 10. wherein said rickettsial pathogen is selected from the group consistingof Rickettsia spp., Ehrlichia spp., Anaplasma spp., and Cowdria spp.

12. The method, according to claim 10. wherein said polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO. 2. SEQ ID NO. 4, SEQ ID NO. 6. SEQ ID NO. 14. SEQ ID NO. 15, SEQ ID NOS. 16-20. SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 26. SEQ ID NO. 28, SEQ ID NO. 30. SEQ ID NO. 32, SEQ ID NO. 34, or homologs thereof and immunogenic fragments thereof.

13. The method, according to claim 10. wherein said polynucleotide has a nucleic acid sequence selected from the group consisting of SEQ ID NO. 1. SEQ ID NO. 3, SEQ ID NO. 5. SEQ ID NO. 7, SEQ IDNO. 8, SEQ ID NOS. 9-13. SEQ ID NO. 21. SEQ ID NO. 22, SEQ ID NO. 25. SEQ ID NO. 27. SEQ ID NO. 29, SEQ ID NO. 31. and SEQ ID NO. 33.

14. The method, accordingto claim 13. wherein said polynucleotidehas the nucleic acid sequence of SEQ ID NO. 1.

15. The method, accordingto claim 13. w herein said polynucleotidehas the nucleic acid sequence of SEQ ID NO. 3.

16. The method, accordingto claim 13. wherein said polynucleotidehas the nucleic acid sequence of SEQ ID NO. 5. 1 17. The method, accordingto claim 10. wherein said nucleic acid further comprises an appropriate nucleic acid vector.

1 18. The method, according to claim 10. wherein said composition further comprises a pharmaceutically acceptable carrier.

1 19. The method, accordingto claim 10. which further comprises administration to said

2 host of said polypeptide encoded by said polypeptide.

1 20. A method for detecting, in a human or animal, antibodies associated with infection

2 by Ehrlichia, wherein said method comprises contacting a biological fluid from said human or

3 animal with a polypeptide selected from the group consisting of SEQ ID NO. 4. SEQ ID NOS.

4 14-20, SEQ ID NOS.23-24. SEQ ID NO.26. SEQ ID NO.28, SEQ ID NO.30. SEQ ID NO.

5 32, SEQ ID NO. 34, and homologs and fragments thereof.

1 21 . A method of detecting the presence of rickettsial nucleic acids comprising

2 contacting a sample suspected of containing rickettsial nucleic acids with a composition

3 comprising a labeled polynucleotide which encodes a polypeptide having the characteristic of

4 eliciting an immune response protective against disease or death caused by a rickettsial

5 pathogen, allowing for the formation of a hybridization complex and detecting said label.

1 22. The composition, according to claim 21. wherein said rickettsial pathogen is

-> selected from the group consisting of Rickettsia spp.. Ehrlichia spp.. Anaplasma spp.. and

-> Cowdria spp.

1 23. The composition. accordingto claim 21. wherein said polypeptide has an amino acid

2 sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6.

3 SEQ ID NO. 14. SEQ ID NO. 15. SEQ ID NOS. 16-20. SEQ ID NO. 23. SEQ ID NO. 24. SEQ

4 ID NO. 26. SEQ ID NO. 28. SEQ ID NO. 30. SEQ ID NO. 32. SEQ ID NO. 34. and homologs

5 and immunogenic fragments thereof.

1 24. The composition, accordingto claim 21. wherein said polynucleotide has a nucleic

2 acid sequence selected from the group consistingof SEQ ID NO. 1. SEQ ID NO. 3. SEQ ID NO.

3 5. SEQ ID NO. 7. SEQ ID NO. 8. SEQ ID NOS. 9-13. SEQ ID NO. 21. SEQ ID NO. 22. . SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29. SEQ ID NO. 31. SEQ ID NO. 33. homologs thereof, and fragments thereof which encode immunogenic polypeptides.