630 Comparison of a Recombinant Endotoxin-Neutralizing Protein with a Human Monoclonal Antibody to Endotoxin for the Treatment of Escherichia coli Sepsis in Rats Nathan Kuppermann, Douglas S. Nelson, Richard A. Saladino, Claudette M. Thompson, Felicia Sattler, Thomas J. Novitsky, Gary R. Fleisher, and George R. Siber Division of Emergency Medicine. Department of Medicine. Children s Hospital. and Laboratory of Infectious Diseases. Dana-Farber Cancer Institute. Harvard Medical School. Boston. and Associates of Cape Cod. Inc.. Woods Hole. Massachusetts A recombinant endotoxin-neutralizing protein (ENP) from Limuluspolyphemus and a mono- clonal IgM anti-lipid A antibody (HA-IA) were compared in a rat model of Escherichia coli sepsis. One hour after intraperitoneal challenge with 10 6 cfu of E. coli 0 18ac K I, animals were sensitized to endotoxin with lead acetate and treated with ENP, HA-IA, or saline, followed by ceftriaxone and gentamicin. Before treatment, 95% of rats had high-grade bacteremia and high serum endotoxin concentrations, which were similar in all treatment groups (P > .60). One hour after treatment, there was no bacterial growth in any blood sample, and endotoxin concentra- tions were significantly lower in the ENP group than in the HA-IA and saline groups (P < .01). At 24 h after challenge, survival in the ENP group was significantly higher than in the HA-IA saline group (P < .001). ENP improved survival in a rat model of E. coli sepsis with high mortality despite effective antibiotic therapy. Despite advances in antibiotic therapy and supportive medical care, bacterial sepsis remains a leading cause of death in the United States, where rates of sepsis in hospital- ized patients have been increasing [I]. One-third of patients with sepsis are infected with gram-negative organisms [2]. In patients with gram-negative infection, release of endotoxin from the bacterial cell wall initiates a series of events leading to clinically evident sepsis [3-5]. Neutralization of endotoxin, as an adjunct to antibiotic therapy, may improve the clinical outcome of patients in- fected with gram-negative bacteria. Immunotherapy with mu- rine [6-8] and human [9, 10] monoclonal antibodies to en- dotoxin has improved survival in animal models of sepsis. However, although several clinical trials have used monoclo- nal antibodies to the lipid A moiety of endotoxin for patients with sepsis [II, 12], improvements in survival have not been demonstrated conclusively [13]. Nonantibody proteins, termed antilipopolysaccharide (anti-LPS) factors, have been isolated from amebocytes of Received 6July 1993; revised II April 1994. Presented: annual meetings. Society for Pediatric Research. Washington. DC May 1993. and Society for Academic Emergency Medicine. San Fran- cisco, May 1993. T. J. Novitsky is an employee of Associates of Cape Cod. which provided the ENP used in this study. Grant support: National Institutes of Health (AI-18125); Associates of Cape Cod. Reprints or correspondence: Dr. George R. Siber, Laboratory of In- fectious Diseases, Dana-Farber Cancer Institute. Harvard Medical School. Boston. MA 02115. The Journal of Infectious Diseases 1994;170:630-5 © 1994 by The University of Chicago. All rights reserved. 0022-1899/94/7003-0019$01.00 horseshoe crabs, Limulus polyphemus [14] and Tachypleus tridentatus [15]. A recombinant version of the anti-LPS fac- tor of L. polyphemus, termed endotoxin-neutralizing protein (ENP), has been expressed in yeast. The limulus anti-Ll'S factor and ENP are 11.8-kDa proteins that inhibit the bio- logic effects of endotoxin in vitro, including the gelation of limulus amebocyte lysate (LAL) [16], mitogenesis of murine splenocytes [17], activation of human endothelial cells [18], and release of tumor necrosis factor (TNF) by human macro- phages (unpublished data). Anti-LPS factor also inhibits the growth of rough gram-negative bacteria [19]. In vivo. these proteins are protective against lethal Escherichia coli endo- toxin challenge in rats [16] and against lethal meningococcal [20] or E. coli [21] endotoxin challenge in rabbits. Recently. ENP was shown to improve survival in rabbits [22] and rats [23] with E. coli sepsis. HA-IA is a human IgM monoclonal antibody directed against endotoxin. It has conferred immunoprotection against gram-negative bacteremia caused by several organ- isms in some laboratory animals [9. 10] but not in others [24. 25]. We compared the efficacy ofENP with that ofHA-IA in a rat model of E. coli sepsis. Materials and Methods Bacteria E. coli. E. coli Ol8ac K I were stored in autoclaved skim milk on glass beads at - 70°C. Each week a bead was inoculated onto trypticase soy agar (Becton Dickinson Microbiology Sys- tems. Cockeysville. MD) and incubated overnight at 37°C. The culture was passaged daily for use on the following day. Each day, a representative colony was transferred into 10 mL of nu- at Penn State University (Paterno Lib) on March 6, 2016 http://jid.oxfordjournals.org/ Downloaded from