Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Binds antigen or epitope whose amino acid sequence is...
Reexamination Certificate
2000-06-20
2004-07-06
Kunz, Gary (Department: 1647)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Binds antigen or epitope whose amino acid sequence is...
C424S141100, C424S164100
Reexamination Certificate
active
06759044
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention concerns vaccines, antibodies, proteins, DNAs and RNAs for diagnosis, prophylaxis and treatment of Cryptosporidium species infections and for detection of Cryptosporidium species. In particular, this invention concerns Cryptosporidium species antigen comprised of a protein, as well as polyclonal and monoclonal antibodies directed against the antigen, DNAs and RNA encoding the Cryptosporidium species antigen and fragments and analogs thereof, and methods for production of recombinant or fusion proteins. This invention also concerns methods for diagnosis, prophylaxis, treatment of Cryptosporidium infections and detection of Cryptosporidium species.
2. Background and Related Disclosures
The genus Cryptosporidium consists of Apicomplexan parasites that invade and develop within epithelial cells of the gastrointestinal, hepatobiliary and respiratory tracts of a wide variety of vertebrates including reptiles, birds and mammals. Cryptosporidium was recognized as a cause of animal disease for several decades before the first cases of human cryptosporidiosis were reported in 1976. However, it was not until 1982 that the magnitude of disease caused by this parasite in both AIDS patients and immunocompetent hosts began to be appreciated. Subsequently, Cryptosporidium has been found to be one of the most common causes of human diarrhea worldwide, and to be an increasingly recognized cause of diarrhea in children, animal care workers, and travelers. (
Cryptosporidium and Cryptosporidiosis in Humans
, Ed. Fayer, R., CRC Press, Boca Raton (1997)).
Large waterborne outbreaks of cryptosporidiosis caused by contaminated municipal water supplies in the US or in the UK have been noted in the last ten years (
N. Enal. J. Med
., 320:1372 (1989), and 33:161 (1994)). The most recent outbreak in Milwaukee in April 1993 involved 400,000 persons and led to the subsequent deaths of more than 100 immunocompromised persons. Like a number of other waterborne outbreaks, the Milwaukee outbreak appears to have been due to contamination from farm or abattoir run-off and specifically to cryptosporidiosis among cows/calves. Nosocomial transmission in hospitals from patients to staff, patient to patient, and contaminated ice to patients and staff have also been well documented (
J. Infect. Dis
., 158:647 (1985)).
Waterborne and nosocomial spread uncovered a number of biological characteristics of oocysts. First, the infectious dose of a parasite is very low. The ID
50
for human volunteers with normal immune systems is 132 oocysts
N. Engl. J. Med
., 332:855 (1995). Second, infected hosts, for example calves, excrete large numbers of oocysts, on the order of 10
10
/day. Third, the oocysts are fully sporulated and ready to infect when excreted. Fourth, the oocysts are environmentally hardy. They remain infectious in cool, moist areas for 3-4 months. They are not killed by chlorine levels achievable in drinking water. Fifth, the oocysts are quite small, 4-6 &mgr;m, and are thus difficult to filter.
The clinical importance of cryptosporidiosis has increased markedly with the recognition of a life-threatening form of the disease in patients with immunodeficiency disorders such as AIDS, hypogammaglobulinaemia, and chemotherapeutic immunosuppression. The prevalence of cryptosporidiosis in AIDS patients in the US is estimated to be 5-10% and in central Africa 40%. Immunodeficient patients may have fulminant cryptosporidial diarrhea that may persist until death, whereas the diarrhea of immunocompetent patients is self-limited and rarely lasts more than 2-4 weeks. Cholera-like diarrhea is common in immunocompromised patients with reported losses of up to 17 liters per day. Hepatobiliary disease may result in severe abdominal pain and nausea. Removal of immunosuppression in chemotherapy patients leads to resolution of the diarrhea. Some AIDS patients with cryptosporidiosis will be able to eliminate the parasite by induction of anti-retroviral therapy (
Am. Intern. Med
., 116:840 (1992)).
Among those who develop disease, a quarter have CD4 counts greater than 209, suggesting that the disease may occur relatively early in the course of HIV disease (
Am. J. Epidemiol
., 144:807 (1996). Unfortunately, few details about the biology and molecular mediators of the disease process have been described and so far no effective therapy has been discovered.
The infective forms of Cryptosporidium, called sporozoites and merozoites, appear to adhere to the host cell and release the contents of anterior organelles (rhoptries, micronemes or dense granules) during the invasion process (
Parasitol. Today
, 8:28(1992)). Proteins involved in these events have in many instances been found to be the target of invasion blocking immunity in vitro and neutralization in vivo (
Infect. Immun
., 56:2538(1988)).
Active and passive immunization studies using malaria and Toxoplasma challenged or infected hosts have shown that certain secreted components of the apical complex organelles are the target of protective antibodies. In some cases, as for example in the case of the circumsporozoite and merozoite surface proteins of malaria, these antigens are under development as vaccines.
While the actual interaction between cryptosporidium and the host's immune system is poorly understood, it is known that disruption of either the cellular or the humoral components can result in protracted cryptosporidiosis (
Parasitol. Today
, 8:24 (1992)). However, specific antibodies alone appear to be enough to neutralize the organism's infectivity. In vitro and in vivo observations indicate that antibodies to
Cryptosporidium parvum
inhibit invasion and intracellular development leading to protection in challenge experiments, or amelioration of infection in established disease (
Infect. Immun
., 59:1172 (1991)).
One source of such antibodies is hyperimmune bovine colostrum (HBC) collected from cows immunized with Cryptosporidium oocysts. Calves challenged with Cryptosporidium oocysts are protected from the development of disease by the administration of HBC (
Infect. Immun
., 61:4079 (1993)). Some immunocompromised AIDS patients infected with Cryptosporidium have also responded to HBC with a reduction in or disappearance of the symptoms of the disease (
Gastroenterology
, 98:486 (1990)). Immunoglobulin from HBC (HBC Ig) has been found to inhibit the ability of the sporozoite to invade and/or develop intracellularly in vitro and it has been used to immunoprecipitate at least 22 different surface radioiodinated proteins of Cryptosporidium sporozoites. Western blot analysis of proteins of whole oocysts which contain sporozoite, indicates that HBC predominantly recognizes two proteins of sizes 250 Kd and >900 Kd (
Infect. Immun
., 61:4079 (1993)).
The use of HBC for human use is problematic. HBC produced using whole oocysts is batch dependent and this may lead to the development of passive immune preparations which are not uniform in immunogenicity and potency. This generates a problem when these immune preparation are to be administered to human patients as such non-uniformity may result in failure of protection. In addition, it would be desirable to allow preparation of large amounts of antigen expressed in heterologous systems than to purify oocyst.
Thus, there is a continuous need for immunogenic agents which are reasonably reproducible and have uniform and controllable immunogenicity and potency which agents would be useful for the immunotherapy of cryptosporidiosis in both uncompromised and immunocompromised subjects, such as AIDS patients, and would allow the prophylaxis and treatment of cryptosporidiosis.
Additionally, there is a need to have available methods for reproducible expression of specific target for Cryptosporidium antigen in large amounts, which antigen would provide a better immunogen. This approach requires that a specific Cryptosporidium antigen is cloned and identified as a potential candidate through its ability to elicit an antibody response that is immunoprotective. B
Huang Jin-Xing
Petersen Carolyn
Gucker Stephen
Kunz Gary
Peters, Verny, Jones & Schmitt, LLP
Verny Hana
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