The present invention relates to parasitic nematode transglutaminase nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins, and inhibitors of such proteins. The present invention also includes therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies, inhibitors, and combinations thereof, as well as the use of these compositions to protect animals from diseases caused by parasitic nematodes.
Parasitic nematode infections in animals, including humans, are typically treated by chemical drugs. One disadvantage with chemical drugs is that they must be administered often. For example, dogs susceptible to heartworm are typically treated monthly. Repeated administration of drugs, however, often leads to the development of resistant nematode strains that no longer respond to treatment. Furthermore, many of the chemical drugs cause harmful side effects in the animals being treated, and as larger doses become required due to the build up of resistance, the side effects become even greater. Moreover, a number of drugs only treat symptoms of a parasitic disease but are unable to prevent infection by the parasitic nematode.
An alternative method to prevent parasitic nematode infection includes administering a vaccine against a parasitic nematode. Although many investigators have tried to develop vaccines based on specific antigens, it is well understood that the ability of an antigen to stimulate antibody production does not necessarily correlate with the ability of the antigen to stimulate an immune response capable of protecting an animal from infection, particularly in the case of parasitic nematodes. Although a number of prominent antigens have been identified in several parasitic nematodes, including in Dirofilaria, there is yet to be a commercially available vaccine developed for any parasitic nematode.
The life cycle of parasitic nematodes generally includes development through four molts, the last two molts taking place in the host animal. Molting is a complex process involving a variety of different mechanisms. However, a lack of understanding of the basic biology, metabolism and biochemistry of parasitic nematodes has resulted in the identification of few targets for chemotherapy or vaccines.
As an example of the complexity of parasitic nematodes, the life cycle of D. immitis, the nematode that causes heartworm, includes a variety of life forms, each of which presents different targets, and challenges, for immunization. Adult forms of the parasite are quite large and preferentially inhabit the heart and pulmonary arteries of an animal. Sexually mature adults, after mating, produce microfilariae which traverse capillary beds and circulate in the vascular system of the dog. One method of demonstrating infection in the dog is to detect the circulating microfilariae. If a dog is maintained in an insect-free environment, the life cycle of the parasite cannot progress. However, when microfilariae are ingested by a female mosquito during blood feeding on an infected dog, subsequent development of the microfilariae into larvae occurs in the mosquito. The microfilariae go through two larval stages (L1 and L2) and finally become mature third stage larvae (L3) which can then be transmitted back to the dog through the bite of the mosquito. It is this L3 stage, therefore, that accounts for the initial infection. As early as three days after infection, the L3 molt to the fourth larval (L4) stage, and subsequently to the fifth stage, or immature adults. The immature adults migrate to the heart and pulmonary arteries, where they mature and reproduce, thus producing the microfilariae in the blood. xe2x80x9cOccultxe2x80x9d infection with heartworm in dogs is defined as that wherein no microfilariae can be detected, but the existence of the adult heartworms can be determined through thoracic examination.
Heartworm not only is a major problem in dogs, which typically cannot even develop immunity upon infection (i.e., dogs can become reinfected even after being cured by chemotherapy), but is also becoming increasingly widespread in other companion animals, such as cats and ferrets. Heartworm infections have also been reported in humans. Other parasitic nematodeic infections are also widespread, and all require better treatment, including a preventative vaccine program. O. volvulus, for example, causes onchocerciasis (also known as river blindness) in humans. Up to 50 million people throughout the world are reported to be infected with O. volvulus, with over a million being blinded due to infection.
Although many investigators have tried to develop vaccines based on specific antigens, it is well understood that the ability of an antigen to stimulate antibody production does not necessarily correlate with the ability of the antigen to stimulate an immune response capable of protecting an animal from infection, particularly in the case of parasitic nematodes. Although a number of prominent antigens have been identified in several parasitic nematodes, including in Dirofilaria and Onchocerca, there is yet to be an effective vaccine developed for any parasitic nematode.
In just the past few years, there has developed an interest in the identification of larval stage-specific enzymes as potential targets for treatment or prevention of nematode diseases. Nematode transglutaminase-catalyzed reactions have recently been identified as possibly important for the growth, development and survival of nematodes, including Acanthocheilonema vitae, Brugia malayi, and Onchocerca volvulus. See, for example, Mehta, 1992, Mol. Biochem. Parasitol., 53, 1-16; Lustigman, 1995, Antimicrobial Agents and Chemother., 39:9, 1913-1919; Lustigman, 1993, Parasitology Today, 9:8, 294-297. However, until now, no compounds or methods based on specific known targets in parasitic nematode development have been designed for treating or preventing parasitic nematode disease.
There remains a need to identify an efficacious composition that protects animals against diseases caused by parasitic nematodes and that, preferably, also protects animals from infection by such nematodes.
The present invention relates to novel products and processes for prevention and treatment of parasitic nematode infection. According to the present invention there are provided parasitic nematode transglutaminase proteins and mimetopes thereof; nematode nucleic acid molecules, including those that encode such proteins; antibodies raised against parasitic nematode transglutaminase proteins (i.e., anti-parasitic nematode transglutaminase antibodies); and other compounds that inhibit parasitic nematode transglutaminase activity (i.e, inhibitory compounds or inhibitors).
The present invention also includes methods to obtain the proteins, mimetopes, nucleic acid molecules, antibodies and inhibitory compounds herein described. Also included in the present invention are therapeutic compositions comprising such proteins, mimetopes, nucleic acid molecules, antibodies, inhibitory compounds, or mixtures thereof, as well as the use of such therapeutic compositions to protect animals from diseases caused by parasitic nematodes.
One embodiment of the present invention is an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a parasitic nematode transglutaminase gene. Preferred parasitic nematode transglutaminase genes of the present invention are transglutaminase genes from Brugia malayi, Dirofilaria immitis, and Onchocerca volvulus. Such nucleic acid molecules are referred to as nematode transglutaminase nucleic acid molecules. A parasitic nematode transglutaminase gene preferably includes at least one of the following nucleic acid sequences: SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or a nucleic acid sequence encoding either the nucleic acid molecule herein designated nOvTG542., or the nucleic acid molecule herein designated nBmTG542.
Another embodiment of the present invention is an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a Dirofilaria immitis (D. immitis) transglutaminase gene; such nucleic acid molecules are referred to as Dirofilaria immitis transglutaminase nucleic acid molecules. A D. immitis transglutaminase gene preferably includes the following nucleic acid sequences: SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.
The present invention also relates to recombinant molecules, recombinant viruses and recombinant cells that include a transglutaminase nucleic acid molecule of the present invention. Also included are methods to produce such nucleic acid molecules, recombinant molecules, recombinant viruses and recombinant cells.
Another embodiment of the present invention includes a Dirofilaria immitis transglutaminase protein, or a protein that includes a Dirofilaria immitis transglutaminase protein. A preferred Dirofilaria immitis transglutaminase protein is capable of eliciting an immune response when administered to an animal and/or of having parasitic nematode transglutaminase activity. A preferred Dirofilaria immitis transglutaminase protein is encoded by a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid molecule including either SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, or SEQ ID NO:14. A preferred Dirofilaria immitis transglutaminase protein includes at least a portion of a protein represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:11.
The present invention also relates to mimetopes of parasitic nematode transglutaminase proteins as well as to isolated antibodies that selectively bind to parasitic nematode transglutaminase proteins or mimetopes thereof. Also included are methods, including recombinant methods, to produce proteins, mimetopes and antibodies of the present invention.
Another embodiment of the present invention is a method to identify a compound capable of inhibiting nematode transglutaminase activity. The method includes the steps of: (a) contacting an isolated nematode transglutaminase protein with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has nematode transglutaminase activity; and (b) determining if the putative inhibitory compound inhibits the nematode transglutaminase activity. Also included in the present invention is a test kit to identify a compound capable of inhibiting nematode transglutaminase activity. Such a test kit includes an isolated nematode transglutaminase protein having nematode transglutaminase activity and a means for determining the extent of inhibition of the nematode transglutaminase activity in the presence of a putative inhibitory compound.
The present invention also includes an inhibitor of nematode transglutaminase activity identified by its ability to inhibit the activity of a nematode transglutaminase and by its inability to substantially inhibit mammalian transglutaminase. Examples of such inhibitors are substrate analogs of nematode transglutaminase, active site inhibitors of nematode transglutaminase, and antibodies that specifically recognize nematode transglutaminase.
Yet another embodiment of the present invention is a therapeutic composition that is capable of protecting an animal from disease caused by a parasitic nematode. Such a therapeutic composition includes an excipient and one or more of the following protective compounds: an isolated nematode transglutaminase protein or a mimetope thereof; an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a nematode transglutaminase gene; an isolated antibody that selectively binds to a nematode transglutaminase protein; an inhibitor of nematode transglutaminase protein activity identified by its ability to (a) inhibit nematode transglutaminase activity, and (b) not substantially inhibit mammalian transglutaminase activity; or any combinations thereof. A preferred therapeutic composition of the present invention also includes an adjuvant, a carrier, or both. Preferred nematode transglutaminase nucleic acid molecule compounds of the present invention include naked nucleic acid vaccines, recombinant virus vaccines and recombinant cell vaccines. Also included in the present invention is a method to protect an animal from disease caused by a parasitic nematode comprising the step of administering to the animal at least one protective compound of the present invention.
Yet another embodiment of the present invention is a method to produce a transglutaminase protein, the method comprising culturing a cell transformed with a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nematode transglutaminase gene.