Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-06-14
2004-10-26
Navarro, Mark (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S320100, C435S325000, C536S023500
Reexamination Certificate
active
06808900
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to protozoan antigens and genes encoding the same. More particularly, the present invention pertains to the cloning, expression and characterization of polypeptides from
Cryptosporidium parvum
(
C. parum
) and antibodies directed to these polypeptides. The invention also pertains to use of the antigenic polypeptides and antibodies in therapeutic and diagnostic compositions.
BACKGROUND
Cryptosporidium parvum
(
C. parum
) is a coccidian protozoan that infects a wide variety of vertebrates, including humans.
C. parvum
can be acquired directly from animal-to-human contact, human-to-human contact or indirectly in fomites, water and sometimes food. Current et al. (1983)
N. Engl. J. Med.
308:1252-1257; Centers of Disease Control (1984) “Cryptosporidiosis among children attending day-care centers—Georgia, Pennsylvania, Michigan, California, New Mexico” 33:599-601; Wolfson et al. (1995)
N. Engl. J. Med.
312:1278-1281. Acquisition of infection occurs by ingestion of oocysts which excyst in the upper small bowel releasing four infective sporozoites. The sporozoites penetrate the lining enterocyte and undergo either sexual or asexual reproduction, gametogony and merogony, respectively. The products of either form of reproduction are capable of sustaining infection in man. Navin et al. (1984)
Rev. Infect. Dis.
6:313-327.
The reservoir of
C. parvum
is in wild and domestic animals, particularly cattle (Tzipori (1983)
Microbiol. Rev.
47:84-96) and this pathogen causes significant economic losses to the farm industry annually. Clinical manifestations of
C. parvum
infection may include watery diarrhea, crampy epigrastric abdominal pain, malabsorption of nutrients and weight loss, anorexia and malaise. The disease is usually self limited in the immunocompetent host but can be life threatening in the immunodeficient host, particularly in human patients with advanced Human Immunodeficiency Virus (HIV) infection. Current et al. (1983), supra; Wolfson et al. (1985)
N. Engl. J. Med.
312:1278-1281; Soave (1988)
Infect. Dis. Clin. N. Amer.
2:485.
The absence of an adequate in vitro culture system has severely limited the investigation of
C. parvum
. Furthermore, use of animal models, particularly mice, is of limited value because adult mice are not normally susceptible to infection with
C. parvum.
Several groups have reported the cloning and characterization of
C. parvum
antigens and genes using a variety of techniques. See, e.g., Jenkins et al. (1993)
Infect. Immun.
61:2377-2382; Jenkins et al. (1995)
Mol. Biochem. Parasitol.
71:149-152; Peterson et al. (1992)
Infect. Immun.
60:2343-2348; Ranucci et al. (1993)
Infect. Immun.
61:2347-2356.
After natural infection, a wide variety of cryptosporidial proteins are recognized by human and animal immune sera. Ortega-Mora et al. (1992)
Infect. Immun.
60:3442-3445; Campbell et al. (1983)
J. Clin. Microbiol.
18:165-169; Whitmire et al. (1991)
Infect. Immun.
59:990-995; Nina et al. (1992)
Infect. Immun.
60:1509-1513; Peeters et al. (1992)
Infect. Immun.
60:2309-2316. The components that constitute protective immunity are unknown although, like most obligate intracellular coccidian protozoa, both the cellular and humoral arms of the immune response are likely to play important roles in the genesis of protective immunity. Lillehoj et al. (1994)
Parasit. Today
10:10-16. Use of recombinant
C. parvum
proteins in vaccine compositions has also been described. See, e.g., U.S. Pat. No. 5,591,434. However, no consistently effective therapy of vaccination exists, perhaps because the antigens used in the vaccines were identified from non-human sources.
There have also been several attempts to identify antibodies that neutralize
C. parvum
infection. In human studies, orally administered hyperimmune bovine colostrum has been found to alter the natural history of
C. parvum
by decreasing the excretion of oocysts, reducing the level of diarrhea, and in a smaller number of cases, clearing the parasite from stool. Tzipori et al. (1986)
Br. Med. J.
293:1276-1277; Nord et al. (1989) “Treatment of AIDS associated cryptosporidiosis with hyperimmune colostrum from cows vaccinated with Cryptosporidium”, Fifth International Conference on AIDS, Montreal, Quebec, May 1989; Ungar et al. (1990)
Gastroenterology
98:486-489. Several antigens that are recognized by sera after natural infection have been characterized and have been shown to be the targets of neutralizing antibody in the murine system. Arrowood et al. (1989)
Infect. Immun.
57:2283-2288; Bjorneby et al. (1991)
Infect. Immun.
59:1172-1176; Doyle et al. (1993)
Infect. Immun.
61:4079-4084; Riggs et al. (1989)
J. Immunol.
143:1340-1345; Peterson et al. (1992)
Infect. Immun.
60:2343-2348. However, animal studies in the murine model of infection indicate only a partially protective role when these antibodies are orally administered.
Thus, there remains a need for the identification of antibodies useful in diagnostic and therapeutic compositions and for the identification of antigens from
C. parvum
that react with human sera and for diagnostic and therapeutic compositions and methods using these antigens.
Disclosure of the Invention
The present invention is based on the discovery of genes encoding
C. parvum
antigenic polypeptides, the characterization of these polypeptides and antibodies that recognize epitopes of these polypeptides. The proteins encoded by the genes have been recombinantly produced and these polypeptides, immunogenic fragments and analogs thereof, and/or chimeric proteins including the same, can be used, either alone or in combination with other
C. parvum
antigens, in novel subunit vaccines to provide protection from cryptosporidial infection in mammalian subjects. Antibodies generated against these proteins, and/or fragments thereof, either alone or in combination with other therapeutic agents, can be used in novel therapeutic agents for mammalian subjects. Furthermore, the antigens and antibodies can be used as diagnostics.
Accordingly, in one embodiment, the subject invention is directed to an isolated nucleic acid molecule comprising a coding sequence for an immunogenic
C. parvum
antigenic polypeptide selected from the group consisting of (a) a
C. parvum
antigenic polypeptide AG1 and (b) a
C. parvum
antigenic polypeptide AG2, or a fragment of the nucleic acid molecule comprising at least 15 nucleotides.
In additional embodiments, the invention is directed to recombinant vectors including the nucleic acid molecules, host cells transformed with these vectors, and methods of recombinantly producing
C. parvum
antigenic polypeptides.
In still further embodiments, the subject invention is directed to vaccine compositions comprising a pharmaceutically acceptable vehicle and an immunogenic
C. parvum
antigenic polypeptide selected from the group consisting of (a) a
C. parvum
antigenic polypeptide AG1, (b) a
C. parvum
antigenic polypeptide AG2 and (c) an immunogenic fragment of (a) or (b) comprising at least 5 amino acids, as well as methods of preparing the vaccine compositions.
In other embodiments, the invention is directed to therapeutic compositions comprising a pharmaceutically acceptable vehicle and an antibody (e.g., monoclonal antibody 1101 or 222) that recognize an immunogenic
C. parvum
antigenic polypeptide selected from the group consisting of (a) a
C. parvum
antigenic polypeptide AG1, (b) a
C. parvum
antigenic polypeptide AG2 and (c) an immunogenic fragment of (a) or (b) comprising at least 5 amino acids, as well as methods of preparing the therapeutic compositions.
In yet other embodiments, the present invention is directed to methods of treating or preventing
C. parvum
infections in a mammalian subject. The method comprises administering to the subject a therapeutically effective amount of the above vaccine or therapeutic compositions.
In additional embodiments, the invention is directed to methods of detecting
C. parvum
antibodies in a biological sample compri
Manitoba, University of
Navarro Mark
Robins & Pasternak LLP
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