Haemophilus influenzae Type b. JOHN R. .... by Porter Anderson andstandard serum supplied by John ..... Newman, S. L., B. Waldo, and R. B. Johnston. 1973.
Vol. 55, No. 11
INFECTION AND IMMUNITY, Nov. 1987, p. 2830-2833 0019-9567/87/112830-04$02.00/0 Copyright © 1987, American Society for Microbiology
NOTES Effect of Complement Depletion on Anticapsular-Antibody-Mediated Immunity to Experimental Infection with Haemophilus influenzae Type b JOHN R. SCHREIBER,'* CHRISTOPHER J. BASKER,2 AND GEORGE R. SIBER2" Laboratory of Infectious Diseases, Dana Farber Cancer Institute, Boston, Massachusetts 021152; Massachusetts Public Health Biologic Laboratories, Boston, Massachusetts 021303; and Department of Pediatrics, Section of Infectious Diseases, Albany Medical College, Albany, New York 12208' Received 18 May 1987/Accepted 27 July 1987
Antibody to the capsular polysaccharide of Haemophilus influenzae type b (Hib) was not protective in infant rats depleted of complement by cobra venom factor (CoVF) even when serum antibody levels were many times the minimum protective level. Partial protection from Hib infection was achieved in CoVF-treated rats only if they were passively hyperimmunized with large doses of immunoglobulin G Hib capsular polysaccharide antibody. In addition, nonimmune CoVF-treated rats had higher mortality and blood bacterial density than nonimmune rats with intact complement systems. Antibody to the capsular polysaccharide (CP) of Haemophilus influenzae type b (Hib) plays a major role in protection from Hib infection. However, anticapsular antibody is bactericidal and opsonic in the presence of complement (7, 17, 23) but is not bactericidal and only poorly opsonic in its absence (11, i2, 15, 18, 19). Thus, complement is also a crucial part of adequate host defense against Hib infection. Unlike most other H. influenzae serotypes, Hib is resistant to complement-mediated bacteriolysis of antibody-free serum at concentrations in serum of up to 90% (22). Thus, both complement and Hib antibody are required for in vitro bacteriolysis of Hib (18, 22). Resistance of Hib to complement-mediated bacteriolysis has been proposed as a major virulence factor of Hib (22). The relative importance of bacteriolysis, complement, and Hib CP antibody in host clearance of Hib, however, is uncertain. For example, complement-depleted infant rats with no Hib CP antibody have higher mortality and blood bacterial density after challenge with Hib than do nondepleted animals (6). The rnoderate protection that complement alone provides nonimmune rats may be due to nonbactericidal functions of complement such as opsonization. Weller et al., for example, have suggested that reticuloendothelial system phagocytosis is the most important method of Hib clearance in vivo and that such clearance, although present in nonimmune animals, is greatly enhanced by the presence of anti-Hib antibody (24). Thus, intravascular bacteriolysis may be less important in host clearante of Hib than of other encapsulated bacteria such as meningococcus, and Fc- and complement-mediated opsonization and phagocytosis may be more important. Since the mechanisms of Hib clearance from the host are only partially understood, the present studies were undertaken to further elucidate the roles of complement and Hib CP antibody in host defense against Hib infection. *
Hib CP antibody was prepared by immunizing an adult volunteer with 50 p.g of purified Hib CP vaccine (lot 764; Merck Sharp and Dohme, West Point, Pa.). Serum was collected 4 weeks after vaccination and was fractionated on an ACA-34 polyacrylamide-agarose sizing column (LKB Instruments, Inc., Rockville, Md.). Fractions were screened by enzyme-linked immunosorbent assay for total immunoglobulin class content and then pooled according to predominant class (18). Fractions rich in immunoglobulin M (IgM) or IgG were purified by affinity chromatography until >99% isotype pure, as previously described (3, 5, 14). An IgG pool was also prepared by Cohn fractionation of plasma from adult volunteers imnmunized with Hib CP vaccine (20). This hyperimmune globulin contained 400 p.g of Hib CP antibody per ml that was >99% IgG. Rat anticapsular antibody was prepared by repeatedly immunizing adult Sprague-Dawley albino rats subcutaneously with a homogenate of equal parts of Freund adjuvant and Hib CP vaccine. Since the Hib CP antibody response was low in four of the five rats, further imrnunizations with diphtheria-Hib CP conjugate vaccine supplied by Porter Anderson (University of Rochester, Rochester, N.Y.) were performed. Rats were then anesthetized and exsanguinated by cardiac puncture, and sera were pooled and made lipid free. Antibody to the capsule of Hib was measured in each globulin preparation and in animal sera by Farr radioactiveantigen-binding assay (1, 16) using tritiated Hib CP supplied by Porter Anderson and standard serum supplied by John Robbins (National Institutes of Health, Bethesda, Md.) (1, 16). The effectiveness of cobra venom factor (CoVF) in depleting complement was confirmed by determinations of the 50% hemolytic complement (CH50) titer by using modifications of standard techniques (9, 13). Dilutions of rat sera, ranging from 1/10 to 1/160 in Veronal-buffered saline containing Ca and Mg, were incubated for 1 h at 37°C with antibody-coated sheep erythrocytes at 108 erythrocytes per ml (Cordis Lab-
Corresponding author. 2830
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TABLE 1. Protection of normal and complement-depleted infant rats from Hib infection by human antibody to Hib CP
TreatmentHib CP Treatment antibody dose of of dose (no. antibody (no. pups) (ng) pups) Saline (22) Saline (19) IgM (5) IgM (6) IgG (7) IgG (5) IgG (4) IgG (7) IgGc (5) IgGc (5) IgG' (9) IgGc (11)
0 0 1,000 1,000 1,350 1,350 5,500 5,500 15,000 15,000 60,000 60,000
Mean serum Hib CP antibody concn
Complement
Bacteremia, day 2 (%)
Meningitis, day 3 (%)
(cumulative)
density (CFU/ml)
