We evaluated the ability of a monoclonal antibody directed against the common H.8 antigen of pathogenic Neisseria sp. to confer passive protection against meningococcal disease in mice. The apparent protection conferred by antibody purified from tissue culture supernatant was actually the result of endotoxin contamination of buffers and tissue culture media. Endotoxin-free anti-H.8 antibody was not protective. The possibility of endotoxin contamination should be considered when evaluating immunity conferred by passively administered antibody in animal models

Barritt, Diana S.

Black, John R.

Cannon, Janne G.

Connell, Terry D.

Woods, Jon P.

Carolina Digital Repository

Vol. 55, No. 8INFECTION AND IMMUNITY, Aug. 1987, p. 1927-19280019-9567/87/081927-02 $02.00/0Copyright © 1987, American Society for MicrobiologyResistance to Meningococcemia Apparently Conferred by Anti-H.8Monoclonal Antibody Is Due to Contaminating Endotoxin and Notto Specific ImmunoprotectionJON P. WOODS, JOHN R. BLACK,t DIANA S. BARRITT, TERRY D. CONNELL, AND JANNE G. CANNON*Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill,North Carolina 27514Received 17 February 1987/Accepted 22 April 1987We evaluated the ability of a monoclonal antibody directed against the common H.8 antigen of pathogenicNeisseria sp. to confer passive protection against meningococcal disease in mice. The apparent protectionconferred by antibody purified from tissue culture supernatant was actually the result of endotoxincontamination of buffers and tissue culture media. Endotoxin-free anti-H.8 antibody was not protective. Thepossibility of endotoxin contamination should be considered when evaluating immunity conferred by passivelyadministered antibody in animal models.Neisseria meningitidis causes significant human diseasefor which there is no completely effective vaccine, in partdue to the marked heterogeneity and variability of meningo-coccal surface structures (6-8). The H.8 antigen is an outermembrane protein possessing several characteristics thatmake it attractive as a meningococcal vaccine component.Anti-H.8 monoclonal antibodies (MAbs) bind to all patho-genic neisseriae, including meningococci of different sero-groups and serotypes (3). The antigen is expressed in vivoand is immunogenic in people during meningococcal infec-tion (2). There is evidence for surface exposure of theantigen in vitro in the closely related gonococcus (3, 9).We tested whether an anti-H.8 MAb would confer passiveprotection in the well-described adult mouse model ofmeningococcal infection (5, 6). Two recent human bloodisolates were used for challenge: FAM18 (serogroup C,serotype 2a) and JB515 (B, nontypable). Meningococci weregrown overnight from frozen stocks on GC medium baseagar (Difco Laboratories) with Kellogg supplements (10).Bacteria were suspended in GCB broth (10) and grown to logphase with constant agitation at 37°C in a 5% CO2 atmo-sphere. A suspension at a concentration of 4 x 108 to 1 x 109CFU/ml (always confirtned by viable counts) was diluted inphosphate-buffered saline, and 0.5 ml was injected intraperi-toneally into female BALB/c mice (Charles River Laborato-ries) 6 to 12 weeks of age. Antibody or control preparations(100 ,ug of affinity-purified antibody, 1.0 mg of ascitesprotein, or an equal volume of buffer) were administeredintraperitoneally in a volume of 0.1 to 0.5 ml 4 h beforemeningococcal challenge. Mice were tail bled 4 h aftermeningococcal challenge as follows: after the tail wascleansed with povidone iodine (Clinidine; Clinipad Corp.)and ethanol, a vein was nicked with a sterilized razor, and 5to 20 ,ul of blood was plated in duplicate on GC agar or wasserially diluted before plating. The level of bacteremia wasexpressed as CFU per milliliter of blood.In initial experiments, MAb was purified from tissueculture supernatant by affinity chromatography (11) usingstaphylococcal protein A-agarose (Sigma Chemical Corp.).* Corresponding author.t Present address: Division of Infectious Diseases, Indiana Uni-versity School of Medicine, Indianapolis, IN 46223.Protein concentrations were determined by using the Bio-Rad assay according to the manufacturer's directions. Anti-H.8 MAb H.101 (immunoglobulin Gl) significantly reducedthe incidence and level of bacteremia caused by bothmeningococcal strains, whereas a control antibody did notprovide such protection (Table 1). These results were re-ported previously (J. R. Black and J. G. Cannon, ProgramAbstr. 24th Intersci. Conf. Antimicrob. Agents Chemother.,abstr. no. 96, 1984).When we attempted to repeat the passive-protection ex-periments using new preparations of antibody and buffer, wefound that we could not duplicate the initial results. Therewas a low incidence of bacteremia in mice receiving anytreatment (buffer, control antibody, or H.101) before injec-tion of meningococci, whereas most mice did become bac-teremic when injected with meningococci alone (data notshown). We suspected that endotoxin, which is a commoncontaminant of even sterile laboratory materials and tissueculture media (1, 15) could be influencing the results, sinceendotoxin is known to increase the nonspecific resistance ofanimals to a number of infectious agents (4, 12). Using theE-toxate Limulus amebocyte lysate assay (Sigma) accordingto the manufacturer's instructions, we determined that bothH.101 and control antibody solutions, as well as diluentHEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid) buffer (United States Biochemical Corp.) and sterilewater from a tissue culture facility, were contaminated withendotoxin. Assay of dilutions of the antibody preparations inpyrogen-free water (Travenol) indicated contamination of atleast 50 to 200 ng/ml, with Shigella flexneri endotoxin usedas a standard (data not shown). Different lots of HEPESprepared in pyrogen-free water (Travenol) all were contam-inated with endotoxin but varied in the concentration ofendotoxin.Purified Shigella endotoxin (Sigma) elicited resistance tomeningococcal infection similar to that seen in the initialpassive-protection experiments. In two experiments, 12mice in groups of three were injected intraperitoneally withstrain FAM18, or with 200 ng of endotoxin followed 4 h laterby strain FAM18. All of these mice became bacteremic, butthe level of bacteremia in endotoxin-injected mice averagedonly 2 and 17% of that in control mice. Reduction inbacteremia caused by strain JB515 was seen after injection19271928 NOTESTABLE 1. Summary of protection experiments usingaffinity-purified, tissue culture-derived MAbsMeanNo. of mice CFU/ml inChallenge MAb No.lofnmic bacteremic blood ofstrain challenged (%) bacteremicmiceJB515a None (buffer) 20 20 (100) 7.8 x 104Control MAb 23 19 (83) 1.8 x 104H.101 18 2 (11) 6.5 x 102FAM18' None (buffer) 28 26 (93) 1.9 X 106Control MAb 16 16 (100) 7.3 x 105H.101 29 16 (55) 8.3 x 103a Inoculum, approximately 9 x 103 CFU.b Inoculum, approximately 3 x 105 CFU.of as little as 50 pg of endotoxin (data not shown). Theprotection provided by injection of purified endotoxin and ofendotoxin-contaminated HEPES buffer was transient, last-ing at least 4 h but less than 36 h (data not shown).Pyrogen-free saline (Travenol), which was endotoxin nega-tive in the Limulus amebocyte lysate assay, did not displaythis protective effect when injected before meningococci.To reassess immunoprotection by the H.101 antibody, weneeded an endotoxin-free preparation of antibody. Attemptsto remove endotoxin completely from the tissue culture-derived preparations, using repeated passages over a Detoxi-Gel affinity column (Pierce Chemical Co.), were unsuccess-ful. Hybridoma-induced ascites fluid (13) was negative in theLimulus amebocyte lysate assay and was used in furtherpassive-protection experiments. Mice injected with ascitesreceived at least as much antibody as the mice in the initialexperiments, and the antibody did enter the blood (data notshown). All mice receiving H.101 or control ascites or salinebecame bacteremic after challenge with strain FAM18 (ca. 3X 104 CFU), and the levels of bacteremia in the three groups(six mice each) were similar (2.0 x 104, 4.6 x 104, and 3.4 x104 CFU/ml, respectively).The results of the passive-protection experiments usingendotoxin-free antibody from ascites indicate that the anti-H.8 MAb did not protect mice from bacteremia caused byintraperitoneally injected meningococci. These results donot disprove a role for the H.8 antigen in pathogenesis, nordo they eliminate the possibility that the antigen can be atarget of an effective immune response. Active immunizationstudies using the H.8 antigen may provide better informationas to the utility of this antigen in a vaccine. Recent progressin the purification of the H.8 antigen (14; unpublished data)should make such experiments possible soon.In these studies, we found that small quantities of endo-toxin could have a dramatic effect on the murine model ofmeningococcal infection. Chong and Huston also detectedtrace amounts of endotoxin in MAb preparations from tissueculture and showed that such amounts could protect micefrom lethal Escherichia coli sepsis (K. Chong and M.Huston, Abstr. Annu. Meet. Am. Soc. Microbiol. 1986,B188, p. 151). Since endotoxin contamination of materialsthat may be used in passive-protection experiments usinganimal models is not unlikely, and since we have shown thatendotoxin can remarkably mimic protective antibody inreducing meningococcal infection, we suggest that the pos-sibility of endotoxin contamination be considered in thedesign of any such experiments and the interpretation ofresults.We thank Carl Frasch for serological identification of meningo-coccal isolates and Lynn Brooks for excellent preparation of themanuscript.This work was supported by Public Health Service awardsA115036 (J.G.C.), A123830 (J.G.C.), and A107001 (J.R.B.) from theNational Institute of Allergy and Infectious Diseases. J.P.W. was aCharles Culpeper Fellow in the Medical Sciences and a HowardHolderness Medical Fellow.LITERATURE CITED1. Bito, L. Z. 1977. Inflammatory effects of endotoxin-like contam-inants in commonly used protein preparations. Science 166:83-85.2. Black, J. R., W. J. Black, and J. G. Cannon. 1985. Neisserialantigen H.8 is immunogenic in patients with disseminatedgonococcal and meningococcal infections. J. Infect. Dis. 151:650-657.3. Cannon, J. G., W. J. Black, I. Nachamkin, and P. W. Stewart.1984. Monoclonal antibody that recognizes an outer membraneantigen common to the pathogenic Neisseria species but not tomost nonpathogenic Neisseria species. Infect. Immun. 43:994-999.4. Cluff, L. E. 1970. Effects of endotoxins on susceptibility toinfections. J. Infect. Dis. 122:205-215.5. Craven, D. E., and C. E. Frasch. 1979. Protection against groupB meningococcal disease: evaluation of serotype 2 proteinvaccines in a mouse bacteremia model. Infect. Immnun. 26:110-117.6. DeVoe, I. W. 1982. The meningococcus and mechanisms ofpathogenicity. Microbiol. Rev. 46:162-190.7. Frasch, C. E. 1979. Noncapsular surface antigens of Neisseriameningitidis. Semin. Infect. Dis. 2:304-337.8. Gotschlich, E. C. 1984. Meningococcal meningitis, p. 237-255.In R. Germanier (ed.), Bacterial vaccines. Academic Press,Inc., New York.9. Hitchcock, P. J., S. F. Hayes, L. W. Mayer, W. M. Shafer, andS. L. Tessier. 1985. Analyses of gonococcal H.8 antigen. Sur-face location, inter- and intrastrain electrophoretic heterogene-ity, and unusual two-dimensional electrophoretic characteris-tics. J. Exp. Med. 162:2017-2034.10. Kellogg, D. S., Jr., W. L. Peacock, Jr., W. E. Deacon, L. Brown,and C. I. Pirkle. 1963. Neisseria gonorrhoeae. I. Virulencegenetically linked to clonal variation. J. Bacteriol. 85:1274-1279.11. Kohler, G. 1981. The technique of hybridoma production, p.285-298. In I. Lefkovits and B. Pernis (ed.), Immunologicalmethods, vol. 2. Academic Press, Inc., New York.12. Morrison, D. C., and R. J. Ulevitch. 1978. The effects ofbacterial endotoxins on host mediation systems. Am. J. Pathol.93:525-617.13. Nachamkin, I., J. G. Cannon, and R. S. Mittler. 1981. Mono-clonal antibodies against Neisseria gonorrhoeae: production ofantibodies directed against a strain-specific cell surface antigen.Infect. Immun. 32:641-648.14. Strittmatter, W., and P. J. Hitchcock. 1986. Isolation andpreliminary biochemical characterization of the gonococcal H.8antigen. J. Exp. Med. 164:2038-2048.15. Weinberg, J. B. 1981. Endotoxin contamination and in vitromonocyte-macrophage function: methods of detecting, detox-ifying, and eliminating endotoxin, p. 139-154. In D. 0. Adams,P. J. Edelson, and H. S. Koren (ed.), Methods for studyingmononuclear phagocytes. Academic Press, Inc., New York.INFECT. IMMUN.

Resistance to meningococcemia apparently conferred by anti-H.8 monoclonal antibody is due to contaminating endotoxin and not to specific immunoprotection.

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Resistance to meningococcemia apparently conferred by anti-H.8 monoclonal antibody is due to contaminating endotoxin and not to specific immunoprotection.

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