Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Monoclonal antibody or fragment thereof
Reexamination Certificate
2000-04-25
2004-01-27
Chin, Christopher L. (Department: 1641)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Monoclonal antibody or fragment thereof
C424S157100, C424S535000, C424S807000, C435S007220, C435S070210, C435S329000, C435S342000, C530S388600, C530S389100, C530S822000, C530S832000
Reexamination Certificate
active
06682737
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to preparations useful for conferring passive or active immunity to the parasite,
Cryptosporidium parvum
. More specifically, the invention relates to a purified glycoprotein constituent of the parasite; use of the glycoprotein in an immunogenic composition; and monoclonal antibodies that bind a particular epitope disposed on the glycoprotein.
BACKGROUND OF THE INVENTION
Cryptosporidium parvum
is a coccidian parasite that causes intestinal disease in humans as well as economically important food animals including calves, lambs, and goat kids. Healthy, immunocompetent adult humans can be infected, but cryptosporidiosis is particularly serious when it occurs in immunodeficient individuals, including neonates, and those who are immunocompromised as a result of medical treatment, or because of other disease, such as infection by human immunodeficiency virus (HIV).
Among domestic animals, cryptosporidiosis is most frequently reported in calves. The ubiquity of
C. parvum
in dairy and beef operations throughout the U.S. and its importance as a cause of calf diarrhea are well documented. For example, Anderson et al. in
Vet. Med. Sm. Anim. Clin
. (June 1981) described well-managed, closed-herd dairies in which
C. parvum
-related morbidity in 1-2 week old calves approached 100%. We conservatively estimate that the combined treatment costs and decreased production losses incurred by the U.S. cattle industry due to cryptosporidiosis alone now exceed $50,000,000 each year.
C parvum
infection begins when sporozoites released from ingested oocysts invade intestinal epithelial cells. Following attachment of the anterior pole of sporozoites to intestinal epithelium, invasion is associated with host cell membrane evagination around the sporozoite and parasitophorous vacuole formation. Vacuole formation in the apical complex of invading sporozoites is thought to represent discharge of invasion mediators from apical organelles. Following invasion, a feeder organelle forms between the parasite and the host cell cytoplasm and increases the interface surface area markedly. This organelle may function in transport of materials between the host cell and developing trophozoite. Two stages of merogony follow trophozoite development. Type 1 merozoites undergo cyclic replication before developing into type 2 merozoites. Type 2 merozoites subsequently give rise to sexual stages. Fertilization follows and results in the production of oocysts which sporulate at the time of passage in feces. Autoinfective sporozoite and merozoite loops in the life cycle may perpetuate infection in immunocompromised hosts.
Since the first cases of human cryptosporidiosis were reported in 1976, Cryptosporidium has become recognized as a common cause of diarrhea in international travelers, children in day-care centers, livestock handlers, and patients with AIDS or other immune deficiency disorders. Among several recent studies that have addressed the prevalence of
C. parvum
infection in AIDS patients with diarrhea, one study identified Cryptosporidium as the most common enteropathogen in diarrheic AIDS patients (Laughon et al.
Gastroenterol
. 94:984 (1988)). Dissemination to extraintestinal sites such as the esophagus, lungs, pancreas and liver has also been shown to occur in immune deficient patients (Soave et al.
Rev. Inf Dis
. 8:1012 (1986); Ungar et al. “Cryptosporidiosis in Humans” pp. 67-75, in J P Dubey, C A Speer and R Fayer (eds.),
Cryptosporidiosis of Man and Animals
, CRC Press (1990)).
Unlike the other major causes of diarrhea, including infection by
E. coli
, rotavirus and coronavirus, there are no effective control measures available for cryptosporidiosis. Despite the evaluation of more than 90 drugs, none has been of consistent value and no immunization regimen is presently available to protect against infection by
C parvum
. Control of
C. parvum
infection therefore depends on achieving an adequate immune response. An index of adequate response comprises resistance to reinfection following recovery and short-term disease in immunocompetent hosts. The disease may persist however in immunodeficient hosts.
While cell-mediated immunity is important to naturally occurring resistance to many coccidial species, the evidence suggests that antibody responses can also be manipulated to control infection with, sporozoan parasites. For example, hyperimmune bovine serum or colostral whey against whole
C. parvum
neutralized sporozoite infectivity and partially protected mice and calves against oocyst challenge. Additionally, oral administration of hyperimmune bovine colostrum to persistently infected immunodeficient patients was followed by cessation of diarrhea and oocyst shedding. Further, monoclonal antibodies (mAbs) reactive with
C parvum
sporozoite and merozoite surface epitopes neutralized their infectivity and partially protected mice against oocyst challenge.
SUMMARY OF THE INVENTION
According to a first aspect of the invention there is provided a monoclonal antibody having the epitope binding specificity of antibody 3E2. In one embodiment, the monoclonal antibody and antibody 3E2 compete with each other for binding to an antigen that is present in a preparation solubilized
C. parvum
sporozoites. This monoclonal antibody, which competes with antibody 3E2 for antigen binding, stimulates a CSP-like reaction after contacting
C. parvum
sporozoites. According to a different embodiment, the monoclonal antibody can have an IgM isotype. In a particular case, the monoclonal antibody is antibody 3E2. The invention also provides a hybridoma that secretes the monoclonal antibody 3E2.
According to a second aspect of the invention there is provided a pharmaceutical composition for administration to a mammal, comprising a monoclonal antibody secreted by hybridoma 3E2 and a pharmaceutically acceptable carrier. The invented composition also can include at least one monoclonal antibody other than the monoclonal antibody secreted by hybridoma 3E2. In a preferred embodiment, this other monoclonal antibody has an epitope binding specificity different from the binding specificity of the monoclonal antibody secreted by hybridoma 3E2. The carrier of the pharmaceutical composition can include at least one stabilizing agent which may be a protease inhibitor, a carrier protein or a pH buffering agent. In one embodiment, the carrier optionally comprises colostrum, for example, bovine colostrum.
A third aspect of the invention relates to a method of providing to a mammal passive immunity against
C parvum
infection, comprising the step of administering to the mammal a composition comprising antibody 3E2, thereby providing passive immunity. In one embodiment, the composition administered to the mammal comprises a
C. parvum
neutralizing amount of monoclonal antibody 3E2. According to a different embodiment, the administered composition includes at least one monoclonal antibody other than antibody 3E2 that specifically binds a
C. parvum
antigen. In a preferred method the composition comprising antibody 3E2 is administered orally in the administering step. In another preferred embodiment of the method, the mammal is a human and in a particularly preferred embodiment the mammal is an immunocompromised human.
A fourth aspect of the invention relates to an isolated circumsporozoite-like antigen of
C parvum
which includes a glycoprotein having a molecular weight of 1,400 kDa and which harbors an epitope specifically recognizable by monoclonal antibody 3E2. The isolated antigen can be isolated by a method that includes centrifugation, more particularly, density gradient centrifugation. Alternatively, the isolated antigen can be isolated by methods that involve immunoprecipitation, isoelectric focusing or preparative polyacrylarnide gel electrophoresis.
A fifth aspect of the invention relates to an immunogenic composition which includes: (a) a substantially purified
C. parvum
antigen specifically recognizable by monoclonal antibody 3E2; and (b) a pharmaceutically acceptabl
Perryman Lance E.
Riggs Michael W.
Chin Christopher L.
Grun James L.
North Carolina State University
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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