Ervin and the staff of the Blood Component Laboratory for plasmapheresis of donors, and James McIver, George Wright, and George Grady and their staff at the ...
INFECTION AND IMMUNITY, Feb. 1983, p. 709-714
Vol. 39, No. 2
0019-9567/83/020709-06$02.00/0 Copyright C 1983, American Society for Microbiology
Efficacy of Human Hyperimmune Globulin in Prevention of Haemophilus influenzae Type b Disease in Infant Rats DONNA AMBROSINO,1 JOHN R. SCHREIBER,2 ROBERT S. DAUM,3 AND GEORGE R. SIBER14' Department ofInfectious Diseases, Sidney Farber Cancer Institute, Harvard Medical School,1 and Department ofPediatrics, Children's Hospital Medical Center,2 Boston, Massachusetts 02115; Department of Pediatrics, Tulane University School of Medicine, New Orleans, Louisiana, 701123; and the Biologic Laboratories of the State Laboratory Institute, Boston, Massachusetts, 021304*
Received 26 July 1982/Accepted 22 October 1982
To determine the protective efficacy of human hyperimmune globulin to Haemophilus influenzae type b disease in an infant rat model, we compared hyperimmune globulin containing 600 ,ug of anti-polyribophosphate (PRP) antibody per ml to conventional immune globulin containing 66 ,ug of anti-PRP antibody per ml. The hyperimmune globulin was fractionated from the pooled plasma of 55 adult donors immunized with PRP, the capsular polysaccharide of H. influenzae type b. The disappearance of passively administered antibody was biphasic, with a linear first-order disappearance curve during the first 7 days. The initial half-life for anti-PRP antibody was 2.38 days in rats nasally colonized but not detectably bacteremic with H. influenzae type b and significantly longer (halflife, 10.3 days; P < 0.01) in noncolonized animals. Hyperimmune globulin afforded 10 times the protection of conventional globulin against bacteremia and meningitis. Globulin depleted of anti-PRP antibody offered no protection. The initial serum antibody levels and the levels during the 8-day observation period predicted protection. Rats maintaining serum antibody levels greater or equal to 50 ng/ml to day 8 had a 10% bacteremia and 5% meningitis incidence in contrast with 95% bacteremia (P < 0.001) and 55% meningitis (P < 0.001) in rats with less than 50 ng of anti-PRP antibody per ml. We conclude that studies of the pharmacology and efficacy of hyperimmune globulin are warranted in high-risk children unable to respond to active immunization.
Haemophilus influenzae type b is a major bacterial pathogen in children, causing a spectrum of diseases which include bacteremia, pneumonia, cellulitis, arthritis, epiglottitis, and meningitis. The risk of invasive disease is especially great in children with sickle cell anemia, asplenia, agammaglobulinemia, and malignancy and in young contacts of H. influenzae type b disease. In addition, certain ethnic groups, such as Navajo Indians and Alaskan Eskimos, are at increased risk for invasive disease (6, 17). Antibody to polyribophosphate (PRP), the capsular polysaccharide of H. influenzae type b, is protective, and purified PRP is immunogenic in older children and adults (15, 19). Unfortunately, children under 18 months old and many older high-risk individuals (14, 15) respond poorly to immunization with purified PRP vaccine. Passive immunization with human immunoglobulin might provide an alternative approach. Robbins et al. have calculated that the minimum protective level of antibody to PRP is 60 to 100 ng/ml (12). Conventional immune globulins must be used frequently and in high doses to approach
We recently produced a human globulin from donors actively immunized with PRP vaccine which contains 10 times the anti-PRP antibody available in conventional immune globulin. We evaluated the protective efficacy and the pharmocology of these preparations in an infant rat model of H. influenzae type b disease. MATERIALS AND METHODS Preparation of immune globulins. Fifty-five healthy adult volunteers were immunized with 0.5 ml of pneumococcal vaccine (Pneumovax, lot 1912B; Merck Sharp and Dohme, West Point, Pa.) combined in the same syringe with 0.5 ml of H. influenzae type b vaccine (lot 764-CF320; Merck Sharp and Dohme) and concurrently with 0.5 ml of meningococcal vaccine (Menomune A/C; Connaught Laboratories, Swiftwater, Pa.) containing polysaccharide of serogroups A and C. Plasma was obtained by plasmapheresis from 1 to 9 months after immunization and was pooled and fractionated by the cold ethanol method of Cohn et al. (5) and Oncley et al. (10) at the Biologic Laboratories of the State Laboratory Institute, Jamaica Plain, Mass. The 52-liter plasma pool yielded 825 ml of hyperimmune serum globulin (HSG). Conventional immune serum globulin (ISG; lot II
these levels. 709
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AMBROSINO ET AL.
GB77) was prepared by the same methods from a 3,353-liter pool obtained from 15,223 U of recovered plasma from healthy blood donors. Both globulins contained 16.5% protein consisting of >95% immunoglobulin G (IgG) and only traces of IgM and IgA. Antibody to PRP was measured by radioimmunoassay (1) with tritiated PRP kindly provided by Porter Anderson, University of Rochester, Rochester, N.Y. The assay was standardized with a standard serum (S. Klein) supplied by John Robbins, Food and Drug Administration, Bethesda, Md. HSG contained 600 ,ug of anti-PRP antibody per ml, and ISG contained 66 ,ug/ml. Absorption of HSG with PRP. A variety of absorption conditions were tried and found to incompletely remove anti-PRP antibody and leave measurable PRP antigen in the supernatant. The best absorption was achieved with the following procedure. PRP purified by the method of Anderson and Smith (2) was added to HSG at five times the concentration giving antigenantibody equilavence in a quantitative precipitin test and incubated at 4°C for 5 days. The mixture was centrifuged at 12,000 x g for 30 min, and the supernatant was dialyzed against 0.02 M K2HPO4, pH 8.0 (starting buffer), and applied to a column of DEAEAffigel Blue (Bio-Rad Laboratories, Richmond, Calif.) equilibrated with starting buffer. The effluent was monitored for absorption at 280 nm, and the proteincontaining fractions eluting with starting buffer were pooled and concentrated with a Minicon-B15 concentrator (Amicon Corp., Danvers, Mass.) to a concentration of 16.5%. The anti-PRP antibody concentration was 5.8 ,ug/ml, which represents
