Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Parasitic organism or component thereof or substance...
Reexamination Certificate
1996-09-13
2001-12-18
Kunz, Gary L. (Department: 1647)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Parasitic organism or component thereof or substance...
C424S085100, C435S258300
Reexamination Certificate
active
06331304
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the field of molecular immunobiology and in particular to immunogenic preparations including vaccines comprising attenuated
BACKGROUND OF INVENTION
Parasite infection is responsible for a number of human and animal diseases.
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 suabsequently 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 forrmalin-killed promastigotes treated with glucan (reviewed in, for example ref. 3). However, none of these approaches have provided efficacious vaccines.
Healing and progression of leishmaniasis are linked to the dissimilar expansion of functionally distinct CD
4
+ lymphocyte responses separated on the basis of their cytokine potential (ref. 4). T helper type 1 (Th1) subset produces interferon (IFN)-, &ggr; and interleukin (IL)-2 and leads to resistance to Leishmania infection, whereas Th2 cells producing IL-4, IL-5 and IL-10 confer susceptibility (ref. 5). In mammalian hosts, Leishmania 35 reside exclusively within mononuclear phagocytes, macrophages and mnonocytes. Cytokines can modulate macrophage differentiation by causing selective changes in macrophage gene expression, leading to alterations on macrophage functions (ref. 6). Macrophages, pre-incubated in vitro with cytokines prior to infection with Leishmania, acquire the capacity to kill the intracellular parasites (refs. 7 to il). Furthermore, cytokines such as IFN-&ggr;, TNF-&agr;, IL-12 and GM-CSF have been used in anti-leishmanial therapy in experimental models (refs. 12 to 17). Expression of the IFN-&ggr;gene has been performed in L. major trypanosomatids. When nude mice were infected with the IFN-&ggr; expressing transfectant, the progression of the disease was considerably slower (ref. 17). The progression of the disease was not retarded in susceptible BALB/c mice however.
GM-CSF is a cytokine with multipontential hetnatopoietic function, stimulating the formation of granulocytes, macrophages, and eosinophils (ref. 18). It activates microphage tumoricidal activity (ref. 19), increases macrophage killing of
Trypanosorna cruzi
(ref. 20) and enhances in vitro killing of
L. donovani
within macrophages (ref. 8).
Differences on the effect of this cytokine have been reported between experimental visceral and cutaneous infections (ref. 27). Although in the
L. donovani
model, GM-CSF demonstrates a clear-cut leishmanicidal activity in vitro and in vivo (refs. 28), the results obtained with
L. major
are conflicting suggesting that GM-CSF may play a positive (refs. 29, 10, 6), neutral (refs. 30, 4) or negative (ref. 31) host defense role.
Parasitic infection of macrophages and, in particular, 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 novel strains of eishmania and other macrophage-infecting parasites, particularly attenuated strains. The novel macrophage-infecting parasites provided herein are useful for the preparation of immunogenic preparations including formulaations for the treatment of hosts infected by Leishmania and 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 a macrophage-infecting parasite expressing a granulocyte macrophage colony stimulating factor (GM-CSF) gene. The parasite may be a strain of Leishmania, including a strain of Leishmania selected from the group consisting of
Leishmania donovani, Leishmania braziliensis, Leishmania tarentolae, Leishmania major, Leishmania mexicana, Leishmania tropica
and
Leishmania aethiopica.
The parasite may be one which is reduced in the ability of the strain to infect or survive in macrophages and hence is attenuated. Further, at least one gene of the parasite contributing to virulence thereof may be functionally disabled. Additionally, the parasite may be further modified to express at least one additional cytokine which may be macrophage-activating.
The GM-CSF may be of murine origin or human origin. Expression of the GM-CSF gene from the parasite may be achieved by providing a plasmid into which the GM-CSF gene is inserted downstream of a promoter. For Leishmania, the intergenic region of the &agr;-tubulin gene of
L. enriettii
may be used and the GM-CSF gene may be inserted into a Leishmania expression vector, which may be a plasmid.
In a further aspect, the present invention provides an immunogenic composition comprising an attenuated form of the parasites as provided herein.
The immnunogenic composition, for the parasite being a strain of Leishmania may be formulated for in vivo administration to a host, such as a primate, including humans, infected by Leishmania to treat such infection.
The immunogenic composition, for the parasite being a strain of Leishmania, 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
Olivier Martin
Ouellette Marc
Papadopoulou Barbara
Gucker Stephen
Kunz Gary L.
Sim & McBurney
Universite Laval
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