Progress 07/01/05 to 09/30/10
Outputs
OUTPUTS: In this project we hypothesized that high affinity anti-C. parvum monoclonal antibodies which have been genetically linked to parasiticidal peptides or enzymes and expressed as recombinant fusions would provide novel, efficacious, and economically feasible products for controlling cryptosporidiosis in calves and other agriculturally important food animal species. Leading parasiticide candidates were expressed as recombinant parasiticide-monoclonal antibody fusion proteins and first evaluated for C. parvum neutralizing activity in vitro. Candidates which demonstrated significant in vitro activity were then evaluated for the ability to reduce intestinal infection in oocyst-challenged neonatal mice - an asymptomatic model of infection levels. These studies identified a subset of monoclonal antibody-parasiticide fusions having anti-cryptosporidial activity in vivo which exceeds that of the individual monoclonals or parasiticides, either alone or in combination, and at significantly lower (up to 50-fold) concentrations than either alone. These observations support the hypothesis that targeted delivery of a parasiticide to the zoite surface, via fusion to a monoclonal antibody, will confer greater neutralizing activity than either the monoclonal or parasiticide, either alone or in combination. The leading fusions identified in mouse studies were then produced in scale-up quantities sufficient to allow efficacy evaluation in neonatal piglets and calves, two clinical models of cryptosporidiosis which develop diarrheal disease. A summary of results of clinical trials is presented in the outcomes/impact section. PARTICIPANTS: PARTICIPANTS: INDIVIDUALS AT THE UNIVERSITY OF ARIZONA: M.W. Riggs, PI; D. A. Schaefer, Senior Research Specialist, Research Staff Collaborator. PARTNER ORGANIZATIONS AND COLLABORATORS: ioGenetics LLC, Madison, WI - M. Imboden, PI; E.J. Homan, Chief Executive Officer; R.D. Bremel, Chief Scientific Officer. TRAINING OR PROFESSIONAL DEVELOPMENT AT THE UNIVERSITY OF ARIZONA: T. Stehmer, pharmacy student; J. Parker, E. Butler, G. Roberts, A. Richert, H. Dineen, M. Cerise - undergraduate or veterinary students; S. Carryn, Post-Doctoral Fellow TARGET AUDIENCES: TARGET AUDIENCES: As described above for training or professional development: undergraduate and veterinary student research experiences; post-doctoral fellow training. Anti-cryptosporidial product development update for veterinarians involved in food animal practice. Private industry disclosures for further product development leading to commercialization. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Dosing trials for a lead recombinant parasiticide-monoclonal antibody fusion were performed in the piglet model using 1) enteric-coated capsules for delivery of a low dose, or 2) solution form for delivery of either low or high dosages. Significant efficacy was observed for one or more evaluation parameters at all dosages and formulations. However, the high dose trial demonstrated the greatest overall efficacy against C. parvum infection and clinical disease based on significant reductions in oocyst shedding and fecal volume, and multi-parameter clinical condition scores when compared to the placebo treated control group. Efficacy of the recombinant parasiticide-monoclonal antibody fusion protein was then assessed in neonatal calves challenged with Cryptosporidium parvum. Significant treatment effects in the fusion protein-treated calves were observed based on reductions in oocyst shedding and clinical scoring parameters. The collective results demonstrate that recombinant parasiticide-monoclonal antibody fusion proteins offer effective new anti-cryptosporidial candidates. Recombinant expression should ultimately make the resulting products economically feasible for agricultural applications. Finally, monoclonal antibody reactivity results identified phenotypic differences between C. hominis and C. parvum within two (glyco)proteins that are involved in parasite gliding motility and attachment/invasion. Such glycan and/or amino acid sequence polymorphisms may play a role in host specificity and will also be important to consider in designing anti-cryptosporidials which are effective against more than one Cryptosporidium species and in different mammalian host species.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: OUTPUTS COMPLETED TOWARDS PROJECT OBJECTIVES: In this project we hypothesize that high affinity anti-Cryptosporidium spp. monoclonal antibodies can be linked to anti-cryptosporidial peptides or enzymes and be expressed as recombinant fusions, and further, that such fusions will provide novel, efficacious, and economically feasible products for controlling cryptosporidiosis in agriculturally important food animal species. In the present reporting period, multiple additional recombinant parasiticide-monoclonal antibody fusion proteins against target epitopes were evaluated for C. parvum sporozoite neutralizing activity in vitro. All fusions which demonstrated significant in vitro activity were then evaluated for the ability to reduce infection in vivo using oocyst challenged neonatal mice, a non-clinical, asymptomatic model of infection. The leading fusions identified in mouse studies were then produced in quantities sufficient to allow efficacy evaluation in neonatal piglets and calves, two clinical models of cryptosporidiosis which develop diarrheal disease. A summary of results of clinical trials conducted during the reporting are presented in outcomes/impacts. DISSEMINATION OF RESULTS: 1) Scientific Presentations at International Meetings: a) The Immunobiology of Cryptosporidiosis, and b) Advances in the Development of Immunologic Control Strategies for Cryptosporidiosis in Calves, 2009 American College of Veterinary Internal Medicine - Canadian Veterinary Medical Association Joint Meeting, Food Animal Forum, Montreal, Quebec, June 4, 2009; and 2) New U.S. Patent Issued - Phospholipases and Cationic Peptide Preparations for Cryptosporidium. US CIP Patent Application Serial #11/254,500 filed 10/20/2005 was allowed and the patent was issued as US Patent #7566447 on 07/28/2009. Inventors: M.W. Riggs, D.A. Schaefer, S. Carryn, M. Imboden, and J. Homan. PARTICIPANTS: INDIVIDUALS AT THE UNIVERSITY OF ARIZONA: M.W. Riggs, PI; D. A. Schaefer, Senior Research Specialist, Research Staff Collaborator. PARTNER ORGANIZATIONS AND COLLABORATORS: ioGenetics LLC, Madison, WI - M. Imboden, PI; E.J. Homan, Chief Executive Officer; R.D. Bremel, Chief Scientific Officer. TRAINING OR PROFESSIONAL DEVELOPMENT AT THE UNIVERSITY OF ARIZONA: T. Stehmer, pharmacy student; G. Roberts, M. Cerise, A. Richert - undergraduate students. TARGET AUDIENCES: As described above for training or professional development: undergraduate student research experiences. Anti-cryptosporidial product development update for veterinarians involved in food animal practice. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Dosing trials for a lead recombinant parasiticide-monoclonal antibody fusion were performed in the piglet model using 1) enteric-coated capsules for delivery of a low dose, or 2) solution form for delivery of either low or high dosages. Significant efficacy was observed for one or more evaluation parameters at all dosages and formulations. However, the high dose trial demonstrated the greatest overall efficacy against C. parvum infection and clinical disease based on significant reductions in oocyst shedding and fecal volume, and multi-parameter clinical condition scores when compared to the placebo treated control group. Efficacy of the recombinant parasiticide-monoclonal antibody fusion protein was then assessed in neonatal calves infected with Cryptosporidium parvum. Significant treatment effects in the fusion protein-treated calves were observed based on reductions in oocyst shedding and clinical scoring parameters. The results demonstrate that recombinant parasiticide-monoclonal antibody fusion proteins offer effective new anti-cryptosporidial candidates.
Publications
- Riggs, M.W. 2009. The Immunobiology of Cryptosporidiosis. ACVIM Food Animal Internal Medicine Forum: 215-217. http://www.vin.com/acvim/2009.
- Riggs, M.W. 2009. Advances in the Development of Immunologic Control Strategies for Cryptosporidiosis in Calves. ACVIM Food Animal Internal Medicine Forum: 218-220. http://www.vin.com/acvim/2009.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: OUTPUTS COMPLETED TOWARDS PROJECT OBJECTIVES: In this project we hypothesize that high affinity anti-Cryptosporidium spp. monoclonal antibodies can be linked to anti-cryptosporidial peptides or enzymes and expressed as recombinant fusions, and further, that such fusions will provide novel, efficacious, and economically feasible products for controlling cryptosporidiosis in agriculturally important food animal species. In the present reporting period, multiple additional recombinant parasiticide-monoclonal antibody fusion proteins were evaluated for C. parvum sporozoite neutralizing activity in vitro. All fusions which demonstrated significant in vitro activity were then evaluated for the ability to reduce infection in vivo using a non-clinical, asymptomatic model of infection: oocyst-challenged neonatal mice. Two of the leading fusions identified in mouse studies were then produced in quantities sufficient to allow efficacy evaluation in clinical models of cryptosporidiosis which develop diarrheal disease: neonatal piglets and calves. DISSEMINATION OF RESULTS: 1) Scientific Presentations to Potential Corporate Partners (April 19-20, 2008): a) The Immunology of Protective Antigens in Cryptosporidiosis - Biology and Applications in Animal Health, M.W. Riggs, and b) Progress on Development of Antibody-Biocide Fusions to Control Cryptosporidium, D.A. Schaefer and M.W. Riggs; 2) New Patent Application Filing: U.S. serial #61/144,299. Targeted Cryptosporidium Biocides. Inventors: E.J. Homan, M. Imboden, M.W. Riggs, D.A. Schaefer; 3) Patents Pending: a) U.S. and Foreign Patent Applications PCT/US05/37709 and US Serial #11/254,500. Phospholipases and Cationic Peptide Biocides for Neutralization of Cryptosporidium Sporozoite Infectivity by Either Parasiticidal or Non-parasiticidal Mechanisms. Inventors: M.W. Riggs, M. Imboden, S. Carryn, and E.J. Homan, and b) U.S. and Foreign Patent Application PCT/US serial #11/545,601. Targeted Biocides. Inventors: M. Imboden, M.W. Riggs, and D.A. Schaefer. PARTICIPANTS: INDIVIDUALS AT THE UNIVERSITY OF ARIZONA: M.W. Riggs, PI; D. A. Schaefer, Senior Research Specialist, Research Staff Collaborator. PARTNER ORGANIZATIONS AND COLLABORATORS: ioGenetics LLC, Madison, WI - M. Imboden, PI; E.J. Homan, Chief Executive Officer; R.D. Bremel, Chief Scientific Officer. TRAINING OR PROFESSIONAL DEVELOPMENT AT THE UNIVERSITY OF ARIZONA: S. Nemeth, doctoral student; T. Stehmer, pharmacy student; J. Parker, E. Butler, G. Roberts, A. Richert, H. Dineen, M. Cerise - undergraduate students. TARGET AUDIENCES: As described above for training or professional development: undergraduate student research experiences. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
These studies have identified an expanded panel of monoclonal antibody-parasiticide fusions having anti-cryptosporidial activity in vitro and in the neonatal mouse model. The activity of fusions has been shown to exceed that of the individual monoclonals or parasiticides, either alone or in combination as separate molecules, when compared on an equimolar basis. These observations indicate that targeted delivery of a parasiticide to the parasite surface, via fusion to a monoclonal antibody, can confer greater neutralizing activity than either alone. Additional dose response trials in mice have demonstrated fusion efficacy at concentrations approximately 50-fold lower than the corresponding parasiticide or monoclonal antibody given as separate molecules. Efficacy against clinical disease caused by C. parvum was demonstrated for one of two fusions evaluated in the neonatal pig model based on reductions in fecal oocyst shedding, diarrhea, and systemic clinical signs. Pilot studies to evaluate this fusion in the neonatal calf model were initiated in the present reporting period. While the number of treated and control calves completed to date is too small to draw conclusions, a trend towards treatment efficacy has been observed based on clinical scores and fecal oocyst counts. Transgenic expression of fusions ultimately shown to have efficacy in clinical models of cryptosporidiosis is expected to make the resulting products economically feasible for agricultural applications.
Publications
- Sturbaum, G.D., Schaefer, D.A., Jost, B.H., Sterling, C.R. and M.W. Riggs. 2008. Antigenic differences in the Cryptosporidium hominis and Cryptosporidium parvum surface proteins P23 and GP900 defined by monoclonal antibody reactivity. Mol. Biochem. Parasitol. 159:138-141.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: EXPERIMENTAL ACTIVITIES: In this project we hypothesize that high affinity anti-Cryptosporidium spp. monoclonal antibodies which have been genetically linked to anti-cryptosporidial peptides or enzymes and expressed as recombinant fusions will provide novel, efficacious, and economically feasible products for controlling cryptosporidiosis in agriculturally important food animal species. In the present reporting period, additional recombinant parasiticide-monoclonal antibody fusion proteins were evaluated for C. parvum neutralizing activity in two in vitro assays to assess their efficacy in killing sporozoites and/or inhibiting sporozoite infectivity. Candidates which demonstrated significant in vitro activity were then evaluated for the ability to reduce intestinal infection in oocyst-challenged neonatal mice. Production of two of the leading candidates identified in these studies was scaled up to allow efficacy evaluation in the first of two clinical models of
cryptosporidiosis to be used - neonatal piglets. In addition, because the biological basis for the host specificity and infectivity patterns of some Cryptosporidium spp. has yet to be fully elucidated, experiments were performed using monoclonal antibodies developed against surface (glyco)proteins of C. parvum (Iowa) to determine reactivity with C. hominis isolates. PATENTS PENDING: 1) U.S. and Foreign Patent Applications PCT/US05/37709 and US Serial #11/254,500. Phospholipases and Cationic Peptide Biocides for Neutralization of Cryptosporidium Sporozoite Infectivity by Either Parasiticidal or Non-parasiticidal Mechanisms. Inventors: M.W. Riggs, S. Carryn, M. Imboden, and E.J. Homan. 2) U.S. and Foreign Patent Application PCT/US serial #11/545,601. Targeted Biocides. Inventors: M.W. Riggs, M. Imboden, and D.A. Schaefer. COLLABORATIONS: M.W. Riggs and D.A. Schaefer at the University of Arizona and M. Imboden, E.J. Homan, and R. Bremel at ioGenetics LLC (Madison, WI).
PARTICIPANTS: INDIVIDUALS AT THE UNIVERSITY OF ARIZONA: M.W. Riggs, PI; D. A. Schaefer, Senior Research Specialist, Research Staff Collaborator; A. C. Balchan, Research Technician. PARTNER ORGANIZATIONS AND COLLABORATORS: ioGenetics LLC, Madison, WI - M. Imboden, PI; E.J. Homan, Chief Executive Officer; R. D. Bremel, Chief Scientific Officer. TRAINING OR PROFESSIONAL DEVELOPMENT AT THE UNIVERSITY OF ARIZONA: S. Carryn, Post-Doctoral Fellow; S. Nemeth, doctoral student; T. Stehmer, undergraduate student; J. Parker, undergraduate student; E. Butler, undergraduate student.
Impacts
These studies have identified multiple additional monoclonal antibody-parasiticide fusions having anti-cryptosporidial activity in vitro and in the neonatal mouse model which exceeds that of the individual monoclonals or parasiticides, either alone or in combination. These observations now allow acceptance of the hypothesis that targeted delivery of a parasiticide to the zoite surface, via fusion to a monoclonal antibody, will confer greater neutralizing activity than either the monoclonal or parasiticide, either alone or in combination. More specifically, a dose response trial in mice demonstrated efficacy at concentrations as low as ~10 micrograms/ml for orally administered fusions. At concentrations up to ~100 micrograms/ml, some fusions demonstrated significantly greater activity against infection levels in mice compared to the corresponding parasiticide or monoclonal antibody given as separate molecules. Based on these observations in mice, clinical model studies
were initiated using neonatal pigs. Thus far, efficacy against clinical disease has been demonstrated for one of the fusions in neonatal pigs based on reductions in fecal oocyst shedding, diarrhea, and systemic clinical signs. Recombinant expression of this and other fusions showing efficacy in clinical models of cryptosporidiosis is expected to make the resulting products economically feasible for agricultural applications. Finally, monoclonal antibody reactivity results identified phenotypic differences between C. hominis and C. parvum within two (glyco)proteins that are involved in parasite gliding motility and attachment/invasion. Such glycan and/or amino acid sequence polymorphisms may play a role in host specificity and will also be important to consider in designing anti-cryptosporidials which are effective against more than one Cryptosporidium species.
Publications
- Sturbaum, G.D., Schaefer, D.A., Jost, B.H., Sterling, C.R., and Riggs, M.W. 2008. Antigenic differences within the Cryptosporidium hominis and Cryptosporidium parvum surface proteins P23 and GP900 defined by monoclonal antibody reactivity. Molecular and Biochemical Parasitology (In press).
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Progress 07/01/06 to 12/31/06
Outputs
In this project we hypothesize that high affinity anti-C. parvum monoclonal antibodies which have been genetically linked to parasiticidal peptides or enzymes and expressed as recombinant fusions will provide novel, efficacious, and economically feasible products for controlling cryptosporidiosis in calves. Two of the leading parasiticide candidates identified in the previous reporting period were expressed as recombinant parasiticide-monoclonal antibody fusion proteins and evaluated for C. parvum neutralizing activity. Seven different candidates were tested in two in vitro assays for their efficacy in killing sporozoites and/or inhibiting sporozoite infectivity. Candidates which demonstrated significant in vitro activity were then evaluated for the ability to reduce intestinal infection in oocyst-challenged neonatal mice. These studies have identified a subset of monoclonal antibody-parasiticide fusions having anti-cryptosporidial activity in vivo which exceeds that
of the individual monoclonals or parasiticides, either alone or in combination. These observations support the hypothesis that targeted delivery of a parasiticide to the zoite surface, via fusion to a monoclonal antibody, will confer greater neutralizing activity than either the monoclonal or parasiticide, either alone or in combination. Production of the leading candidates identified in these studies is currently being scaled up to allow efficacy evaluation in a clinical model of cryptosporidiosis.
Impacts
Fusion of selected parasiticidal peptides and enzymes to high affinity anti-C. parvum monoclonal antibodies is expected to allow targeted delivery to the parasite surface and result in superior neutralizing activity at theoretically lower concentrations than either parasiticide or monoclonal antibody alone. Recombinant expression is expected to make the resulting products economically feasible for agricultural applications.
Publications
- No publications reported this period
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