Recombinant immunogenic actinomycetale

Details Recombinant immunogenic actinomycetale Recombinant immunogenic actinomycetale

1994-07-26

2001-05-22

Minnifield, Nita (Department: 1645)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S252100, C424S248100, C424S204100, C424S188100, C536S023100

Reexamination Certificate

active

06235518

ABSTRACT:

This invention relates to a strain of a species of Actinomycetale (the “Actinomycetale strain”), particularly a strain of mycobacteria, which: a) has been transformed with at least one gene or a DNA fragment thereof (an “antigen-encoding gene”) that codes for all or at least part of an antigen, particularly at least one epitope thereof (a “desired antigen”, preferably a “immunizing antigen”); and b) can be used to inoculate mammals, particularly humans, to immunize them, preferably protect them (e.g., from a pathogen carrying all or part of the desired antigen). In this regard, the term “desired antigen” comprises any molecule which is capable of inducing an immune response, including a protein, glycoprotein, glycolipoprotein, peptidoglycolipid, etc., or an immunogenic fragment thereof, and which can, for example, come from, or be derived from, a pathogen against which the transformed Actinomycetale strain is to provide an immunity, preferably a protection.
BACKGROUND OF THE INVENTION
Various types of vaccines have been developed against pathogens. When a humoral immune response is able to confer protection, subunit or killed vaccines are efficient. However, in the case of tuberculosis and certain other infectious diseases, killed pathogens are not protective.
The vaccine currently used to protect against tuberculosis,
Mycobacterium bovis
BCG (Bacille Calmette-Guerin) or “BCG”, is the unique live bacterial vaccine in use.
M. bovis
BCG and all other mycobacteria survive in macrophages which are antigen-presenting cells and initiate the humoral and T-cell mediated immune response (EDWARDS and KIRKPATRICK, 1986). This might explain the stimulant activities of this vaccine.
M. bovis
BCG offers many advantages for development of a recombinant polyvalent vaccine vector expressing antigens from a wide variety of pathogens, particularly those in which cell-mediated immunity is important for protection (BLOOM, 1986; JACOBS et al, 1988). It is an attenuated
M. bovis
strain which has been used without major side effects to vaccinate more than two billion people, it is produced at low cost, and it can be given at birth as a single dose and is then able to confer long term immunity. It also has stimulant activities and has been used as an adjuvant in various protocols of immunization.
The recent development of genetic tools for transforming mycobacteria has enabled the cloning of foreign genes in both fast-growing (
M. smegmatis
) and slow-growing (
M. bovis
BCG) strains. Several phasmid-and plasmid-based vectors have been reported, for example in PCT publication WO88/06626. Starting from pAL5000, a plasmid from
M. fortuitum
whose entire nucleotide sequence has been determined (RAUZIER et al, 1988), various
E. coli
-mycobacteria shuttle plasmids have been constructed which stably replicate in mycobacteria, including a “mini” mycobacterial replicon, pRR3 (RANES et al, 1990). Foreign genes have been cloned on these vectors and on other integrative vectors described in STOVER et al (1991) and shown to be expressed in mycobacteria using their own control elements or as fused genes (MATSUO et al, 1990).
SUMMARY OF THE INVENTION
This invention provides an immunogenic Actinomycetale strain, particularly a strain of mycobacteria, transformed with the following, operably linked, chimaeric DNA sequence, comprising an antigen-encoding gene that: a) is foreign to the Actinomycetale strain; b) encodes a desired antigen, preferably an immunizing antigen; c) is under the control of a promoter foreign to the Actinomycetale strain, preferably a promoter from another strain of Actinomycetale, especially a promoter from another species of Actinomycetale, particularly a Streptomyces promoter, quite particularly a stress-responsive (e.g., heat-shock) promoter; and d) is preferably associated with a ribosome binding site (“RBS”) foreign to the Actinomycetale strain, particularly foreign to Actinomycetale and/or synthetic.
This invention also provides the aforementioned chimaeric DNA sequence, especially wherein the antigen-encoding gene codes for an HIV-1 protein (European patent publications (“EP”) 0 178 978 and 201,540) or an HIV-2 protein (EP 0 239 425), particularly a Nef protein (e.g., the Nef 1 protein of HIV-1 or Nef 2 protein of HIV-2), or an antigenic fragment or an epitope thereof which can induce an immune response in a mammal, particularly a T or B response, quite particularly wherein the antigen-encoding gene is under the control of the
S. albus
groES/groEL1 promoter.
This invention further provides a system, such as a plasmid, capable of transforming a Actinomycetale strain. This system comprises a foreign promoter, a foreign ribosome binding site and a DNA fragment coding for an antigen foreign to the Actinomycetale strain, particularly the aforementioned chimaeric DNA sequence, quite particularly pWRIP17.
This invention yet further provides: a) an immunogenic composition, particularly a vaccine, comprising the aforementioned transformed Actinomycetale strain, particularly a strain of mycobacteria, quite particularly BCG, and if necessary, a suitable carrier, as well as, for example, an adjuvant for human use; and b) a method of immunizing a mammalian host, particularly a human, against, for example, a pathogen, comprising the step of administering to the host the aforementioned composition.
This invention also provides: a) a process to produce the desired antigen by culturing the transformed Actinomycetale strain; and b) a process for using the desired antigen or the transformed Actinomycetale strain, for example, as a diagnostic agent and/or an immunogenic agent. An antigenic preparation containing the desired antigen or the transformed Actinomycetale strain can induce the synthesis of specific antibodies or cellular reactions in in vitro and in vivo diagnostic tests and immunogenic treatments.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with this invention, a recombinant immunogenic strain of a species of Actinomycetale, such as Actinomycetaceae and Mycobacteriacae, especially Corynebacteria, Streptomyces and Mycobacteria, particularly Mycobacteria, can be obtained by transforming the Actinomycetale strain with the operably linked chimaeric DNA sequence of this invention.
The antigen-encoding gene of the chimaeric DNA sequence or expression cassette, used to transform the Actinomycetale strain in accordance with this invention, can be any gene or DNA fragment thereof which encodes a desired antigen as hereinbefore defined, whose expression products can be used, for example, to induce an immune response, preferably in a vaccine administered to a mammalian host, preferably a human, to immunize and preferably protect the host, for example, against a pathogen. Pathogens, whose immunizing antigens can be encoded by the antigen-encoding gene, include, for example, viral, parasitic and bacterial, particularly viral, pathogens such as
M. leprae, M. tuberculosis, M. intracellulare, M. africanum, M. avium
, plasmodium sporozoites and merozoites, diphtheria toxoid, tetanus toxoids, Leishmania, Salmonella, some Treponema, pertussis toxin and other antigenic determinants and viruses, including measles, mumps, herpes, influenza, Schistosoma, Shigella, Neisseria, Borrelia, rabies, polio virus, hepatitis virus, human immunodeficiency viruses (HIV), HTLV-I, HTLV-II, and Simian immunodeficiency virus (SIV), as well as oncogenic viruses. Alternatively, the antigen-encoding gene can encode an immunizing antigen from other than a pathogen, such as a snake or insect venom. In this regard, this invention is not limited to the expression of desired antigens for immunizing a mammal against a pathogen but also includes the use of the transformed Actinomycetale strain of this invention for the development of other kinds of immunotherapy treatments, as well as for the production of molecules of interest. For example, the transformed Actinomycetale strain, cloned with a gene coding for the synthesis of a hormone, could be used in an anti-fertility vaccine, or the transformed Actinomycetale strain,

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