This proposal is the second revised competing renewal for continuation of NIH Award #2 R01-A1-41326. Our objective is to develop an immunotherapeutic formulation for the treatment and prevention of complications associated with Stx-producing Escherichia coli (STEC) infections. Clinical isolates of STEC are known to predominantly produce Stxl, Stx2, and/or Stx2c. Children are particularly susceptible to development of Stx-mediated HUS. Our hypothesis is that administration of Stx-specific antibodies will prevent or modify the outcome of infection for individuals at risk of developing HUS. In the earlier awards, we have generated a panel of human monoclonal antibodies (Hu-mAbs) specific for Stx 1 or Stx2. Using the gnotobiotic piglet model, we have shown that Stx-specific Hu-mAbs neutralize Stx and prevent development of Stx-mediated complications. We now wish to determine which Hu-mAbs should be included in a formulation suitable for clinical evaluation. Based on superior efficacy, four Hu-mAbs specific for Stx2 (3 against the A subunit and 1 against the B subunit), and 2 for Stxl (both against B subunit) have been selected as candidates. The next step is to determine which combination of Hu-mAbs, is both compatible and highly effective. In this proposal we plan to define the structural and functional characteristics, which facilitate protective efficacy of Stx-1 and Stx2-specific Hu-mAbs (Specific Aim 1). Affinity and efficacy of each HumAb will then be studied against their respective toxin (Specific Aim 2). The efficacy of protection of a given antibody dose will then be determined in terms of time after bacterial infection (Specific Aim 3). Finally, combinations of Hu-mAbs specific for B subunit of Stx 1 and A or B subunits of Stx2, will be examined for relative efficacy and compatibility, to determine which is the most effective and thus suitable for clinical evaluation (Specific Aim 4). At the conclusion of these experiments we will have determined the components, and optimized the formulation of Hu-mAbs which will be recommended for testing in human patients. The Hu-mAbs will first be characterized and ranked according to their efficacy, affinity and compatibility with each other. The optimal amount of each Hu-mAb in the formulation required to provide the longest protection after bacterial infection will also be established. This is not a hypothesis-driven proposal, but an essential segment for the characterization of a promising therapy for HUS, against which currently there is no effective treatment.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI041326-07
Application #
6796701
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Schmitt, Clare K
Project Start
1996-09-23
Project End
2008-02-29
Budget Start
2004-03-01
Budget End
2005-02-28
Support Year
7
Fiscal Year
2004
Total Cost
$356,625
Indirect Cost
Name
Tufts University
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
039318308
City
Boston
State
MA
Country
United States
Zip Code
02111
Akiyoshi, Donna E; Sheoran, Abhineet S; Rich, Curtis M et al. (2010) Evaluation of Fab and F(ab')2 fragments and isotype variants of a recombinant human monoclonal antibody against Shiga toxin 2. Infect Immun 78:1376-82
Jeong, Kwang-Il; Tzipori, Saul; Sheoran, Abhineet S (2010) Shiga toxin 2-specific but not shiga toxin 1-specific human monoclonal antibody protects piglets challenged with enterohemorrhagic Escherichia coli producing shiga toxin 1 and shiga toxin 2. J Infect Dis 201:1081-3
Krautz-Peterson, Greice; Chapman-Bonofiglio, Susan; Boisvert, Karen et al. (2008) Intracellular neutralization of shiga toxin 2 by an a subunit-specific human monoclonal antibody. Infect Immun 76:1931-9
Sheoran, Abhineet S; Chapman-Bonofiglio, Susan; Harvey, Barrett R et al. (2005) Human antibody against shiga toxin 2 administered to piglets after the onset of diarrhea due to Escherichia coli O157:H7 prevents fatal systemic complications. Infect Immun 73:4607-13
Akiyoshi, D E; Rich, C M; O'Sullivan-Murphy, S et al. (2005) Characterization of a human monoclonal antibody against Shiga toxin 2 expressed in Chinese hamster ovary cells. Infect Immun 73:4054-61
Tzipori, Saul; Sheoran, Abhineet; Akiyoshi, Donna et al. (2004) Antibody therapy in the management of shiga toxin-induced hemolytic uremic syndrome. Clin Microbiol Rev 17:926-41, table of contents
Sheoran, Abhineet S; Chapman, Susan; Singh, Pradeep et al. (2003) Stx2-specific human monoclonal antibodies protect mice against lethal infection with Escherichia coli expressing Stx2 variants. Infect Immun 71:3125-30
Mukherjee, Jean; Chios, Kerry; Fishwild, Dianne et al. (2002) Production and characterization of protective human antibodies against Shiga toxin 1. Infect Immun 70:5896-9
Mukherjee, Jean; Chios, Kerry; Fishwild, Dianne et al. (2002) Human Stx2-specific monoclonal antibodies prevent systemic complications of Escherichia coli O157:H7 infection. Infect Immun 70:612-9
Donohue-Rolfe, A; Kondova, I; Mukherjee, J et al. (1999) Antibody-based protection of gnotobiotic piglets infected with Escherichia coli O157:H7 against systemic complications associated with Shiga toxin 2. Infect Immun 67:3645-8