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Title:  Vaccine against mycobacterial infections

United States Patent:  6,492,145

Issued:  December 10, 2002

Inventors:  Lowrie; Douglas Bruce (London, GB)

Assignee:  Medical Research Council (London, GB)

Appl. No.:  737487

Filed:  November 15, 1996

PCT Filed:  November 23, 1995

PCT NO:  PCT/GB95/01119

371 Date:  May 18, 1995

Abstract

A naked nucleic acid construct comprising a coding sequence which encodes a mycobacterial stress protein or proline-rich antigen or an antigenically effective fragment thereof operably linked to a promoter capable of expressing the said coding sequence in a mammalian host cell is useful as a vaccine against a mycobacterial infection such as tuberculosis and leprosy.

Description of the Invention

This invention relates to vaccines against mycobacterial infections such as tuberculosis and leprosy.

Despite its central position in classical immunology surprisingly little is known of how a protective cell-mediated immune response is either acquired or expressed against tuberculosis or leprosy. It is not know why vaccination with live bacille Calmette-Guerin (BCG) is highly protective in only some human populations or why, in contrast to live BCG, injections of dead BCG or antigenic components, even in large amounts and with adjuvants, confer only slight protection in animals.

In an attempt to develop an alternative vaccine based on the Mycobacterium leprae 65 kDa heat shock protein (MLhsp65) antigen (Mehra et al (1986): Proc. Natl. Acad. Sci. USA; 83, 7014-7017), we have now stably transfected bone marrow cells with an expression vector encoding this antigen. When the transfected bone marrow cells were injected into mice, the mice were found to be resistant to infection by Mycobacterium tuberculosis, the causative agent of tuberculosis. Further, we have injected mice with naked DNA encoding MLhsp65 or the Mycobacterium leprae 36 kDa proline rich-antigen (Thole et al, Infection and Immunity (1990) 58, 80-87). These mice were also found to be resistant to infection by Mycobacterium tuberculosis.

These findings have general applicability. Accordingly, the present invention provides use of a naked nucleic acid construct comprising a coding sequence which encodes a mycobacterial stress protein or proline-rich antigen or an antigenically effective fragment thereof operably linked to a promoter capable of expressing the said coding sequence in a mammalian host cell, in the manufacture of a medicament for use as a vaccine against a mycobacterial infection.

The invention also provides:

such a naked nucleic acid construct for use as a vaccine against a mycobacterial infection;

a vaccine composition comprising such a naked nucleic acid construct and an acceptable carrier or diluent;

a method of vaccinating a mammalian host against a mycobacterial infection, which method comprises administering to the host an effective amount of such a naked nucleic acid construct;

bone marrow cells transfected with a nucleic acid construct comprising a coding sequence which encodes a mycobacterial stress protein or proline-rich antigen or an antigenically effective fragment thereof operably linked to a promoter capable of expressing the said coding sequence in bone marrow cells;

a method of vaccinating a mammalian host against a mycobacterial infection, which method comprises administering to the host an effective amount of such transfected bone marrow cells; and

a naked nucleic acid construct as above wherein the coding sequence encodes a mycobacterial proline-rich antigen or an antigenically effective fragment thereof.

The naked nucleic acid construct comprises a coding sequence which encodes a mycobacterial stress protein or a mycobacterial proline rich-antigen or an antigenically effective fragment thereof operably linked to a promoter capable of directing expression of the said coding sequence in a mammalian host cell. Nucleic acid encoding at least one further mycobacterial protein or fragment thereof operably linked to a promoter may be included in the construct. Typically, the thus encoded further mycobacterial protein or fragment thereof will be an antigenic protein or an antigenic fragment thereof. The further mycobacterial protein or fragment thereof may be a further mycobacterial stress protein or proline-rich antigen or antigenic fragment thereof.

The naked nucleic acid construct is typically cell-free and virus-free. It is typically in isolated form. It may be purified. Although it is preferred that a construct is DNA, it may also be RNA or a modified nucleic acid. The nucleic acid may contain modifications in its backbone and possibly additions at either the 5' or 3', or both, ends of the molecule in the case of linear, as opposed to circular, constructs. This may assist in prolonging the life of the nucleic acid when taken up by host cells, for example, muscle cells which may enhance the potency of the construct. Known modifications to nucleic acid molecules include the provision of methylphosphonate and phosphorothioate backbones and addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.

The mycobacterial stress protein encoded by the nucleic acid constructs of the present invention is generally one whose expression increases substantially when the mycobacterium from which it is derived is placed under environmental stress. Typically, the mycobacterial stress protein is a heat shock protein, for example a protein whose expression increases substantially when the bacterium from which it is derived is subjected to a high temperature, for example 42oC. or greater.

The mycobacterial stress protein is typically derived from Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis, Mycobacterium avium or Mycobacterium vaccae. Suitable proteins include the 70, 65 and 10 kDa heat shock proteins of Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis, Mycobacterium avium or Mycobacterium vaccae. Of these, the 65 kDa heat shock proteins of Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium bovis are preferred, the heat shock proteins of Mycobacterium leprae being particularly preferred.

The mycobacterial proline-rich antigen may be a proline-rich antigen of Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis, Mycobacterium avium or Mycobacterium vaccae. A suitable proline-rich antigen is the 36 kDa proline-rich antigen of Mycobacterium leprae.

An antigenic fragment of a mycobacterial stress protein or proline-rich antigen preferably contains a minimum of five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty or fifty amino acids. The fragment may be up to ten, twenty, thirty, forty or fifty amino acids long. Alternatively, up to twenty or up to ten amino acid residues may have been omitted from the amino- and/or carboxy-terminus of the stress protein or proline-rich antigen.

The antigenic sites of the mycobacterial stress protein or proline-rich antigen may be identified using standard procedures. These may involve fragmentation of the polypeptide itself using proteolytic enzymes or chemical agents and then determining the ability of each fragment to bind to antibodies or to provoke an immune response when inoculated into an animal or suitable in vitro model system (Strohmaier et al, J.Gen.Virol., 1982, 59, 205-306).

Alternatively, the DNA encoding the mycobacterial stress protein or proline-rich antigen may be fragmented by restriction enzyme digestion or other well-known techniques and then introduced into an expression system to produce fragments. These fragments may be fused to a polypeptide usually a polypeptide of bacterial origin. The resulting fragments are assessed as described previously (Spence et al, J.Gen.Virol., 1989, 70, 2843-51; Smith et al, Gene, 1984, 29, 263-9).

Another approach is to chemically synthesise short peptide fragments (3-20 amino acids long; conventionally 6 amino acids long) which cover the entire sequence of the full-length polypeptide with each peptide overlapping the adjacent peptide. This overlap can be from 1-10 amino acids but ideally is n-1 amino acids where n is length of the peptide; Geysen et al, Proc. Natl. Acad. Sci, 1984, 81, 3998-4002. Each peptide is then assessed as described previously except that the peptide is usually first coupled to some carrier molecule to facilitate the induction of an immune response.

Finally, there are predictive methods which involve analysis of the sequence for particular features, e.g. hydrophilicity, thought to be associated with immunologically important sites (Hopp and Woods, Proc. Natl. Acad. Sci., 1981, 78, 3824-8; Berzofsky, Science, 1985, 229, 932-40). These predictions may then be tested using the recombinant polypeptide or peptide approaches described previously.

The nucleic acid sequence encoding the mycobacterial shock protein or proline-rich antigen or fragment thereof is typically included within a replicable expression vector. Such an expression vector comprises an origin of replication so that the vector can be replicated in a host cell such as a bacterial host cell, a promoter for the expression of the nucleic acid sequence and optionally a regulator of the promoter. The vector may contain one or more selectable marker genes, for example an ampicillin resistance gene for the identification of bacterial transformants or a neomycin resistance gene for the identification of mammalian cell transformants. optionally, the nucleic acid construct may also comprise an enhancer for the promoter. The construct may also comprise a polyadenylation signal operably linked 3' to the nucleic acid encoding the functional protein. The construct may also comprise a terminator 3' to the sequence encoding the mycobacterial stress protein or fragment thereof. The construct may also comprise one or more introns or other coding sequences 3' to the sequence encoding the mycobacterial stress protein or fragment thereof. The intron or introns may be from the host organism to which the construct is to be administered or from another eukaryotic organism.

In the nucleic acid constructs the nucleic acid sequence encoding the mycobacterial stress protein or proline-rich antigen or antigenic fragment thereof is operably linked to a promoter capable of expressing the sequence. "Operably linked" refers to a juxtaposition wherein the promoter and the nucleic acid sequence encoding the mycobacterial stress protein or proline-rich antigen or fragment thereof are in a relationship permitting the coding sequence to be expressed under the control of the promoter. Thus, there may be elements such as 5' noncoding sequence between the promoter and coding sequence.

These elements may be native either to the organism from which the promoter sequence is derived or to the organism from which the mycobacterial stress protein or proline-rich antigen or fragment thereof is derived. Alternatively, the said element or elements may be native to neither the organism from which the promoter sequence is derived nor the organism from which the mycobacterial stress protein or proline-rich antigen or fragment thereof is derived. Such sequences can be included in the construct if they enhance or do not impair the correct control of the coding sequence by the promoter.

The expression vector may be of any type. For example, the vector may be in linear or circular form. It is preferred that the construct is incorporated into a plasmid vector, since it has been found that covalent closed circle (CCC) plasmid DNA can be taken up directly by muscle cells but that the DNA does not integrate into the genomic DNA of the cells (Ascadi et al, (1991): The New Biologist; 3, 71-81). Those of skill in the art will be able to prepare suitable vectors comprising nucleic acid sequences encoding mycobacterial stress proteins or proline-rich antigens or fragments thereof starting with widely available vectors which will be modified by genetic engineering techniques such as those described by Sambrook et al, (Molecular Cloning: A Laboratory Manual, 1989). Two suitable starting vectors are the plasmids pCDM8 (Invitrogen; Seed and Aruffo, Proc. Natl. Acad. Sci. USA (1987) 84, 3365-3369) and pHMG (Gautier et al, Nucl. Acids Res. (1989) 17, 8389).

Any promoter capable of directing expression of the sequence encoding the mycobacterial stress protein or proline-rich antigen or fragment thereof may be operably linked to that sequence. Particularly suitable promoters are those that direct expression in a mammalian cell. For example, promoters from viral genes that are expressed in mammalian cells such as the cytomegalovirus (CMV) immediate early gene promoter are suitable. Also suitable are promoters from mammalian genes that are expressed in many or all mammalian cell types such as the promoters of housekeeping genes. For example, the p-hydroxymethyl-CoA-reductase (HMG) promoter (Gautier et al (1989)) is particularly suitable. Also suitable are promoters and other regulatory elements of genes selectively expressed in antigen-presenting mammalian cells such as macrophages and dendritic cells.

The nucleic acid constructs are useful for gene therapy. In particular, they are useful for naked DNA vaccination of mammalian hosts against mycobacterial infections such as those caused by Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium bovis. Accordingly, constructs comprising nucleic acid from any mycobacterial species may be prepared. Owing to the degree of conservation of some mycobacterial stress proteins, it is not always necessary to use a nucleic acid sequence from a particular species to vaccinate against infection by that species. For example, live BCG (Vacille Calmette-Guerin) cells of Mycobacterium bovis have long been used to vaccinate humans against Mycobacterium tuberculosis.

In the present invention, therefore, a nucleic acid construct encoding a stress protein or proline-rich antigen of Mycobacterium tuberculosis, Mycobacterium leprae or Mycobacterium bovis may be used to vaccinate against infection by any of these three species of Mycobacterium. For example, the present inventors have shown that constructs encoding the Mycobacterium leprae 65 kDa hsp or Mycobacterium leprae 36 kDa proline-rich antigen act as effective vaccines against Mycobacterium tuberculosis in mice.

A range of mammalian species can be vaccinated against mycobacterial infection using the nucleic acid constructs of the present invention. However, vaccination of humans against Mycobacterium tuberculosis is particularly desirable. Also desirable is the vaccination of cattle or deer against Mycobacterium bovis. Also desirable is the vaccination of badgers against Mycobacterium bovis as badgers can transmit the bacteria to cattle.

The naked nucleic acid constructs of the invention may be administered to mammals including humans by any route appropriate. Suitable routes include oral and parenteral, including subcutaneous, intramuscular, intravenous and intradermal routes.

Preferred routes of administration are oral delivery and injection, typically intramuscular or intradermal injection. Injection of the vaccine composition into the skeletal muscle or the skin of the human or animal subject is particularly preferred. Another mode of delivery of a vaccine composition according to the invention is by a biolistic or "particle gun" method.

The naked nucleic acid constructs of the invention may be administered to the subject alone or in a liposome or associated with other delivery molecules. The effective dosage depends on many factors such as whether a delivery molecule is used, the route of delivery and the size of the mammal being vaccinated. Typical doses are from 0.1-1000 .mu.g of the nucleic acid construct per dose, for example 1-500 .mu.g, 50-500 .mu.g, such as 50-75 .mu.g, and 100-500 .mu.g per dose.

Dosage schedules will vary according to, for example, the route of administration, the species of the recipient and.the condition of the recipient. However, single doses and multiple doses spread over periods of days, weeks or months are envisaged. Single doses typically comprise 0.1-1000 .mu.g, for example 100-500 .mu.g, of nucleic acid and multiple doses typically comprise 0.1-1000 .mu.g, for example 100-500 .mu.g, of nucleic acid each, preferably in a form suitable for intramuscular or intradermal injection. Also, single or multiple nucleic acid pellets comprising a construct according to the invention, for example pellets comprising 100-500 .mu.g of DNA can be implanted into the recipient intramuscularly or intradermally. If the construct is administered by a biolistic method, doses will generally be at the lower end of the above mentioned ranges, owing the hight efficiency of this route. Such doses may comprise, for example, 0.1-10 .mu.g, such as 0.1-1 .mu.g, of the construct.

While it is possible for the naked nucleic acid constructs of the invention to be administered alone it is preferable to present them as pharmaceutical formulations. The formulations of the present invention comprise at least one active ingredient, a nucleic acid construct according to the invention, together with one or more acceptable carriers thereof and optionally other therapeutic ingredients. The carrier or carriers must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipients thereof. Liposomes may be used. Suitable liposomes include, for example, those comprising the positively charged lipid (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA), those comprising dioleoylphosphatidylethanolamine (DOPE), and those comprising 3.beta.-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (Dc-Chol).

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, bactericidal antibiotics and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injections, immediately prior to use. Injection solutions and suspensions may be prepared extemporaneously from sterile powders, granules and tablets of the kind previously described.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question. Of the possible formulations, sterile pyrogen-free aqueous and non-aqueous solutions are preferred. Also preferred are formulations in which the nucleic acid constructs of the invention are contained in liposomes.

Effective vaccines against mycobacterial infection by Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium bovis can also be prepared from bone marrow cells transfected with a nucleic acid construct which encodes a mycobacterial stress protein or proline-rich antigen or fragment thereof operably linked to a promoter capable of directing expression of the said coding sequence in the said bone marrow cell.

The bone marrow cells may be transfected by any suitable method. For example the nucleic acid constructs may be packaged into infectious viral particles, for example retroviral particles. This may be done using the methodology described by Silva et al (1992): Microb. Pathogen. 12, 27-38). The constructs may also be introduced by electroporation, calcium phosphate precipitation, biolistic methods or by contacting naked nucleic acid constructs with the bone marrow cells in solution.

In the said nucleic acid constructs with which the bone marrow cells are infected, the nucleic acid may be DNA or RNA, preferably DNA.

The nucleic acid constructs with which the bone marrow cells are transfected may be of any suitable type.

Typically, the constructs will be in the form of an expression vector, such as a retrovival vector or a naked DNA expression vector as defined herein. For example, the construct may be in the form of retrovival shuttle vector derived from the widely available pZipNeo vector or from a plasmid vector as defined herein, for example pCDM8 or pHMG.

The constructs with which the bone marrow cells are transfected may comprise a coding sequence encoding at least one further mycobacterial protein or fragment thereof operably linked to a promoter capable of directing expression of the coding sequence in the mammalian cell. Typically, the thus encoded further mycobacterial protein or fragment thereof will be an antigenic protein or an antigenic fragment thereof. The further mycobacterial protein or fragment thereof may be a further mycobacterial stress protein or proline-rich antigen or antigenic fragment thereof.

The constructs with which the bone marrow cells are transfected may include any suitable promoter. Particularly suitable promoters are those that direct expression in a mammalian cell. For example, promoters from viral genes that are expressed in mammalian cells such as the cytomegalovirus (CMV) immediate early gene promoter are suitable. Also suitable are promoters from mammalian genes that are expressed in many or all mammalian cell types such as the promoters of housekeeping genes. For example, the p-hydroxymethyl-CoA-reductase (HMG) promoter (Gautier et al (1989)) is particularly suitable.

Bone marrow cells transfected with the said constructs may be administered by any suitable method, such as parenteral injection, preferably intravenous injection.

Any effective amount of bone marrow cells transfected with the said nucleic acid constructs may be administered to the recipient. Typically, from about 1x104 to about 1x108 bone marrow cells are administered, for example about from 105 to 107, for example 1x106 bone marrow cells.

The transfected bone marrow cells administered to the recipient may be of any type that is compatible with the recipient's immune system. Typically, as for any transplantation of cells or tissue, the major tissue transplantation antigens of the administered bone marrow cells will match the major tissue transplantation antigens of the recipient's cells. The administered bone marrow cells may be derived from the recipient individual.

Claim 1 of 5 Claims

What is claimed is:

1. A method of vaccinating a mammalian host against a mycobacterial infection, which method comprises administering to the host an effective amount of a naked nucleic acid construct consisting of a coding sequence which encodes the 65 kDa heat shock protein of Mycobacterium tuberculosis, Mycobacterium leprae or Mycobacterium bovis operably linked to a promoter capable of expressing said coding sequence in mammalian host cell, wherein the effective amount results in an immunogenic response.
 


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