Attenuated strains of Leishmania and uses thereof

Details Attenuated strains of Leishmania and uses thereof Attenuated strains of Leishmania and uses thereof

2000-07-17

2002-07-30

Navarro, Mark (Department: 1645)

Chemistry: molecular biology and microbiology

Treatment of micro-organisms or enzymes with electrical or...

Modification of viruses

C435S258300

Reexamination Certificate

active

06426203

ABSTRACT:

FIELD OF INVENTION
The present invention relates to the field of molecular immunobiology and in particular to immunogenic preparations including vaccines comprising attenuated strains of Leishmania.
BACKGROUND OF INVENTION
Parasitic protozoa of the order Kinetoplastidae are the causative agents of several tropical diseases including sleeping sickness by
Trypanosoma brucei
, Chagas by
Trypanosoma cruzi
, visceral (kala-azar) and cutaneous (oriental sore) Leishmaniasis by
Leishmania donovani
and
Leishmania major
respectively. In particular Leishmania protozoans are the causative agents of human leishmaniasis, which includes a spectrum of diseases ranging from self-healing skin ulcers to fatal visceral infections. Human leishmaniasis is caused by at least thirteen different species and subspecies of parasites of the genus Leishmania. Leishmaniasis has been reported from about eighty countries and probably some 400,000 new cases occur each year. Recently the World Health Organization has reported 12 million people to be infected (ref. 1. Throughout this application various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure. A listing of the references appears at the end of the disclosure).
Untreated visceral leishmaniasis is usually fatal and mucocutaneous leishmaniasis produces mutilation by destruction of the naso-oropharyngeal cavity and, in some cases, death.
In addition a major health problem has been created in areas of high infection when blood is collected for transfusion purposes. Since blood is a carrier of the parasites, blood from an infected individual may be unknowingly transferred to a healthy individual.
The Leishmania protozoans exist as extracellular flagellated promastigotes in the alimentary tract of the sandfly in the free-living state, and are transmitted to the mammalian host through the bite of the insect vector. Once introduced, the promastigotes are taken up by macrophages, rapidly differentiate into non-flagellated amastigotes and start to multiply within the phagolysosomal compartment. As the infected cells rupture, amastigotes subsequently infect other macrophages giving rise to the various symptoms associated with leishmaniasis (refs. 1 and 2).
Leishmaniasis is, therefore, a serious disease and various types of vaccines against the disease have been developed, including live parasites; frozen promastigotes from culture; sonicated promastigotes; gamma-irradiated live promastigotes; and formalin-killed promastigotes treated with glucan (reviewed in, for example ref. 3). However, none of these approaches have provided efficacious vaccines.
Trypanosomatids, among many other metabolic peculiarities, maintain the redox balance of the cell by a mechanism that is completely different from that of their mammalian host. They lack glutathione reductase which in nearly all organisms is responsible for the maintenance of an intracellular reducing environment important for the reduction of disulphides, the detoxification of peroxides and the synthesis of DNA precursors (refs. 4 and 5). Instead, they possess a unique system using as a main thiols, the N2, N8-bis(glutathionyl)spermidine, also named trypanothione [T(SH)2] and the monoglutathionyl spermidine. These conjugates are kept in the reduced state by trypanothione reductase (TR) (refs. 4, 6 and 7). TR is a member of a NADPH-dependent flavoprotein oxidoreductase family and it is structurally and mechanistically related to the human glutathione reductase (GR) (refs. 6 and 8).
Leishmania, during its infective cycle, must survive the rigours of the host's oxidative phagocytic macrophages producing toxic oxygen intermediates. TR and thiols play a vital role in maintaining an intracellular reducing environment and in protecting these parasites against oxidative damage, arising both internally as a result of their aerobic metabolism and externally by the immune response of the mammalian host (refs. 4 and 6).
Leishmania infection may lead to serious disease. It would be advantageous to provide attenuated strains of Leishmania and methods of production thereof, for use as antigens in immunogenic preparations, including vaccines, and the generation of diagnostic reagents.
SUMMARY OF THE INVENTION
The present invention is directed towards the provision of attenuated strains of Leishmania. The attenuated strains are useful for the preparation of immunogenic preparations including vaccines against disease caused by infection by a virulent Leishmania strain and as tools for the generation of immunological and diagnostic reagents.
In accordance with one aspect of the present invention, there is provided an attenuated strain of Leishmania wherein at least one gene of the strain contributing to virulence thereof and expressed in both the promastigote and amastigote stages of the life cycle of the strain has been functionally disabled by, for example, a deletion of at least a portion of the gene or by mutagenesis.
In another aspect of the invention, there is provided an attenuated strain of Leishmania wherein both wild-type copies of a gene of the strain contributing to virulence thereof have been functionally disabled and a third copy of the wild-type gene is present in the chromosome of the strain. The gene contributing to the virulence of the strain in this aspect of the invention may be one expressed in both the promastigote and amastigote stages of the life cycle of the strain.
The gene may contribute to the ability of the strain to infect or survive. within macrophages and, in a particular embodiment, may encode trypanothione reductase. The attenuated Leishmania strain may be selected from the group consisting of
Leishmania donovani, Leishmania braziliensis, Leishmania tarentolae, Leishmania major, Leishmania mexicana, Leishmania tropica and Leishmania aethiopica.
In a further aspect, the present invention provides an immunogenic composition comprising the attenuated strains as provided herein. The immunogenic composition may be formulated as a vaccine for in vivo administration to a host, such as a primate including humans, to confer protection against disease caused by a virulent strain of Leishmania, including
Leishmania donovani, Leishmania braziliensis, Leishmania tarentolae, Leishmania major, Leishmania mexicana, Leishmania tropica and Leishmania aethiopica.
In an additional aspect, the invention provides a method of generating an immune response in a host, such as, a primate including humans, comprising administering thereto an immunoeffective amount of the immunogenic composition, as provided herein. In a particular aspect, the immunogenic composition may be formulated as a vaccine for in vivo administration to the host to confer protection against disease caused by a virulent strain of Leishmania, including
Leishmania donovani, Leishmania braziliensis, Leishmania tarentolae, Leishmania major, Leishmania mexicana, Leishmania tropica
and
Leishmania aethiopica.
In yet an additional aspect, there is provided a method for producing a vaccine for protection against a disease caused by infection by a virulent strain of Leishmania, including
Leishmania donovani, Leishmania braziliensis, Leishmania tarentolae, Leishmania mexicana, Leishmania tropica
and
Leishmania aethiopica
, and comprising administering the immunogenic composition as provided herein to a test host to determine an amount and frequency of administration thereof to confer protection against disease caused by infection by the Leishmania parasite and formulating the immunogenic composition in a form suitable for administration to a treated host, including humans, in accordance with said determined amount and frequency of administration.
In a further aspect of the invention, there is provided a method of forming an attenuated strain of Lei

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