Vaccine against mycobacterial infections

Details Vaccine against mycobacterial infections Vaccine against mycobacterial infections

1996-11-15

2002-12-10

Navarro, Mark (Department: 1645)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S093000, C514S04400A

Reexamination Certificate

active

06492145

ABSTRACT:

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 42° C. 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 d

Affiliated with

Also associated with

Rating

Vaccine against mycobacterial infections does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Vaccine against mycobacterial infections, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Vaccine against mycobacterial infections will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2968596