Title of Invention	MYCOBACTERIUM TUBERCULOSIS SPECIFIC PROTEIN AND GENE, AND ITS APPLICATIONS
Abstract	The gene represented by open reading frame Rv3881c of M. tuberculosis has been sequenced and the sequence of the protein encoded by this gene deduced. The DNAs and their encoded polypeptides can be used for vaccines. Combinations of this gene in different vectors including naked DNA, live attenuated poxvirus and live M. bovis BCG can be used for improved vaccines for bacterial pathogens and parasites.

Title of Invention

MYCOBACTERIUM TUBERCULOSIS SPECIFIC PROTEIN AND GENE, AND ITS APPLICATIONS

Abstract

The gene represented by open reading frame Rv3881c of M. tuberculosis has been sequenced and the sequence of the protein encoded by this gene deduced. The DNAs and their encoded polypeptides can be used for vaccines. Combinations of this gene in different vectors including naked DNA, live attenuated poxvirus and live M. bovis BCG can be used for improved vaccines for bacterial pathogens and parasites.

Full Text	This invention relates to a polypeptide and fiosion proteins of mycobacterium tubercuJosis and otber bacterial pathogens whose antigenicity is not caused by a single protein or component, and parasites, including preparation of vaccines and also to a method of preparing the said polypeptide and fusion proteins. BACKGROUND: Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), currently the world's largest killer from any single agent. The identification of mycobacterial antigens that induce protective T-cell responses and/or stimulate humoral immunity during tubercular infection of humans is therefore an area of investigation that has received increasing emphasis. Since protective immunity against tuberculosis relies exclusively on cell mediated immune responses, antigens in the former class constitute potential candidates for the development of effective vaccines, while those in the latter group can be tested as new, improved tools for diagnosis of TB. Similarly, numerous other bacterial pathogens have pathogenicity that, as with TB, is not caused by a single protein, as is the case also with parasites generally. Antigens produced by these pathogens are also potential candidates for the development of effective vaccines. The majority of M tuberculosis proteins that induce strong human cell mediated immune (CMI) responses are actively secreted by M. tuberculosis, making them valuable components of effective vaccines. The unique ability of live, dividing mycobacteria, but not dead bacilli to efficiently induce protective immunity (7, 22) led to the hypothesis that proteins that are actively secreted by M. tuberculosis during growth are key in generating protective T-cell responses (4, 23). Indeed, e}q>erimental vaccines based on culture filtrate proteins have been shown to induce some levels of protective immunity in animal models of TB (5, 14, 15, 26). Secreted protems of M. tuberculosis are also potent inducers of antibody production (13). Bioinfonnatics ^proaches have been used to predict the existence of more than 700 secreted proteins ofM. tuberculosis and 2 D gel electrophoresis has been used to resolve as many as 620 individual members, and the identity of ] 14 of these was established. ID addition to their immunostimulatory roles m directly mteracting with the innate immune system [7], secreted mycobacterial protems show great promise as potential vaccine targets by virtue of their role as important T-cell antigens in the ad^tive immune response [8 -10]. The immunological characterization of individual components of M tuberculosis culture filtrates is a crucial step toward understanding the role of the secreted proteins in inducing immune responses during the course of TB. Earlier work had identified approximately ten actively secreted proteins using antibodies from immunized animals (1), most of whom were characterized by gene cloning and nucleotide sequencing (2, 6, 9, 11, 17-20, 29, 34). Some of the known secreted proteins induce cellular immune responses (35); however, the target antigens in the secreted protein fi-actions of the strongest human T-cell responses are yet to be characterized (S, 29). An aspect of this invention is an isolated DNA sequence coding for the polypeptide sequence of the Rv38Slc antigen, a protein secreted by M. tuberculosis, that is specific for some mycobacterial species that belong to the M. tuberculosis complex, as well as recombinant polypeptide sequences encoded by that DNA. Another aspect of this invention is a DNA cocktails for vaccines as well as a "cocktail" of purified natural and recombinant protein antigens or polypeptides for immunodiagnostics or vaccines. SUMMARY OF INVENTION: The gene for the protein Rv3881c has been isolated and sequenced (SEQ ID N0:2). That gene can be incorporated into a plasmid and expressed in E. coli to produce purified Rv3881c protein, whose sequence (SEQ ID N0:1) has been deduced. Additional e7q}ression systems will be apparent to persons skilled in the art Rv3881c is a protein secreted by M. tuberculosis and is specific to M. tuberculosis and M. bovis. Homologues of this gene or protein have not been detected m any other mycobacteria including M. simiae, M. scrojulaceum, M. intracellulare, M. szulgai, M. kansasii, M. fortuitum, M.parafortuitum, M. phlei, M. vaccae, M. flavescens, M. chitae, M. leprae, hi. gordonae, M. chelonae, M. smegmatis.M. avium . This invention includes the Rv3881c amino acid sequence shown in FIG. 1 (SEQ ID NO: 1). A preferred embodiment is the mature recombinant Rv3881c protein which is the polypeptide extending from the T in the underlined TQSQT to the C-terminal K in FIG. 1. Also preferred are antigoiic polypeptides derived from the sequences shown in FIG. 1, whether produced by natural, recombinant or synthetic (including chemical synthesis) means or other means known in the art. The invention also includes variants of these polypeptides that retain their antigenic and immunogenic properties. This invention includes an isolated nucleic acid having the sequence shown in FIG. 1 {SEQ ID N0:2). Other embodiments can be derived by making silent substitutions, those that do not change the amino acid sequence encoded by the nucleic aci4 in the nucleic acid sequence. In preferred embodiments these nucleic acids are made by modifying the sequence by mut^enesis, recombination or synthetic (including chemical synthesis) means or other means known in the art. Also preferred are embodiments wherein the nucleic acid does not contain the entire nucleic acid sequence shown in PIG. 1 (SEQ ID N0:2), with or without silent substitutions. This invention also includes vaccines that contain a recombinant Rv3881c polypqitide. In preferred embodiments the vaccine includes the Rv38S 1 c gene. In preferred embodunents the vaccine includes the mature recombinant protein. In preferred onbodiments the vaccine includes a live vector organism such as the M.bovis BCG vaccine strain or other attenuated variants of M bovis or M. tuberculosis. In preferred embodiments the vaccine includes a live attenuated poxvirus. This invention includes a method of eliciting an immune response and/or protective immunity against M. tuberculosis or another member of the M. tuberculosis complex in a vertebrate, said method including administering to the vertebrate a recombinant Rv3881c polypeptide, whereby said polypeptide elicits immune responses against the Mycobactraium in the vertebrate. A DNA vaccine according to this invention includes a vector, preferably a plasmid vector, and one or more isolated nucleotide sequences each ^tcoding the Rv388lc polypeptide, and transcriptional and translational regulatory sequences operably linked to the isolated nucleotide sequences for expression in a cell of a vertebrate. The DNA vaccine may include the regulatory sequences of CMV immediate-early promoter and/or intron A, or other non-retroviral sequences. A recombinant BCG vaccine according to this invention includes live M. bovis-BCG vector carrying the Rv388ic gene under regulatory elements that can function inside mycobactCTia. The said BCG vector is attenuated for virulence in humans and vertebrates. A recombinant poxvirus vaccine according to this invention includes live attenuated poxvirus vector carrying the Rv3881c gene under poxvirus regulatory elements that can function inside eukaryotic cells infected with the said poxvirus. This invention also includes combinations of the above DNA vaccine, recombinant BCG vaccine and recombinant poxvirus vaccine in all possible permutations and combinations. This invention also includes methods of eliciting an immune response and/or protective immunity by administering to a vertebrate such a DNA vaccine, recombinant BCG vaccine, recombinant poxvirus vaccine or their various permutations and combinations whereby expression of said Rv3881c nucleotide sequences in said celt eUcits immune responses against the Mycobacterium. This invention also includes methods for enhancing immime responses using various adjuvants such as interleukins, along with the Rv388 Ic nucleotide sequence or polypeptide. In preferted methods of this invention the vertebrate is a human. In preferred metiiods of this invOTtion the vertebrate is also a bovine or any species known to suffer from M. tuberculosis- or M. 6ov«-mediated disease. A DNA vaccine according to this invention may be administered to a vertebrate through a route of administration selected from the group consisting of inhalation, intravenous, intramuscular, intrqieritoneal, intradermal, and subcutaneous. A preferred embodiment is a method wherein die DNA vaccine is administered by contacting the DNA vaccine with a mucosal surface of the vertebrate. A preferred embodiment is a method wherein the DNA vaccine is microsphere encapsulated, and is administered by contacting the microsphere-enc^qisulated DNA vaccine with a mucosal surface of the vertebrate. A prefored embodiment is a method wherein the DNA vaccine is coated onto gold beads for administration to the vertdirate by particle bombardment delivery. A preferred embodiment is a method wherein the gold beads are approximately 1 pm to 2 pm in diameter. A preferred embodimoit is a method wherein the protective immunity is homologous, homotypic, heterotypic, or heterologous. A recombinant BCG or recombinant poxvirus vaccine according to this invention may be administered to a vertebrate through a route of administration selected from the groi^ consisting of inhalation, intravenous, intramuscular, intr^qieritoneal, intradermal, and subcutaneous. This invention includes the use of mature Rv3881c polypeptide or a fragment(s) thereof in diagnostic tests for the detection in a patient of an immune response to M. tuberculosis or another member of the M. tuberculosis complex. A diagnostic test can be perfoimed in the format of the commonly used Mantoux or Tine test for the detection of an immune reaction in the skin. This invention also includes the use of mature Rv3881c polypeptide, or a fragment(s) thereof, to bind antibody in human or animal sera in an ELISA, or any other solid-phase immunoassay. Several formats of solid-phase immunoassays are known in the art and can be ad^ted for use in this invention. This invention includes the use of a nucleic acid sequence of this invention, as a probe for the detection of M. tuberculosis and M. bovis, the only 2 mycobacterial strains so far found to contain the Rv3881c gene. Nucleic acid detection assays are well known to those skilled in the art. Assays can involve direct or indirect detection of the target sequence. Amplification of the target sequence can also be performed prior to or as a part of detection. Amplification can be performed with ligase chain reaction, polymerase chain reaction, self-sustained sequence reaction, NASBA and Q-beta amplification. Specific primers for the amplification of the Rv3881c gene can be derived fi-om the nucleic acid of the present invention by standard procedures {and tests for specificity). Such primers can be selected simply by testing 15 to 50 nucleotide long sequences derived from the gene for specific hybridization to and specific amplification of the gene in the presence of various nucleic acids e^qiected to be present in a sample. This invention includes mixtures of antigens, or antigen "cocktails", that include at least three and as many as six or even more M. tuberculosis antigens and/or peptides thereof, at least two of which and preferably all of which are ^ecific to the M. tuberculosis complex. The mixture should preferably include at least two purified proteins or polypeptides that are highly immunologically active in an antibody system or in T-cell recognition, for use in serodiagnosis and skin tests, respectively. Similar cocktails can be made of protein or polypeptide antigens expressed by other bacterial pathogens, such as Listeria, Shigella and Salmonella, or by parasites, such as Plasmodium, Leishmania and Trypanosoma. This invention also includes vaccines, both protein-based and DNA-based. The vaccines may comprise cocktails of purified proteins or polypeptide antigens, ofM. tuberculosis or another bacterial pathogen or a parasite. The vaccines may comprise cocktails of DNAs encoding such antigens. Bach member of the cocktail must induce a protective immune response. For vaccines, specificity to the pathogen is not required. Preferably, the cocktail comprises the most protective antigens available. DETAILED DESCRIPTION OF THE DRAWINGS : FIG. 1 shows the amino acid sequence (SEQ ID N0:1) of the Rv3881c protein, a polypeptide of 460 aa having a calculated Mr of 47594 Daltons. FIG. 2 shows the nucleotide sequence (SEQ ID N0:2) of the gene encoding the Rv3881c protein ofM. tuberculosis and the deduced amino acid sequence of the protein (SEQ ID N0:1). The sequence (SEQ ID N0:2) contains an open reading frame of 1380 nucleotides that encodes the Rv3881c protein, whose deduced amino acid sequence is also shown below die nucleotide sequence. The first codon (ATG) is shown m bold. The underlmed pent^eptide (TQSQT in SEQ ID N0:1) marks the start of the mature protein. FIG. 3. We performed Southern blot analysis using EcoRI plus Sall-digested DNA from the different mycobacterial strains and a 311-bp BamHI-Smal fragment representing the 3'end of the Rv3881c gene to detect the Rv3881c gene in the M. tuberculosis genome. A smgle band of approximately 7 kb was visimlized after hybridization, mdicatmg that the Rv3881c gene is presumably present in a single copy in the bacterial chromosome. The same positive signal was detected using DNAs exfracted from other reference strains (H37Ra, H37Rv). M. bovis and M. bovis BCG also gave positive signals, with the size of the band detected in the latter being only 5 kb due to a genomic deletion close to the Rv3881c gene. No restriction fr^ment loigth polymorphism was observed in DNAs that tested positive. In confrast, no hybridization signal was detected in DNAs exfracted from unrelated mycobacterial species (M simiae, M. scrofitlaceum, M. intracellulare, M. szulgai, M. kansasii, M. fortuitum, M.parafortuitum, M. phlei, M. vaccae, M. flavescens, M. chitae). The Rv 3881c gene has also been rqwrted to be absent in M. leprae, M. gordonae, M. chelonae, M. smegmatis ,and M. avium (Lodes et. al., 2001). These hybridization results suggest that the Rv 3881c gene is conserved in only in M. tuberculosis and M. bovis of the M. tuberculosis complex, while it is absent in all other mycobacteria. Purified protein and polypeptide antigens can be made using all or part of tiie sequences shown in FIG. 1 (SEQ ID NO; 1 and SEQ ID N0:2> by methods well known in the art. The DNA sequence shown in FIG. 1 (SEQ ID N0;1). a°d portions thereof, can be obtained by methods well known in the art. M. tuberculosis genomic DNA and primers derived from FIG. 1 (SEQ ID NO.l) can be used to ampliiy the entire gene sequaice OT portions thereof by polymerase chain reaction amplification, for example. Libraries of M. tuberculosis genomic DNA and DNA probes derived from FIG. 1 (SEQ ID NO:l) can also be used to clone the Rv3881c gene or portions thereof. To obtain pure recombinant Rv3881c protein in large amounts the sequence encoding the mature Rv3S81c protein (extending from nucleotide 196 to nucleotide 588, FIG. 1) was subcloned in the E. coli plasmid pET20b+ (Novagen) as a fusion protein bearing a short polyhistidise tract at its carboxy terminus. The ta^ed protein, having an ^^arent molecular weight of SO Kda was purified by affinity chromatogr^hy using nickel columns (25). The nickel-affinity purified terminally tagged protein is referred to as recombinant (rec) Rv3881c. Tufile 1; M. tuberculosis challenge bacilli recovered fl'om ^leois of guinea pigs immunized using tiie said experimental vaccines. DNA coding for the polypeptide referred to as recombinant 213 (claim 1) was expressed under the transcriptional control of the cytomegalovirus (CMV) promoter. Guinea pigs were immunized using this DNA or pJW vector DNA as control. Other animals received as immunizing antigen either BCG or recombinant BCG (REC-BCG) which expresses the 213 polypeptide of M. tuberculosis. Another groi^ of animals were immunized with pJW213 DNA followed by REC-BCG. All animals received the immunogens intradennally. Control refers to animals that received no immunization whatever. All animals were challenged intramuscularly using 5 loglO viable bacilli of the virulent M. tuberculosis sti-ain NTI83949. Load of challenge bacilli m the spleens of animals was enumerated 6 weeks later. Statistical treatment of the above data revealed that BCG and pJW213 were not significantly different from each other in their protective ability. A "DNA transcription unit" is a polynucleotide sequence, bounded by an initiation site and a termination site, that is transcribed to produce a primary transcript. As used herein, a "DNA transcription unit" includes at least two components: (1) antigen-encoding DNA, and (2) a transcriptional promoter element or elements operatively linked for expression of the antigen coding DNA. Antigen-coding DNA can encode one or multiple antigens, such as antigens fi-om two or more different Mycobacterial proteins. The DNA transcription unit can additionally be inserted into a vector which includes sequences for expression of the DNA transcription unit. A DNA transcription unit can optionally include additional sequences such as enhancer elements, splicing signals, tennination and polyadenylation signals, viral replicons, and bacterial plasmid sequences. In the present method, a DNA transcription unit (i.e., one type of transcription unit) can be administered individually or in combination with one or more other types of DNA transcription units. DNA transcription units can be produced by a numb^ of known methods. For example, DNA encoding the desired antigen can be inserted into an expression vector (see, for example, Sambrook et al.. Molecular Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press (1989)). With the availability of automated nucleic acid synthesis equipment, DNA can be synthesized directly when the nucleotide sequence is known, or by a combination of polymerase chain reaction (PCR), cloning, and fermentation. Moreover, when the sequence of the deshed polypq}tide is known, a suitable coding sequence for the polynucleotide can be inferred. The DNA transcription unit can be administered to an individual, or inoculated^ in the presence of adjuvants or other substances that have the capability of promoting DNA uptake or recruiting immune system cells to the site of the inoculation. It should be understood that the DNA transcription unit itself is expressed in the host cell by transcription factors provided by the host cell, or provided by a DNA transcription unit The desired antigen can be any antigen or combination of antigens from a Mycobacterium. The antigen or antigens can be naturally occurring, or can be mutated or specially modified. The antigen or antigens can represent different forms, such as strains of Mycobacteria. These antigens may or may not be structural components of a Mycobacterium. The encoded antigens can be translation products or polypeptides. The polypeptides can be of various lengths. In addition, they can be designated to undergo intracellular, extracellular, or cell-surface expression. Furthermore, they can be designed to undergo release from cells. An individual can be inoculated through any parenteral route. For example, an individual can be inoculated by intravenous, intr^eritoneal, intradermal, subcutaneous, inhalation, or intramuscular routes, or by particle bombardment using a gene gun. Muscle is a useful site for the delivery and expression of DNA transcription unit-encoding polynucleotide, because animals have a proportionately large muscle mass which is conveniently access^ by dir^t injection through the skin. A comparatively large dose of polyaucleotide can be deposited into muscle by multiple and/or repetitive mjections, for example, to extend immunization over long periods of time. Muscle cells are injected with polynucleotide encodmg polypeptides, and these polypeptides are presented by muscle cells in the context of antigens of the major histocompatibility complex to provoke a selected immune response {see, e.g., Feigner, et al. WO90/11092, herein incorporated by reference). The epidermis is another useftil site for the delivery and expression of polynucleotide, because it is conveniently accessed by direct injection or particle bombardment. A comparatively large dose of polynucleotide can be deposited in the epidermis by multiple injections or bombardments to extend therqiy over long periods of tune. In immunization strategies of the invention, skin cells are injected with polynucleotide coding for antigenic or immunogenic polypeptides, and these polypeptides are presented by skin cells in the context of antigens of the major histocompatibility complex to provoke a selected immune response against the immunogen. In addition, an individual can be inocidated by a mucosal route. The DNA transcription unit can be administered to a mucosal surface by a variety of methods including DNA-containing nose-drops, inhalants, si^positories, microsphere encapsulated DNA, or by bombardment with DNA coated gold particles. For example, the DNA transcription unit can be administered to a respiratoiy mticosal surface, such as the nares or the trachea. Any appropriate physiologically compatible medium, such as saline for injection, or gold particles for particle bombardment, is suitable for introducmg the DNA transcription unit into an individual. Intradennal administration of DNA by particle bombardmrat can be used to deliver DNA for expression of a M. tuberculosis Rv3881c polypeptide in skin cells. The Accell particle bombardment device ("gene gun"; Agracetus, Middleton, Wis.) or similar devices obtained from other companies can be employed to deliver DNA-coated gold beads to the epidermis. Vertebrates can be immunized with gold particles coated with the DNA v^cine using particle bombardment technology as presented in the following articles: Yang, M. S. et al., (1990) Pioc. Natl. Acad. Sci. USA 87:9568-9572; YangN.-S. (1992) CRC Crit. Rev. Biotechnol. 12:335-356; and Cheng, L. et al. (1993) Proc. NaU. Acad. Sci. USA 90:4455-4459. The beads deliver DNA mto cells, where the DNA dissolves and can be expressed (Yang, M. S. et al. (1991) Proc. Natl. Acad. Sci. USA 88: 2726-2730). Expression is transient, with most of the expression being lost within 2-3 days due to the normal sloughing of the epidermis (Williams, R. S. et al., Proc. NaU. Acad. Sci. USA 88: 2726-2730(1991)). These particle bombardment techniques can be easily adapted for use in human patients using M. tuberculosis Rv3881c DNA vaccines. Administration of a DNA vaccine to a human can be perfonned by any one or more of several routes selected from the following: intravenous, intramuscular, intr^^jedtoneal, intradennal, inhalation, and subcutaneous. For example, inti^ermal administration by gene gun is a preferred route. The site of administration is chosen for the convenience of the patient. The dose is between 1 and 50 pg of DNA vaccine per kg body weight, preferably 10-25 .about.g per kg body weight. For a human infant, two inoculations are given at a 4 week interval. A human of any age who is caring for an infected infant or is inimimocompromised due to ilhiess, drug treatment, or other cause puttmg him or her at risk of infection is inoculated with the DNA vaccine by gene gun delivery for at least 2 inoculations at 4 week intervals. Mucosal routes of DNA inoculation involve the administration of microsphere-encapsulated DNA to raise protective responses against a M. tuberculosis challenge. MPT63 DNA vaccines can be encapsulated in microspheres. Each patient receives a primary inoculatioii and a boost. The patiaits receive approximately 1-50 .aboutg/kg body weight of microsphere-encapsulated DNA for both the primary and boost inoculations. Each administration of enc^Miulated DNA is delivered in 100 pi of water intranasally. A safe, effective vaccine that protects against infection by Mycobacterium is important in both human and veterinary medicine. A Mycobacterium DNA vaccine of the invention is useful in providing protection against Mycobacterium infection humans, human infants, caretakers of infected infants, and immunocompFomised humans. A bovine and porcine DNA vaccines of the invention are useful to prevent Mycobacterium infections in cattle and piglets thereby allowing the animals to dirive for increased agricultural benefit. A DNA vaccine against any human or animal Mycobacterium can be constructed and used according to the invention. Such vaccines are useiul in providing homologous protection against a specific strain of Mycobacterium. The DNA vaccine of the invention is also useful in providing heterologous protection in that a DNA vaccine derived fixtm one species-specific Mycobacterium, serotype, or strain can be used to induce protective immunity against a Mycobacterium fixim a different species-specific Mycobacterium, serotype, or strain. Broad protection against multiple strains within a given serotype is possible according to die invention by inoculating the human or animal with a DNA vaccine encoding a protection inducing protein from a Mycobacterium stram of the same serotype. Thus, a single DNA vaccine of the invention is useful in providing protection against multiple strains of Mycobacterium. The recombinant Rv3881c protein, as well as polypetide portions thereof which are immunogenic or can be made immunogenic by techniques known in the art, are useful as components of a protein-based or subunit vaccine. Preparation and administration of such vaccines are well known in the art. This invention also includes combinations, or "cocktails" of purified protein and polypeptide antigens or DNAs oicoding them, and the use of such cocktails for vaccines and immunodiagnostics. Cocktails according to the invention include antigens (and DNAS) of bact«ial pathogens whose pathogenicity (unlike, for example, cholera) is not primarily caused by a single protein or virulence factor, and of parasites generally. The inventjon has been disclosed and described with reference to its preferred embodiments. The t»t results are provided as examples of the utility of the invention and are not intsided to limit die scope of the invention, which wj]} be understood to include derivative DNAs, proteins, polypeptides and vaccines set forth above. In particular, the invention is to be understood to include all modiHcations within the scope of the qjpended claims. References: 1. A. Rama Rao and S. Vijaya (1996). Analysis of a genomic DNA expression library of Mycobacterium tuberculosis using tubeculosis patient sera: evidence for modulation of host immune reqwnse. Infect. Immun. 64: 3765-3771. 2. A. Rama Rao and Vijaya Satchidanandam (1997) Differential inununogenicity of novel Mycobacterium tuberculosis antigens derived from live versus dead bacilU. Infect. Immun. 65: 4880-4882. 3. Rama Rao, A., Shanti, S., and Vijaya Satchidanandam (1998). Characterization of novel immunodominant antigens of M. tuberculosis. Microbiology IM:-1197-1203. 4. Vijaya Satchidanandam. 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Purification and characteriz^on of a low-moJecuIar-mass T-eeil antigen secreted by Mycobacterium tijberculosis. Infeet. Immun. 63:1710-1717. 34. 30. Stratagene. 1993. Immunosereening protocol. Stratagene, La Jolla, Calif. 35. 31. Stratagene. 1993. Lambda Zap II Library. A Protocol. Stiatagene, La Jolla, calif. 36. 32. W^ott, M. E. E. 19S4. Compilation of published signal sequences. Nuel. Aeids Res 12:4154-5164. 37. 33. Wiker, H. G., Harboe, M., and Nagai, S. 1991. A localization index for distinction between extracellular and intracellular antigens of Mycobacterium tuberculosis. J. Gen. Microbiol. 137: 875-884. 38. 34. Yamaguehi, R., Matsuo, K., Yamaazaaki, A., Abe, C, Nagai, S., Teresaka, K., and Yamada, T. 1989. Cloning and characterization of the gene for immunogenic protein MPB64 of Mycobacterium bovis BCG. Infeet. Immun. 57: 283-288. 39. 35. Young, D. B., Kauftnann, S. H. E., Hermans, P. W. M., and Thole, J. E. R. 1992. Mycobacteria] protein antigens: a compilation. Molec. Microbiol. 6: 133-145. 40. Lodes, M. J., Davin C. Dillon, Raodoh Mohamath, Craig H. Day, Darin R. Baison, Lisa D. Reynolds, Patricia Mcneill, Diana Pedral Sampaio, Yasir A. W. Skeiky, Roberto Badaro, David H. Persing, Steven G. Reed, And Raymond L. Houghton (2001). Serological ExpresEdon Cloning and Immunological Evaluation of MTB48, a Novel Mycobacterium tuberculosis Antigen. J. Clin. Microbiol. 39; 2485-2493. I Claim: 1. A purified polypeptide, wherein the polypeptide comprises of SEQ ID NO: 1. 2. A purified polypeptide, wherein the polypeptide comprises amino acid residues 65 to 460 of SEQ ID NO:l. 3. A purified polypeptide as claimed in claim 1 comprising amino acid residues 86 to460ofSEQIDNo:l. 4. A purified polypeptide, wherein the polypeptide is a mature recombinant Rv 3881 C protein extending from the T in the underlined TQSQT to the C-terminal K in Fig.l 5. A fusion protein comprising two domains, wherein the first domain comprises: (a) aRv3881cpolypeptide(SEQIDNO:l);or (b) a fragment of the polypeptide of sequence ID No.l shorter than full-length sequence ID No. 1 polypeptide and having antigenic and immunogenic properties. 6. The fusion protein of claim 5, wherein the second domain comprises a polyhistidine tag. 7. A purified nucleic acid having the sequence shown in Fig.2 (SEQ ID No.2) 8. A purified nucleic acid as claimed in claim 7, wherein the said acid does not contain the entire nucleic acid sequence shown in Fig.2 along with or without silent substitutions. 9. A vaccine comprising of a recombinant RV 3881C polypeptide. 10. A vaccine as claimed in claim 9, wherein the vaccine includes the RV 3881 C gene. 11. A vaccine as claimed in claim 9 or 10, wherein the vaccine includes the mature recombinant protein. 12. A vaccine as claimed in any one of claims 9 to 11, wherein the vaccine includes a live vector organism such as M. bovis BCG vaccine strain or other alternated variants of M. bovis or M.tuberculosis. 13. A vaccine as claimed in any one of claims 9 to 12, wherein the vaccine includes a live attenuated poxvirus. 14. A vaccine as claimed in any one of claims 9 to 13, wherein the vaccine is a DNA vaccine, recombinant BCG vaccine or a recombinant poxvirus vaccine, or a combination of the said vaccines. 15. A vaccine as claimed in claim 14, wherein the vaccine is a DNA vaccine including a vector, preferably a plasmid vector, and one or more isolated nucleotide sequences each encoding the Rv3881c polypeptide, and transcriptional and translational regulatory sequences operably linked to the isolated nucleotide sequences for expression in a cell of a vertebrate. The DNA vaccine may include the regulatory sequences of CMV immediate-early promoter and/or mtron A, or other non-retroviral sequences. 16. A vaccine as claimed in claim 15, wherein the DNA vaccine may include other immunostimulatory adjuvant sequences such as those encoding interleukins 2,12, 7,15 or 18. 17. A vaccine as claimed in claim 14, wherein the vaccine is a BCG vaccine that includes live M, bovis-BCG vector carrying the Rv3881c gene under regulatory elements that can function inside mycobacteria, the said BCG vector is attenuated for virulence in humans and vertebrates. 18. A vaccine as claimed in claim 14, wherein the vaccine is a recombinant pox virus vaccine that includes live attenuated poxvirus vector carrying the Rv3881c gene under poxvirus regulatory elements that functions inside eukaryotic cells infected with the said poxvirus. 19. A diagnostic composition comprising mature Rv 3881c polypeptide or a fragment (s) thereof. 20. A diagnostic composition as claimed in claim 19, wherein the said composition includes mixtures of antigens, or antigen "cocktails", that include at least three and as many as six or even more M, tuberculosis antigens and/or peptides thereof, at least two of which and preferably all of which are specific to the M, tuberculosis complex. 21. A diagnostic composition as claimed in claim 17, wherein the mixture preferably includes at least two purified proteins or polypeptides that are highly immunologically active in an antibody system or in T-cell recognition, for use in serodiagnosis and skin tests, respectively 22. A probe for the detection of M.tuberculosis and M.bovis comprising the nucleic acid sequence derived from the Rv3881c gene. 23. A process for the preparation of a purified polypeptide by known methods characterized in that the polypeptide obtained comprises of SEQ ID No. 1. 24. A process as claimed in claim 23, wherein the polypeptide comprises amino acid residues 65 to 460 of SEQ ID NO: 1. 25. A process for preparing pure recombinant Rv3881c protein in large quantities comprising the steps of sub cloning the sequence encoding the mature Rv3881c protein (extending from nucleotide 196 to nucleotide 588, Fig.l) in E.Coli plasmid pET20b+ (Novagen) as a fusion protein bearing a short polyhistidine tract at its carboxy terminus and purifying the tagged protein so obtained by affinity chromatography using divalent metal ion columns. 26. A process as claimed in claim 25, wherein the tagged protein obtained has an apparent molecular weight of 50Kda. 27. A process as claimed in claim 25 or 26, wherein the tagged protein obtained is a recombinant (rec) Rv3881c.

Full Text

This invention relates to a polypeptide and fiosion proteins of mycobacterium tubercuJosis and otber bacterial pathogens whose antigenicity is not caused by a single protein or component, and parasites, including preparation of vaccines and also to a method of preparing the said polypeptide and fusion proteins.
BACKGROUND:
Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), currently the world's largest killer from any single agent. The identification of mycobacterial antigens that induce protective T-cell responses and/or stimulate humoral immunity during tubercular infection of humans is therefore an area of investigation that has received increasing emphasis. Since protective immunity against tuberculosis relies exclusively on cell mediated immune responses, antigens in the former class constitute potential candidates for the development of effective vaccines, while those in the latter group can be tested as new, improved tools for diagnosis of TB.
Similarly, numerous other bacterial pathogens have pathogenicity that, as with TB, is not caused by a single protein, as is the case also with parasites generally. Antigens produced by these pathogens are also potential candidates for the development of effective vaccines.
The majority of M tuberculosis proteins that induce strong human cell mediated immune (CMI) responses are actively secreted by M. tuberculosis, making them valuable components of effective vaccines. The unique ability of live, dividing mycobacteria, but not dead bacilli to efficiently induce protective immunity (7, 22) led to the hypothesis that proteins that are actively secreted by M. tuberculosis during growth are key in generating protective T-cell responses (4, 23). Indeed, e}q>erimental vaccines based on culture filtrate proteins have been shown to induce some levels of protective immunity in animal models of TB (5, 14, 15, 26). Secreted protems of M. tuberculosis are also potent inducers of antibody production (13).

Bioinfonnatics ^proaches have been used to predict the existence of more than 700 secreted proteins ofM. tuberculosis and 2 D gel electrophoresis has been used to resolve as many as 620 individual members, and the identity of ] 14 of these was established. ID addition to their immunostimulatory roles m directly mteracting with the innate immune system [7], secreted mycobacterial protems show great promise as potential vaccine targets by virtue of their role as important T-cell antigens in the ad^tive immune response [8 -10]. The immunological characterization of individual components of M tuberculosis culture filtrates is a crucial step toward understanding the role of the secreted proteins in inducing immune responses during the course of TB. Earlier work had identified approximately ten actively secreted proteins using antibodies from immunized animals (1), most of whom were characterized by gene cloning and nucleotide sequencing (2, 6, 9, 11, 17-20, 29, 34). Some of the known secreted proteins induce cellular immune responses (35); however, the target antigens in the secreted protein fi-actions of the strongest human T-cell responses are yet to be characterized (S, 29). An aspect of this invention is an isolated DNA sequence coding for the polypeptide sequence of the Rv38Slc antigen, a protein secreted by M. tuberculosis, that is specific for some mycobacterial species that belong to the M. tuberculosis complex, as well as recombinant polypeptide sequences encoded by that DNA.
Another aspect of this invention is a DNA cocktails for vaccines as well as a "cocktail" of purified natural and recombinant protein antigens or polypeptides for immunodiagnostics or vaccines.
SUMMARY OF INVENTION:
The gene for the protein Rv3881c has been isolated and sequenced (SEQ ID N0:2). That gene can be incorporated into a plasmid and expressed in E. coli to produce purified Rv3881c protein, whose sequence (SEQ ID N0:1) has been deduced. Additional e7q}ression systems will be apparent to persons skilled in the art
Rv3881c is a protein secreted by M. tuberculosis and is specific to M. tuberculosis and M. bovis. Homologues of this gene or protein have not been detected m any other

mycobacteria including M. simiae, M. scrojulaceum, M. intracellulare, M. szulgai, M. kansasii, M. fortuitum, M.parafortuitum, M. phlei, M. vaccae, M. flavescens, M. chitae, M. leprae, hi. gordonae, M. chelonae, M. smegmatis.M. avium .
This invention includes the Rv3881c amino acid sequence shown in FIG. 1 (SEQ ID NO: 1). A preferred embodiment is the mature recombinant Rv3881c protein which is the polypeptide extending from the T in the underlined TQSQT to the C-terminal K in FIG. 1. Also preferred are antigoiic polypeptides derived from the sequences shown in FIG. 1, whether produced by natural, recombinant or synthetic (including chemical synthesis) means or other means known in the art. The invention also includes variants of these polypeptides that retain their antigenic and immunogenic properties.
This invention includes an isolated nucleic acid having the sequence shown in FIG. 1 {SEQ ID N0:2). Other embodiments can be derived by making silent substitutions, those that do not change the amino acid sequence encoded by the nucleic aci4 in the nucleic acid sequence. In preferred embodiments these nucleic acids are made by modifying the sequence by mut^enesis, recombination or synthetic (including chemical synthesis) means or other means known in the art. Also preferred are embodiments wherein the nucleic acid does not contain the entire nucleic acid sequence shown in PIG. 1 (SEQ ID N0:2), with or without silent substitutions.
This invention also includes vaccines that contain a recombinant Rv3881c polypqitide. In preferred embodiments the vaccine includes the Rv38S 1 c gene. In preferred embodunents the vaccine includes the mature recombinant protein. In preferred onbodiments the vaccine includes a live vector organism such as the M.bovis BCG vaccine strain or other attenuated variants of M bovis or M. tuberculosis. In preferred embodiments the vaccine includes a live attenuated poxvirus.
This invention includes a method of eliciting an immune response and/or protective immunity against M. tuberculosis or another member of the M. tuberculosis complex in a vertebrate, said method including administering to the vertebrate a recombinant Rv3881c

polypeptide, whereby said polypeptide elicits immune responses against the Mycobactraium in the vertebrate.
A DNA vaccine according to this invention includes a vector, preferably a plasmid vector, and one or more isolated nucleotide sequences each ^tcoding the Rv388lc polypeptide, and transcriptional and translational regulatory sequences operably linked to the isolated nucleotide sequences for expression in a cell of a vertebrate. The DNA vaccine may include the regulatory sequences of CMV immediate-early promoter and/or intron A, or other non-retroviral sequences.
A recombinant BCG vaccine according to this invention includes live M. bovis-BCG vector carrying the Rv388ic gene under regulatory elements that can function inside mycobactCTia. The said BCG vector is attenuated for virulence in humans and vertebrates. A recombinant poxvirus vaccine according to this invention includes live attenuated poxvirus vector carrying the Rv3881c gene under poxvirus regulatory elements that can function inside eukaryotic cells infected with the said poxvirus.
This invention also includes combinations of the above DNA vaccine, recombinant BCG vaccine and recombinant poxvirus vaccine in all possible permutations and combinations.
This invention also includes methods of eliciting an immune response and/or protective immunity by administering to a vertebrate such a DNA vaccine, recombinant BCG vaccine, recombinant poxvirus vaccine or their various permutations and combinations whereby expression of said Rv3881c nucleotide sequences in said celt eUcits immune responses against the Mycobacterium. This invention also includes methods for enhancing immime responses using various adjuvants such as interleukins, along with the Rv388 Ic nucleotide sequence or polypeptide.
In preferted methods of this invention the vertebrate is a human. In preferred metiiods of this invOTtion the vertebrate is also a bovine or any species known to suffer from M. tuberculosis- or M. 6ov«-mediated disease. A DNA vaccine according to this invention

may be administered to a vertebrate through a route of administration selected from the group consisting of inhalation, intravenous, intramuscular, intrqieritoneal, intradermal, and subcutaneous. A preferred embodiment is a method wherein die DNA vaccine is administered by contacting the DNA vaccine with a mucosal surface of the vertebrate. A preferred embodiment is a method wherein the DNA vaccine is microsphere encapsulated, and is administered by contacting the microsphere-enc^qisulated DNA vaccine with a mucosal surface of the vertebrate. A prefored embodiment is a method wherein the DNA vaccine is coated onto gold beads for administration to the vertdirate by particle bombardment delivery. A preferred embodiment is a method wherein the gold beads are approximately 1 pm to 2 pm in diameter. A preferred embodimoit is a method wherein the protective immunity is homologous, homotypic, heterotypic, or heterologous.
A recombinant BCG or recombinant poxvirus vaccine according to this invention may be administered to a vertebrate through a route of administration selected from the groi^ consisting of inhalation, intravenous, intramuscular, intr^qieritoneal, intradermal, and subcutaneous.
This invention includes the use of mature Rv3881c polypeptide or a fragment(s) thereof in diagnostic tests for the detection in a patient of an immune response to M. tuberculosis or another member of the M. tuberculosis complex. A diagnostic test can be perfoimed in the format of the commonly used Mantoux or Tine test for the detection of an immune reaction in the skin. This invention also includes the use of mature Rv3881c polypeptide, or a fragment(s) thereof, to bind antibody in human or animal sera in an ELISA, or any other solid-phase immunoassay. Several formats of solid-phase immunoassays are known in the art and can be ad^ted for use in this invention.
This invention includes the use of a nucleic acid sequence of this invention, as a probe for the detection of M. tuberculosis and M. bovis, the only 2 mycobacterial strains so far found to contain the Rv3881c gene. Nucleic acid detection assays are well known to those skilled in the art. Assays can involve direct or indirect detection of the target

sequence. Amplification of the target sequence can also be performed prior to or as a part of detection.
Amplification can be performed with ligase chain reaction, polymerase chain reaction, self-sustained sequence reaction, NASBA and Q-beta amplification. Specific primers for the amplification of the Rv3881c gene can be derived fi-om the nucleic acid of the present invention by standard procedures {and tests for specificity). Such primers can be selected simply by testing 15 to 50 nucleotide long sequences derived from the gene for specific hybridization to and specific amplification of the gene in the presence of various nucleic acids e^qiected to be present in a sample.
This invention includes mixtures of antigens, or antigen "cocktails", that include at least three and as many as six or even more M. tuberculosis antigens and/or peptides thereof, at least two of which and preferably all of which are ^ecific to the M. tuberculosis complex. The mixture should preferably include at least two purified proteins or polypeptides that are highly immunologically active in an antibody system or in T-cell recognition, for use in serodiagnosis and skin tests, respectively.
Similar cocktails can be made of protein or polypeptide antigens expressed by other bacterial pathogens, such as Listeria, Shigella and Salmonella, or by parasites, such as Plasmodium, Leishmania and Trypanosoma.
This invention also includes vaccines, both protein-based and DNA-based. The vaccines may comprise cocktails of purified proteins or polypeptide antigens, ofM. tuberculosis or another bacterial pathogen or a parasite. The vaccines may comprise cocktails of DNAs encoding such antigens. Bach member of the cocktail must induce a protective immune response. For vaccines, specificity to the pathogen is not required. Preferably, the cocktail comprises the most protective antigens available.

DETAILED DESCRIPTION OF THE DRAWINGS :
FIG. 1 shows the amino acid sequence (SEQ ID N0:1) of the Rv3881c protein, a polypeptide of 460 aa having a calculated Mr of 47594 Daltons.
FIG. 2 shows the nucleotide sequence (SEQ ID N0:2) of the gene encoding the Rv3881c protein ofM. tuberculosis and the deduced amino acid sequence of the protein (SEQ ID N0:1). The sequence (SEQ ID N0:2) contains an open reading frame of 1380 nucleotides that encodes the Rv3881c protein, whose deduced amino acid sequence is also shown below die nucleotide sequence. The first codon (ATG) is shown m bold. The underlmed pent^eptide (TQSQT in SEQ ID N0:1) marks the start of the mature protein.
FIG. 3. We performed Southern blot analysis using EcoRI plus Sall-digested DNA from the different mycobacterial strains and a 311-bp BamHI-Smal fragment representing the 3'end of the Rv3881c gene to detect the Rv3881c gene in the M. tuberculosis genome. A smgle band of approximately 7 kb was visimlized after hybridization, mdicatmg that the Rv3881c gene is presumably present in a single copy in the bacterial chromosome. The same positive signal was detected using DNAs exfracted from other reference strains (H37Ra, H37Rv). M. bovis and M. bovis BCG also gave positive signals, with the size of the band detected in the latter being only 5 kb due to a genomic deletion close to the Rv3881c gene. No restriction fr^ment loigth polymorphism was observed in DNAs that tested positive. In confrast, no hybridization signal was detected in DNAs exfracted from unrelated mycobacterial species (M simiae, M. scrofitlaceum, M. intracellulare, M. szulgai, M. kansasii, M. fortuitum, M.parafortuitum, M. phlei, M. vaccae, M. flavescens, M. chitae). The Rv 3881c gene has also been rqwrted to be absent in M. leprae, M. gordonae, M. chelonae, M. smegmatis ,and M. avium (Lodes et. al., 2001). These hybridization results suggest that the Rv 3881c gene is conserved in only in M. tuberculosis and M. bovis of the M. tuberculosis complex, while it is absent in all other mycobacteria.

Purified protein and polypeptide antigens can be made using all or part of tiie sequences shown in FIG. 1 (SEQ ID NO; 1 and SEQ ID N0:2> by methods well known in the art.
The DNA sequence shown in FIG. 1 (SEQ ID N0;1). a°d portions thereof, can be obtained by methods well known in the art. M. tuberculosis genomic DNA and primers derived from FIG. 1 (SEQ ID NO.l) can be used to ampliiy the entire gene sequaice OT portions thereof by polymerase chain reaction amplification, for example. Libraries of M. tuberculosis genomic DNA and DNA probes derived from FIG. 1 (SEQ ID NO:l) can also be used to clone the Rv3881c gene or portions thereof.
To obtain pure recombinant Rv3881c protein in large amounts the sequence encoding the mature Rv3S81c protein (extending from nucleotide 196 to nucleotide 588, FIG. 1) was subcloned in the E. coli plasmid pET20b+ (Novagen) as a fusion protein bearing a short polyhistidise tract at its carboxy terminus. The ta^ed protein, having an ^^arent molecular weight of SO Kda was purified by affinity chromatogr^hy using nickel columns (25). The nickel-affinity purified terminally tagged protein is referred to as recombinant (rec) Rv3881c.
Tufile 1; M. tuberculosis challenge bacilli recovered fl'om ^leois of guinea pigs immunized using tiie said experimental vaccines. DNA coding for the polypeptide referred to as recombinant 213 (claim 1) was expressed under the transcriptional control of the cytomegalovirus (CMV) promoter. Guinea pigs were immunized using this DNA or pJW vector DNA as control. Other animals received as immunizing antigen either BCG or recombinant BCG (REC-BCG) which expresses the 213 polypeptide of M. tuberculosis. Another groi^ of animals were immunized with pJW213 DNA followed by REC-BCG. All animals received the immunogens intradennally. Control refers to animals that received no immunization whatever. All animals were challenged intramuscularly using 5 loglO viable bacilli of the virulent M. tuberculosis sti-ain NTI83949. Load of challenge bacilli m the spleens of animals was enumerated 6 weeks later.

Statistical treatment of the above data revealed that BCG and pJW213 were not significantly different from each other in their protective ability.
A "DNA transcription unit" is a polynucleotide sequence, bounded by an initiation site and a termination site, that is transcribed to produce a primary transcript. As used herein, a "DNA transcription unit" includes at least two components: (1) antigen-encoding DNA, and (2) a transcriptional promoter element or elements operatively linked for expression of the antigen coding DNA. Antigen-coding DNA can encode one or multiple antigens, such as antigens fi-om two or more different Mycobacterial proteins. The DNA transcription unit can additionally be inserted into a vector which includes sequences for expression of the DNA transcription unit.

A DNA transcription unit can optionally include additional sequences such as enhancer elements, splicing signals, tennination and polyadenylation signals, viral replicons, and bacterial plasmid sequences. In the present method, a DNA transcription unit (i.e., one type of transcription unit) can be administered individually or in combination with one or more other types of DNA transcription units.
DNA transcription units can be produced by a numb^ of known methods. For example, DNA encoding the desired antigen can be inserted into an expression vector (see, for example, Sambrook et al.. Molecular Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press (1989)). With the availability of automated nucleic acid synthesis equipment, DNA can be synthesized directly when the nucleotide sequence is known, or by a combination of polymerase chain reaction (PCR), cloning, and fermentation. Moreover, when the sequence of the deshed polypq}tide is known, a suitable coding sequence for the polynucleotide can be inferred.
The DNA transcription unit can be administered to an individual, or inoculated^ in the presence of adjuvants or other substances that have the capability of promoting DNA uptake or recruiting immune system cells to the site of the inoculation. It should be understood that the DNA transcription unit itself is expressed in the host cell by transcription factors provided by the host cell, or provided by a DNA transcription unit
The desired antigen can be any antigen or combination of antigens from a Mycobacterium. The antigen or antigens can be naturally occurring, or can be mutated or specially modified. The antigen or antigens can represent different forms, such as strains of Mycobacteria. These antigens may or may not be structural components of a Mycobacterium. The encoded antigens can be translation products or polypeptides. The polypeptides can be of various lengths. In addition, they can be designated to undergo intracellular, extracellular, or cell-surface expression. Furthermore, they can be designed to undergo release from cells.

An individual can be inoculated through any parenteral route. For example, an individual can be inoculated by intravenous, intr^eritoneal, intradermal, subcutaneous, inhalation, or intramuscular routes, or by particle bombardment using a gene gun. Muscle is a useful site for the delivery and expression of DNA transcription unit-encoding polynucleotide, because animals have a proportionately large muscle mass which is conveniently access^ by dir^t injection through the skin. A comparatively large dose of polyaucleotide can be deposited into muscle by multiple and/or repetitive mjections, for example, to extend immunization over long periods of time. Muscle cells are injected with polynucleotide encodmg polypeptides, and these polypeptides are presented by muscle cells in the context of antigens of the major histocompatibility complex to provoke a selected immune response {see, e.g., Feigner, et al. WO90/11092, herein incorporated by reference).
The epidermis is another useftil site for the delivery and expression of polynucleotide, because it is conveniently accessed by direct injection or particle bombardment. A comparatively large dose of polynucleotide can be deposited in the epidermis by multiple injections or bombardments to extend therqiy over long periods of tune. In immunization strategies of the invention, skin cells are injected with polynucleotide coding for antigenic or immunogenic polypeptides, and these polypeptides are presented by skin cells in the context of antigens of the major histocompatibility complex to provoke a selected immune response against the immunogen.
In addition, an individual can be inocidated by a mucosal route. The DNA transcription unit can be administered to a mucosal surface by a variety of methods including DNA-containing nose-drops, inhalants, si^positories, microsphere encapsulated DNA, or by bombardment with DNA coated gold particles. For example, the DNA transcription unit can be administered to a respiratoiy mticosal surface, such as the nares or the trachea.
Any appropriate physiologically compatible medium, such as saline for injection, or gold particles for particle bombardment, is suitable for introducmg the DNA transcription unit into an individual.

Intradennal administration of DNA by particle bombardmrat can be used to deliver DNA for expression of a M. tuberculosis Rv3881c polypeptide in skin cells. The Accell particle bombardment device ("gene gun"; Agracetus, Middleton, Wis.) or similar devices obtained from other companies can be employed to deliver DNA-coated gold beads to the epidermis.
Vertebrates can be immunized with gold particles coated with the DNA v^cine using particle bombardment technology as presented in the following articles: Yang, M. S. et al., (1990) Pioc. Natl. Acad. Sci. USA 87:9568-9572; YangN.-S. (1992) CRC Crit. Rev. Biotechnol. 12:335-356; and Cheng, L. et al. (1993) Proc. NaU. Acad. Sci. USA 90:4455-4459. The beads deliver DNA mto cells, where the DNA dissolves and can be expressed (Yang, M. S. et al. (1991) Proc. Natl. Acad. Sci. USA 88: 2726-2730). Expression is transient, with most of the expression being lost within 2-3 days due to the normal sloughing of the epidermis (Williams, R. S. et al., Proc. NaU. Acad. Sci. USA 88: 2726-2730(1991)).
These particle bombardment techniques can be easily adapted for use in human patients using M. tuberculosis Rv3881c DNA vaccines. Administration of a DNA vaccine to a human can be perfonned by any one or more of several routes selected from the following: intravenous, intramuscular, intr^^jedtoneal, intradennal, inhalation, and subcutaneous. For example, inti^ermal administration by gene gun is a preferred route. The site of administration is chosen for the convenience of the patient. The dose is between 1 and 50 pg of DNA vaccine per kg body weight, preferably 10-25 .about.g per kg body weight. For a human infant, two inoculations are given at a 4 week interval. A human of any age who is caring for an infected infant or is inimimocompromised due to ilhiess, drug treatment, or other cause puttmg him or her at risk of infection is inoculated with the DNA vaccine by gene gun delivery for at least 2 inoculations at 4 week intervals.
Mucosal routes of DNA inoculation involve the administration of microsphere-encapsulated DNA to raise protective responses against a M. tuberculosis challenge.

MPT63 DNA vaccines can be encapsulated in microspheres. Each patient receives a primary inoculatioii and a boost. The patiaits receive approximately 1-50 .aboutg/kg body weight of microsphere-encapsulated DNA for both the primary and boost inoculations. Each administration of enc^Miulated DNA is delivered in 100 pi of water intranasally.
A safe, effective vaccine that protects against infection by Mycobacterium is important in both human and veterinary medicine. A Mycobacterium DNA vaccine of the invention is useful in providing protection against Mycobacterium infection humans, human infants, caretakers of infected infants, and immunocompFomised humans. A bovine and porcine DNA vaccines of the invention are useful to prevent Mycobacterium infections in cattle and piglets thereby allowing the animals to dirive for increased agricultural benefit. A DNA vaccine against any human or animal Mycobacterium can be constructed and used according to the invention. Such vaccines are useiul in providing homologous protection against a specific strain of Mycobacterium. The DNA vaccine of the invention is also useful in providing heterologous protection in that a DNA vaccine derived fixtm one species-specific Mycobacterium, serotype, or strain can be used to induce protective immunity against a Mycobacterium fixim a different species-specific Mycobacterium, serotype, or strain.
Broad protection against multiple strains within a given serotype is possible according to die invention by inoculating the human or animal with a DNA vaccine encoding a protection inducing protein from a Mycobacterium stram of the same serotype. Thus, a single DNA vaccine of the invention is useful in providing protection against multiple strains of Mycobacterium.
The recombinant Rv3881c protein, as well as polypetide portions thereof which are immunogenic or can be made immunogenic by techniques known in the art, are useful as components of a protein-based or subunit vaccine. Preparation and administration of such vaccines are well known in the art.

This invention also includes combinations, or "cocktails" of purified protein and polypeptide antigens or DNAs oicoding them, and the use of such cocktails for vaccines and immunodiagnostics. Cocktails according to the invention include antigens (and DNAS) of bact«ial pathogens whose pathogenicity (unlike, for example, cholera) is not primarily caused by a single protein or virulence factor, and of parasites generally. The inventjon has been disclosed and described with reference to its preferred embodiments. The t»t results are provided as examples of the utility of the invention and are not intsided to limit die scope of the invention, which wj]} be understood to include derivative DNAs, proteins, polypeptides and vaccines set forth above. In particular, the invention is to be understood to include all modiHcations within the scope of the qjpended claims.
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40. Lodes, M. J., Davin C. Dillon, Raodoh Mohamath, Craig H. Day, Darin R. Baison, Lisa D. Reynolds, Patricia Mcneill, Diana Pedral Sampaio, Yasir A. W. Skeiky, Roberto Badaro, David H. Persing, Steven G. Reed, And Raymond L. Houghton (2001). Serological ExpresEdon Cloning and Immunological Evaluation of MTB48, a Novel Mycobacterium tuberculosis Antigen. J. Clin. Microbiol. 39; 2485-2493.

I Claim:
1. A purified polypeptide, wherein the polypeptide comprises of SEQ ID NO: 1.
2. A purified polypeptide, wherein the polypeptide comprises amino acid residues 65 to 460 of SEQ ID NO:l.
3. A purified polypeptide as claimed in claim 1 comprising amino acid residues 86 to460ofSEQIDNo:l.
4. A purified polypeptide, wherein the polypeptide is a mature recombinant Rv 3881 C protein extending from the T in the underlined TQSQT to the C-terminal K in Fig.l
5. A fusion protein comprising two domains, wherein the first domain comprises:

(a) aRv3881cpolypeptide(SEQIDNO:l);or
(b) a fragment of the polypeptide of sequence ID No.l shorter than full-length sequence ID No. 1 polypeptide and having antigenic and immunogenic properties.

6. The fusion protein of claim 5, wherein the second domain comprises a polyhistidine tag.
7. A purified nucleic acid having the sequence shown in Fig.2 (SEQ ID No.2)
8. A purified nucleic acid as claimed in claim 7, wherein the said acid does not contain the entire nucleic acid sequence shown in Fig.2 along with or without silent substitutions.
9. A vaccine comprising of a recombinant RV 3881C polypeptide.

10. A vaccine as claimed in claim 9, wherein the vaccine includes the RV 3881 C gene.
11. A vaccine as claimed in claim 9 or 10, wherein the vaccine includes the mature recombinant protein.
12. A vaccine as claimed in any one of claims 9 to 11, wherein the vaccine includes a live vector organism such as M. bovis BCG vaccine strain or other alternated variants of M. bovis or M.tuberculosis.
13. A vaccine as claimed in any one of claims 9 to 12, wherein the vaccine includes a live attenuated poxvirus.
14. A vaccine as claimed in any one of claims 9 to 13, wherein the vaccine is a DNA vaccine, recombinant BCG vaccine or a recombinant poxvirus vaccine, or a combination of the said vaccines.
15. A vaccine as claimed in claim 14, wherein the vaccine is a DNA vaccine including a vector, preferably a plasmid vector, and one or more isolated nucleotide sequences each encoding the Rv3881c polypeptide, and transcriptional and translational regulatory sequences operably linked to the isolated nucleotide sequences for expression in a cell of a vertebrate. The DNA vaccine may include the regulatory sequences of CMV immediate-early promoter and/or mtron A, or other non-retroviral sequences.
16. A vaccine as claimed in claim 15, wherein the DNA vaccine may include other immunostimulatory adjuvant sequences such as those encoding interleukins 2,12, 7,15 or 18.
17. A vaccine as claimed in claim 14, wherein the vaccine is a BCG vaccine that includes live M, bovis-BCG vector carrying the Rv3881c gene under regulatory

elements that can function inside mycobacteria, the said BCG vector is attenuated for virulence in humans and vertebrates.
18. A vaccine as claimed in claim 14, wherein the vaccine is a recombinant pox virus vaccine that includes live attenuated poxvirus vector carrying the Rv3881c gene under poxvirus regulatory elements that functions inside eukaryotic cells infected with the said poxvirus.
19. A diagnostic composition comprising mature Rv 3881c polypeptide or a fragment (s) thereof.
20. A diagnostic composition as claimed in claim 19, wherein the said composition includes mixtures of antigens, or antigen "cocktails", that include at least three and as many as six or even more M, tuberculosis antigens and/or peptides thereof, at least two of which and preferably all of which are specific to the M, tuberculosis complex.
21. A diagnostic composition as claimed in claim 17, wherein the mixture preferably includes at least two purified proteins or polypeptides that are highly immunologically active in an antibody system or in T-cell recognition, for use in serodiagnosis and skin tests, respectively
22. A probe for the detection of M.tuberculosis and M.bovis comprising the nucleic acid sequence derived from the Rv3881c gene.
23. A process for the preparation of a purified polypeptide by known methods characterized in that the polypeptide obtained comprises of SEQ ID No. 1.
24. A process as claimed in claim 23, wherein the polypeptide comprises amino acid residues 65 to 460 of SEQ ID NO: 1.

25. A process for preparing pure recombinant Rv3881c protein in large quantities
comprising the steps of sub cloning the sequence encoding the mature Rv3881c
protein (extending from nucleotide 196 to nucleotide 588, Fig.l) in E.Coli
plasmid pET20b+ (Novagen) as a fusion protein bearing a short polyhistidine
tract at its carboxy terminus and purifying the tagged protein so obtained by
affinity chromatography using divalent metal ion columns.
26. A process as claimed in claim 25, wherein the tagged protein obtained has an
apparent molecular weight of 50Kda.
27. A process as claimed in claim 25 or 26, wherein the tagged protein obtained is a
recombinant (rec) Rv3881c.

Documents:

457-CHE-2004 CORRESPONDENCE OTHERS 10-03-2011.pdf

457-che-2004 correspondence others 30-11-2010.pdf

457-che-2004 abstract.pdf

457-che-2004 claims.pdf

457-che-2004 correspondence others.pdf

457-che-2004 correspondence po.pdf

457-CHE-2004 CORRESPONDENCE-OTHERS 09-09-2009.pdf

457-che-2004 description (complete).pdf

457-che-2004 drawings.pdf

457-che-2004 form-1.pdf

457-che-2004 form-19.pdf

457-che-2004 form-26.pdf

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Patent Number

234756

Indian Patent Application Number

457/CHE/2004

PG Journal Number

29/2009

Publication Date

17-Jul-2009

Grant Date

15-Jun-2009

Date of Filing

14-May-2004

Name of Patentee

INDIAN INSTITUTE OF SCIENCE,

Applicant Address

BANGALORE-560 012, KARANATAKA,

Inventors:

#	Inventor's Name	Inventor's Address
1	MRS, SACHIDANANDAM VIJAYA	DEPT OF MICROBIOLOGY AND CELL BIOLOGY SIR, C.V. RAMAN AVENUE, INDIAN INSTITRE OF SCIENCE BANGALORE 5600 12.

PCT International Classification Number

A61K39/02

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA