Samuel R. Wilson

Intravenous immunoglobulin in neonatal group B streptococcal disease: Pharmacokinetic and safety studies in monkeys and humans

Numerous studies have suggested that opsonic antibody is important in neonatal immunity to group B streptococci. Immunoglobulin G is primarily transferred from the mother to the fetus across the placenta in the last few weeks of pregnancy. Premature babies may, therefore, not acquire sufficient opsonic antibody to protect them from infection with group B streptococci. Although maternal immunization may provide adequate maternal opsonic antibody, premature infants with antibody deficiency may remain susceptible to infection. Intravenous immunoglobulin administered to term pregnant rhesus monkeys did not provide reliable levels of serum opsonic activity to group B streptococci in their offspring. Pharmacokinetic and safety studies were also performed in human neonates. Significant elevations in group B streptococcal-specific IgG did occur in human neonates given 500 mg/kg intravenous immunoglobulin and the infusions appeared safe and well tolerated. The availability of intravenous immunoglobulin with functional activity against group B streptococci may provide a rapid and effective method of delivering opsonic antibody to neonates.

MODIFIED HUMAN IMMUNE SERUM GLOBULIN FOR INTRAVENOUS ADMINISTRATION: IN VITRO OPSONIC ACTIVITY AND IN VIVO PROTECTION AGAINST GROUP B STREPTOCOCCAL DISEASE IN SUCKLING RATS

ABSTRACT. Human immune serum globulin (ISG) preparations were tested in an in vivo suckling rat protection assay and an in vitro opsonophagocytic assay against various types and strains of Group B streptococci (GBS). Standard ISG provided minimal protection in suckling rats against type III GBS sepsis, whereas preparations of ISG modified for intravenous administration (MISG) provided significant protection against all strains of type III, type II and type Ia GBS tested. Although less protection was obtained against type la strains, the survival in suckling rats challenged with all types of GBS varied from 73 % to 91% with MISG therapy, as compared with 5% to 12% survival in untreated animals. In this in vivo model, MISG was protective even when administered after bacterial challenge, but had to be administered within 5 h of infection. MISG also had high in vitro opsonic activity against GBS types III and II, but was less effective with some type Ia strains. Just as MISG was more protective than ISG in vivo, it also was more opsonic in vitro. A detailed comparison of one lot of MISG with its parent ISG revealed that the modified preparation actually contained less IgG. When equivalent concentrations of affinity‐purified IgG from both preparations were tested, the IgG from MISG was significantly more opsonic. Since the affinity purification procedure eliminated the possibility that IgM or substances introduced in the modification process were actually responsible for the enhanced bactericidal activity, it appears that the individual IgG molecules in MISG may be more effective. These studies suggest that MISG which has been modified by reduction and alkylation for intravenous administration may provide a valuable adjunct to chemotherapy in the treatment of GBS disease in the neonate.

Functional characteristics of a modified immunoglobulin preparation for intravenous administration: summary of studies of opsonic and protective activity against group B streptococci.

Group B streptococci (GBS) remains a major cause of morbidity and mortality in newborn babies, despite antibiotic therapy. Recent studies suggest that phagocytosis and killing of GBS is ineffective due to a deficiency in anti-GBS antibody. Using an opsonophagocytic bacterial assay and a suckling rat model of GBS sepsis, we analyzed a modified human immunoglobulin for opsonic and protective antibody. Immune globulin (IGIV) prepared for intravenous use (Gamimune®, Cutter Laboratories, Inc.) was highly protective in this experimental GBS model. Using the opsonophagocytic assay, antibody activity to several strains of types Ia, II, and III GBS were also demonstrated with IGIV. To ensure that the IGIV activity was in fact antibody, globulin from IGIV was isolated and analyzed. Purified IgG retained opsonic activityin vitro and also provided protection in experimental GBS disease. Variation in the quantity of IgG necessary for protection was observed in different GBS strains. Since IGIV has opsonic and protective activity against several strains and serotypes of GBS, its intravenous administration may provide a valuable adjunct to standard antibiotic therapy for neonatal GBS infections.

Chronic helminth infection does not exacerbate Mycobacterium tuberculosis infection.

Background Chronic helminth infections induce a Th2 immune shift and establish an immunoregulatory milieu. As both of these responses can suppress Th1 immunity, which is necessary for control of Mycobacterium tuberculosis (MTB) infection, we hypothesized that chronic helminth infections may exacerbate the course of MTB. Methodology/Principal Findings Co-infection studies were conducted in cotton rats as they are the natural host for the filarial nematode Litomosoides sigmodontis and are an excellent model for human MTB. Immunogical responses, histological studies, and quantitative mycobacterial cultures were assessed two months after MTB challenge in cotton rats with and without chronic L. sigmodontis infection. Spleen cell proliferation and interferon gamma production in response to purified protein derivative were similar between co-infected and MTB-only infected animals. In contrast to our hypothesis, MTB loads and occurrence and size of lung granulomas were not increased in co-infected animals. Conclusions/Significance These findings suggest that chronic filaria infections do not exacerbate MTB infection in the cotton rat model. While these results suggest that filaria eradication programs may not facilitate MTB control, they indicate that it may be possible to develop worm-derived therapies for autoimmune diseases that do not substantially increase the risk for infections.

Functional Antibacterial Activity of a Human Intravenous Immunoglobulin Preparation: in vitro and in vivo Studies

Abstract. The functional antibacterial activity of an intravenous immunoglobulin preparation (Swiss Red Cross Immunoglobulin, SRK‐Ig) was tested both in vitro and in vivo. Using type III group B streptococci as a model system, the intravenous immunoglobulin preparation was shown to enhance phagocytosis and killing of the bacteria by human neutrophils. There was good activity to several type III strains, and for efficient opsonization both antibody and complement were required. In an animal model of neonatal group B streptococcal sepsis, passively administered immunoglobulin enhanced survival with 27/37 (73%) treated animals surviving compared to only 3/16 (19%) controls. These studies demonstrate that the intravenous immunoglobulin preparation SRK‐Ig has retained functional activity and may have potential value for prophylaxis or therapy of certain bacterial infections.

Type III group B streptococcal strain differences in susceptibility to opsonization with human serum.

Summary: Human serum opsonins to type III Group B streptococci (GBS) were studied in an in vitro opsonophagocytic assay. Two type III GBS test strains were susceptible (893 and IIINor) and two resistant (891 and 892) to opsonization by the majority of sera from 15 healthy adults. Four individuals with undetectable or low opsonic liters to the test strains were immunized with pneumococcal vaccine; immunization with pneumococcal vaccine induced a titer rise in all but one instance when susceptible GBS strains were tested. In contrast, only a single titer rise was detected when resistant GBS strains were employed in the test. These results indicate that immunization with a cross–reacting antigen (identical to core antigen of type III GBS) fails to induce opsonic antibody to all strains of type III GBS. A resistant strain was made highly susceptible to neutrophil killing in vitro by exposure to neuramindase prior to incubation with opsonic serum. Using a fluorescent lectin–binding assay, this enzyme appeared to remove surface sialic acid, suggesting that sialic acid is an antiphagocytic factor. However, the possibility that other surface moieties may act as antiphagocytic factors cannot be ruled out. Both opsonic susceptible and resistant strains absorbed opsonic antibody from serum, which suggests that the GBS antiphagocytic factors do not prevent binding of antibody to resistant bacteria.These findings indicate that demonstration of serum opsonic activity to one strain of type III GBS may not accurately depict opsonic activity to other strains. In addition, immunization with core antigen did not enhance opsonic activity against all GBS strains. These data also point out the need to use assays which measure functional antibody, since demonstration of antibody binding may not reflect its ability to facilitate bacterial phagocytosis and killing.

Characterization of late tuberculosis infection in Sigmodon hispidus

We previously described primary tuberculosis in Sigmodon hispidus cotton rats up to 6 months following a pulmonary challenge. At that time, we observed fewer animals demonstrating disease as time from exposure progressed. We hypothesized that some cotton rats may control a primary infection to latency in a similar fashion to humans. The current experiment was designed to examine the natural progression of disease in S. hispidus at a later timepoint following a respiratory challenge with Mycobacterium tuberculosis (Mtb). An additional objective was to test whether cotton rats may become latently infected, and to determine whether latent disease might be activated by cyclophosphamide induced immune suppression. Thirty-four percent of the inoculated cotton rats died prior to 9 months following the challenge. However, 50% of immunocompetent animals surviving past 9 months demonstrated positive lung tissue cultures for Mtb without histologic evidence of disease. None of the immunosuppressed animals demonstrated this pattern. These findings are consistent with the development of latent tuberculosis infection in some cotton rats. Furthermore, it appears reactivation of disease occurs with cyclophosphamide induced immunosuppression. Cotton rats may serve as a model for latent as well as active tuberculosis infection.

INTRAVENOUS IMMUNOGLOBULIN (IVIG) LOTS USED IN CLINICAL TRIALS TO PREVENT LATE-ONSET INFECTION IN THE HIGH RISK NEONATE CONTAINED VARIABLE STAPHYLOCOCCUS EPIDERMIDIS ANTIBODY ACTIVITY. † 1806

Three large multi-center randomized double-blind placebo controlled clinical trials in the U.S. have observed and published variable results following IVIG prophylaxis for late-onset infection in the high risk neonate.Staphylococcus epidermidis was the major cause of infection in these studies. We hypothesized that these differences might in part be due to variable S. epidermidis antibody activity contained in the IVIG lots infused. We were able to obtain bottles of each of the twelve original lots of IVIG used in these trials. All IVIG lots were diluted to 5 grams% with normal saline. Each was then. evaluated to determine the amount of antibody present against a clinical (ATCC) strain of S. epidermidis (Haywood, serotype 2) using a modification of an in-vitro assay of neutrophil mediated opsonophagocytosis previously reported (Fischer et al, Journal of Infectious Diseases 1994;169:324-9). Antibody levels are the geometric mean of two assays and represent the largest reciprocal dilution which promoted ≥ 90% bacterial killing. No bacterial killing was noted in negative controls which were without IVIG, complement and both. The total IgG, measured by radial immunodiffusion, was similar for all the samples tested. The S. epidermidis opsonic activity of the IVIG lots ranged from undetectable to 20-1. Two of the IVIG lots (one with opsonic activity at 20-1 and the other at 5-1) and normal saline were then evaluated for in-vivo activity against the same strain of S. epidermidis by determining the per cent survival of lethally infected suckling rats (Fischer et al, ibid.) treated with each lot. Suckling rat survival was significantly increased (p 0.5) than those treated with IVIG containing low levels of S. epidermidis opsonic activity. We conclude that the IVIG lots used in the clinical trials evaluating IVIG prophylaxis in the high risk neonate had significantly variable S. epidermidis antibody activity. Further analysis will be necessary to correlate the clinical and antibody variability. If so, the development of screened or selected products with high titers of activity against neonatal specific pathogens would be warranted.

1002 QUANTITATIVE PROTECTION STUDIES IN A SUCKLING PAT MODEL OF GROUP B STREPTCCOCCAL SEPSIS

Group B streptococci (GBS) are a major cause of sepsis and meningitis in the newborn. Neonatal susceptibility to GBS infection appears to be associated with a deficiency of opsonic antibody to GBS. The present studies were designed to determine if human antibodies protective for one type III GBS strain provide uniform protection for other type III strains. IgG was affinity purified from pooled human immunoglobulin using a staphylococcal protein A immunoabsorbant. The relative titers of IgG antibodies to group B and type III (native and core) antigens were determined. To evaluate protection, suckling rats were challenged S.Q. with each of 4 different clinical isolates of GBS type III followed immediately by various concentrations of purified IgG given IP. With 5 or 10 μg of IgG significant survival occurred in animals challenged with one strain (17/20, 85%), whereas this amount of antibody afforded no protection against the other 3 strains (0/27). Administration of 20 μg of IgG provided some protection for 2 of these strains (7/23, 3/9), but uniform lethality occurred with the fourth strain even when more than 40 μg IgG was given. These studies demonstrate that four type III GBS strains require different amounts of human IgG for protection. Strain variability must be examined in future investigations of vaccine induced or passively acquired IgG for protection against GBS disease.

1310 KINETIC AND METABOLIC STUDIES OF AN INTRAVENOUSLY ADMINISTERED IMMUNOGLOBULIN PREPARATION IN A NEONATAL LAMB MODEL

Bacterial sepsis and meningitis persist as major neonatal problems despite a wide variety of effective antibiotics. Recent studies have shown that a deficiency of opsonic antibody is associated with increased risk to developing group B strep-tococcal (GBS) sepsis and meningitis. We have previously shown that a new human immunoglobulin preparation modified for intravenous administration (MISG) protected suckling rats from lethal GBS infection. The present studies were designed to evaluate the kinetics and metabolic effects of MISG in the neonatal lamb. MISG (0.5 to 1.0 gm/kg) was given IV over 15 min and blood samples were obtained at 15, 30 and 60 min, and at 4 and 24 hr. No overt toxic or anaphylactic effects were observed. IgG levels peaked at about 800 mg/dl (0.5 gm/kg/dose) and 1500 mg/dl (1.0 gm/kg/dose). Changes in serum osmolality and oncotic pressure were minimal, but serum glucose levels were elevated at 30 and 60 min after infusion. Post infusion Na, K and Cl were not different from baseline levels. These data show that IV administration of MISG in a neonatal model elevates serum IgG levels without inducing serious side effects. Passive administration of human IgG to neonates may provide a valuable adjunct to standard antibiotic therapy of bacterial disease and future studies appear warranted.