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Use of Capsular Polysaccharide–Tetanus Toxoid Conjugate Vaccine for Type. II Group B .... tailed for GBS type Ia and Ib conjugate vaccine production [11].
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Use of Capsular Polysaccharide–Tetanus Toxoid Conjugate Vaccine for Type II Group B Streptococcus in Healthy Women Carol J. Baker,1 Lawrence C. Paoletti,2 Marcia A. Rench,1 Hilde-Kari Guttormsen,2 Vincent J. Carey,2 Melissa E. Hickman,1 and Dennis L. Kasper2

1

Section of Infectious Diseases, Departments of Pediatrics and Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas; 2Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital, and Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts

An estimated 15% of invasive group B streptococcal (GBS) disease is caused by type II capsular polysaccharide (II CPS). In developing a pentavalent vaccine for the prevention of GBS infections, individual GBS CPSs have been coupled to tetanus toxoid (TT) to prepare vaccines with enhanced immunogenicity. Type II GBS (GBS II) vaccine was created by direct, covalent coupling of II CPS to TT by reductive amination. In 2 clinical trials, 75 healthy nonpregnant women 18–45 years old were randomized to receive II CPS–TT (II-TT) conjugate (dose range, 3.6–57 mg of CPS component) or uncoupled II CPS vaccine. Both vaccines were well tolerated. II CPS–specific IgG serum concentrations (as well as IgM and IgA) peaked 2 weeks after immunization, being significantly higher in recipients of conjugated vaccine than in recipients of uncoupled CPS. Immunological responses to conjugate were dose dependent and correlated with opsonophagocytosis in vitro. These results support inclusion of II-TT conjugate when preparing a multivalent GBS vaccine.

For ∼3 decades, group B Streptococcus (GBS) has been a dominant pathogen in newborns and their mothers, and, more recently, the prominence of invasive infection among adults with underlying medical conditions has been recognized. However, the incidence of early-onset neonatal GBS infection has decreased by 65%, from 1.7 per 1000 live births in 1993 to 0.6 per 1000 in 1998, through the implementation of a policy for the use of maternal intrapartum antibiotic prophylaxis [1]. An estimated 3900 early-onset infections and 200 neonatal deaths were prevented in 1998. Despite this success in preventing earlyonset disease, as well as maternal bacteremic infections, the projected number of cases of invasive GBS disease in the United States in 1998 was 16,888, with ∼80% of these cases occurring in individuals 13 months old. The case-fatality rate is even more significant in adults (12%) than in neonates (4%). Widespread use of antimicrobial prophylaxis has obvious Received 2 May 2000; revised 7 July 2000; electronically published 8 September 2000. Presented in part: First Annual Conference on Vaccine Research, Washington, DC, 1988 (abstract P-16). Financial support: National Institute of Allergy and Infectious Diseases, National Institutes of Health (contract AI-75326 and merit award AI-23339 to D.L.K.). The contents of this publication do not necessarily reflect the views or politics of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the US government. Reprints or correspondence: Dr. Carol J. Baker, Dept. of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Rm. 302A, Houston, TX 77030 ([email protected]). The Journal of Infectious Diseases 2000; 182:1129–38 q 2000 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2000/18204-0016$02.00

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limitations in its application as a strategy for preventing GBS disease. Concerns include adverse events associated with drug administration, the development of antibiotic resistance among GBS, the emergence of gram-negative enteric pathogens, the need to use prophylaxis with each pregnancy, and the cost of such an intervention. By contrast, the development of a multivalent polysaccharide-protein GBS conjugate vaccine, theoretically, would extend protection against invasive GBS disease to pregnant women, adults with defined underlying chronic medical conditions, the elderly, and, by placental transport of maternal antibodies, infants with late-onset (17 days old) infection, while providing a permanent and cost-effective prevention method [2]. More than a decade ago, purified capsular polysaccharides (CPS) from GBS serotypes Ia, II, and III were assessed as potential vaccine candidates. Although these preparations were safe in healthy adults, their immunogenicity was variable, and better vaccine candidates were needed [3]. In the 1990s, preparation of GBS protein–CPS conjugate vaccines, by coupling CPS from serotypes Ia, Ib, II, III, and V GBS to tetanus toxoid (TT), was accomplished. In experimental models, each of the CPS-TT conjugate vaccines was more immunogenic than was its uncoupled counterpart [4–9]. Administration of CPS-TT conjugates to healthy nonpregnant women confirmed that immunogenicity in response to conjugated CPS was significantly better than that to the uncoupled CPS. For example, 90% of adults receiving type III CPS coupled to TT (III-TT) had a 11 mg/mL III CPS–specific antibody response compared to only 50% of those receiving the uncoupled CPS [10]. When serotypes Ia and Ib were similarly assessed, 195% and 190% of the subjects receiving type Ia-TT

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or type Ib-TT conjugate vaccines, respectively, had a Ia CPS or Ib CPS–specific IgG concentration 11 mg/mL 8 weeks after immunization. Conversely, one-third of subjects receiving uncoupled Ia or Ib CPS vaccines were vaccine nonresponders (serum concentrations of CPS-specific IgG !1 mg/mL and !4fold increase in specific IgG after immunization) [11]. We envision that a multivalent GBS polysaccharide-conjugate vaccine would include CPSs from serotypes Ia, Ib, II, III, and V. Taken together, these capsular types account for 95%–100% of isolates from invasive infections of newborns, young infants, and adults. Among them, serotype II strains cause ∼15% of invasive infections in pregnant women and nonpregnant adults, as well as an estimated 10% of infant infections, primarily in those with early-onset disease [12–14]. The purpose of the present investigation was to determine the safety and immunogenicity of type II GBS CPS coupled to TT (II-TT) and to compare this with that of uncoupled II CPS in healthy, nonpregnant adults. Results with this monovalent candidate are an important prelude to the phase I and II testing of a pentavalent vaccine and, ultimately, to an efficacy trial.

Materials and Methods Synthesis of experimental type II conjugate vaccines. CPS of GBS type II (strain 18RS21) was purified by methods described elsewhere for the purification of III CPS [5]. Mild, selective oxidation of the CPS was performed with sodium meta-periodate to formaldehyde groups on 31%, 57%, and 73% of total sialic acid residues. The percentage of residues that were oxidized was verified with gas chromatography and/or mass spectrometry on trimethylsyl derivatives of coded samples, as described elsewhere [5]. For conjugation to TT, each of the oxidized II CPS (∼5–7 mg) was combined with an equal amount of monomeric TT in 0.25 mL of 0.2 M sodium phosphate (pH 7.3). Sodium cyanoborohydride (∼12 mg) was added to each vial, and the mixtures were incubated at 377C. Analysis of the progress of the conjugation reactions, purification of the resulting conjugates, and protein and carbohydrate analysis on the purified vaccines were performed by methods detailed for GBS type Ia and Ib conjugate vaccine production [11]. The 3 conjugate vaccines prepared—II31%-TT, II57%-TT, and II73%TT—contained 30%, 34%, and 61% carbohydrate by weight, respectively, with the remaining material as TT. Vaccination of female mice with experimental II-TT vaccines. The protective efficacy of conjugate vaccines was evaluated in a neonatal mouse model of GBS infection [15]. In brief, female CD1 mice were vaccinated intraperitoneally with 2 mg of II31%-TT, II57%TT, or II73%-TT, II CPS, or 0.9% saline in a total volume of 0.5 mL. Conjugate vaccines, uncoupled II CPS, and saline were mixed 1:1 with 2% alum (Alhydrogel Superfos Biosector). A booster dose of conjugate vaccine, II CPS, or saline was given 3 weeks after the primary dose. Mice were then bred, and their offspring were challenged with GBS within 48 h of birth. Because of a technical error with litters born after the first mating, mice were rebred (29 days after the booster dose) without revaccination, and these second litters were challenged. Mouse pups were challenged with a single

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intraperitoneal injection volume of 0.05 mL containing type II strain 18RS21 (5.5 3 10 6 to 7.5 3 10 6 cfu per pup or a 70-fold LD50), and survival was recorded 48 h later. Survival of pups from the second mating are presented. Elsewhere we have shown that maternal mouse vaccination with III CPS-TT GBS vaccine was highly efficacious in protection against GBS challenge in 3 successive mouse litters [9]. Sera from dams were obtained after delivery of the second litters (84 days after the primary vaccination), and II CPS–specific IgG concentrations were determined by use of a quantitative ELISA, as described elsewhere for the quantitation of murine antibody to III CPS [16]. Microtiter plates were coated with 0.5 mg/well of II CPS—human serum albumin conjugate as the target antigen. Separate wells were coated with goat antimouse IgG as the capture antibody to which dilutions of mouse IgG were added to generate a standard curve. Goat antimouse IgG–alkaline phosphatase conjugate was used as the secondary antibody at a 1: 3000 dilution. Sera were serially diluted beginning at a dilution of 1:200. Preparation of II CPS and II-TT vaccines for clinical trials. II CPS was purified from 100-L fermenter cultures of GBS strain 18RS21. Growth parameters and purification methods were similar to those detailed elsewhere for III CPS [5]. Component sugar content of purified II CPS was determined with a high-anion-exchange chromatography system (Dionex, Sunnyvale, CA), as described elsewhere [17]. Purified II CPS was composed of 36.0% (w/w) galactose, 25.5% (w/w) glucose, 20.3% (w/w) N-acetylglucosamine, and 18.2% (w/w) sialic acid; it contained !2% (w/w) protein, !6 mg/mg nucleic acid, and had a Mr of 580,000. Purified II CPS was periodate-oxidized before coupling, which resulted in the formation of aldehydes on 35% of the total sialic acid residues. Bulk TT (lot 48; Statens Seruminstitut, Copenhagen, Denmark; provided by AMVAX, Beltsville, MD) was applied onto a S-300 HR gel filtration column to isolate monomeric TT, which had a Mr of 150,000. Periodate-oxidized II CPS and monomeric TT were covalently coupled by reductive amination with use of sodium cyanoborohydride and were purified by gel filtration chromatography by use of methods described elsewhere for the manufacture of type Ia and Ib conjugate vaccines [11]. The composition of bottled GBS II–TT conjugate vaccine (lot 94-2a) was determined by the methods of Dubois et al. [18], with purified II CPS as the standard, and of Larson et al. [19], with TT as the standard. GBS II–TT conjugate vaccine (lot 94-2a) was bottled at the Salk Institute (Swiftwater, PA) as a lyophilized preparation with a sucrose excipient in multidose vials at a II CPS concentration of 57 mg/0.5 mL. Uncoupled II CPS (lot 94-5S) was bottled at the Experimental Vaccine Preparation Laboratory (Program Resources, Rockville, MD) in single dose vials at a CPS concentration of 45 mg/0.5 mL in phosphate-buffered saline (PBS), pH 7.2, containing 0.01% thimerosal. All vaccines were stored at 47C–87C before use. GBS II vaccines were tested in adherence to Good Manufacturing Practices regulations 21 CFR, part 58. Bottling and testing of the vaccines conformed to the Guidelines on Sterile Drug Products Produced by Aseptic Processing (June 1987), as developed by Center for Drug and Biologicals and Office of Regulatory Affairs (US Food and Drug Administration [FDA]). Final container tests performed on these vaccines adhered to the guidelines established by the FDA for general biological products standards: 21 CFR,

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parts 610.11 (general safety), 610.12 (microbial sterility), and 610.13 (pyrogenicity). GBS II CPS and II-TT vaccines satisfactorily passed these tests. Clinical trials with GBS II vaccines. We performed 2 separate phase I and II trials. Study participants were women who met each of the following criteria: 18–45 years old; good health without acute or chronic illness; use of an acceptable birth control method throughout the study; negative serum pregnancy test at study enrollment; not breast feeding; no TT immunization within the prior 12 months; no prior immunization with a GBS II vaccine; and no allergy to the preservative thimerosal. For the 30 women enrolled in the first trial, vital signs were recorded at study entry. Type II vaccines were evaluated in 2 clinical trials. In the first trial, the safety and immunogenicity of II-TT conjugate and uncoupled II CPS vaccine were assessed. Subjects were randomized to receive 1 of the 2 vaccines (n p 15 per group), either II-TT conjugate (57-mg dose of CPS component) or II CPS (45-mg dose), both at a volume of 0.5 mL in PBS containing 0.01% thimerosal. The second study further assessed the safety and immunogenicity of II-TT conjugate vaccine while determining the optimal dose of conjugate to elicit an immune response. Subjects were randomized to receive 1 of 3 4-fold decreasing doses of II-TT conjugate (n p 15 per dose group). The CPS doses of conjugate given to these 3 groups were 57 mg, 14.3 mg, and 3.6 mg, respectively, each delivered in a final volume of 0.5 mL in PBS and 0.05% thimerosal. All IITT CPS–containing vaccines and the saline placebo were delivered as a single 0.5-mL intramuscular injection in the deltoid region. Of the 75 women enrolled in these 2 studies, 50 (66.7%) were white, 9 (12%) were Hispanic, 8 (10.7%) were Asian, 7 (9.3%) were black, and 1 (1.3%) was of other or unknown race. For assessment of reactogenicity, subjects and study personnel, except for the nurse administering the injections, were blinded to vaccine group assignment. Subjects were observed by study personnel in the clinic for 15–30 min after vaccination. They were interviewed by telephone the following day and were examined in the clinic 2 days after vaccination. When present, systemic symptoms, including temperature and injection site signs and symptoms, were recorded by subjects daily for 8 days. Two subjects inadvertently received II-TT conjugate dose of 14.3 mg 16 weeks after entry into the first trial, in which they were immunized with uncoupled II CPS. Reactogenicity data are included for these subjects, but they are excluded from analysis of the immunogenicity data for the dose-response trial, which thus reflects a total of 13, rather than 15, subjects at the 14.3-mg/mL dose. One subject failed to develop an immune response to either vaccine, and the other had high concentrations of II CPS–specific IgG after immunization with both vaccines. Serological methods. Blood samples were obtained immediately before and at 2, 4, 8, and 26 weeks after vaccination. Fifteen subjects in the first trial also had blood specimens collected 104 weeks after vaccination. II CPS–specific IgG, IgA, and IgM in serum samples were measured by ELISA with II CPS covalently linked to human serum albumin (HSA) as the coating antigen. Methods for these ELISAs were essentially the same as described elsewhere for the quantitative determination of III CPS–specific IgG [20]. Competitive inhibition experiments indicated that the conjugation of II CPS to HSA does not alter epitope specificity,

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because the concentration of uncoupled II CPS and II-HSA conjugate that inhibited 50% of the binding of II CPS–specific IgG to II-HSA–coated plates was identical. For the IgA and IgM ELISAs, the quantitation was accomplished using F(ab’)2 fragments of goat antihuman IgA (Chemicon, Temecula, CA) or IgM (Southern Biotechnology, Birmingham, AL) as the coating antigen, and a standard curve was derived, using purified standard human IgA or IgM (Sigma Chemical, St. Louis, MO), respectively. In each assay, a standard human reference serum known to contain 23.1 mg/mL of IgM CPS–specific and 4.8 mg/mL of IgA CPS–specific antibody was included as a control. The lower limit of detection for the II CPS–specific IgG ELISA was 0.06 mg/mL. For IgA, the lower limit of detection was 0.02 mg/mL, and, for IgM, it was 0.05 mg/mL. The results were expressed as geometric mean concentrations (GMCs) of II CPS–specific IgG, IgA, or IgM, ranges, and 95% confidence intervals (CIs). A radioactive antigen-binding assay (RABA) employing purified II CPS extrinsically labeled (but not structurally altered) with tritium was used to quantify CPS-specific antibodies in sera, as described elsewhere [21]. Opsonophagocytic assays. Pre- and 4-week–postimmunization sera from selected vaccine recipients of II-TT conjugate vaccine (n p 8) and of uncoupled II CPS vaccine (n p 8) were tested in vitro for their ability to promote killing of GBS II strain 78-471. This strain contains both the a and b C proteins and is sensitive to opsonophagocytic killing only in the presence of specific immune sera. Sera were processed within 1 h of collection, to preserve endogenous complement, and were tested in the presence of adult polymorphonuclear leukocytes at a final concentration of 10% in the reaction mixture [22]. Results were expressed as the differences between the number of colony-forming units of GBS before and after incubation for 40 min at 377C and as the mean log10 reduction. Correlation coefficients (r) between the log10 decrease in colonyforming units and the CPS-specific IgG concentrations in these sera were determined by Spearman’s rank correlation. Statistical analysis. Because the clinical trials for GBS II used the same vaccine lot, performance statistics for each vaccine preparation were derived from measurements in all recipients of that preparation. Natural logarithms of antibody concentrations were assumed to be normally distributed. Antibody concentrations less than the lower limits of detection by the IgG, IgA, or IgM ELISAs were designated as censored (i.e., their exact values [mg/mL] are in the intervals 0.00–0.06, 0.00–0.20, and 0.00–0.50, respectively). Maximum likelihood estimation of antibody GMCs and associated CIs was based on the results of experiments in which some observations were censored (S-Plus version 3.4; Mathsoft, Cambridge, MA). Vaccine response distributions were illustrated with the reverse cumulative distribution plot [23]. Geometric mean distributions of antibody concentrations by vaccine group were compared nonparametrically by a modification of the Wilcoxon rank sum test and by the repeated measures Wilcoxon [24]. Differences between proportions of vaccine recipients exhibiting certain response characteristics (reactogenicity or 4-fold increase in antibody concentrations at intervals after vaccination) were evaluated by Fisher’s exact test. The GMC of II CPS–specific IgG among recipients of the lowest II-TT, compared with that of recipients of uncoupled CPS, was determined by a global test that contrasted average effect over all postvaccine visits [24].

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Figure 1.

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Schematic of the repeating unit of the type II capsular polysaccharide of group B Streptococcus

cacious, with average survival rates of 54% and 43%, respectively. On the basis of these data, we sought to create for clinical use a II-TT vaccine with II CPS that had between 30% and 60% of its sialic acid residues converted to aldehydes as sites for binding to protein. GBS II-TT vaccine lot 94-2a was manufactured using a CPS with 35% sialic acid oxidation, which resulted in a vaccine that was composed of 44% (w/w) carbohydrate and 56% (w/w) protein. The structure of the type II polysaccharide is shown schematically in figure 1. Clinical testing of type II conjugate vaccine. The type II conjugate and the uncoupled II CPS vaccines were well tolerated when given to 75 healthy women of childbearing age as single intramuscular doses. No serious adverse effects were reported. Only 1 subject, a recipient of the II-TT vaccine at the 14.3-mg/mL dose, had a systemic reaction (table 1). Seventeen hours after vaccination, this individual developed chills, malaise, headache, and temperature to 37.87C. These symptoms resolved after 36 h and may have been related to vaccine administration. The most frequent injection site reaction was mildto-moderate pain, which occurred more frequently in women receiving a conjugate vaccine at 1 of the 2 higher doses than among recipients of the lower dose of conjugate vaccine or the uncoupled CPS. These small differences, however, were not statistically significant. Most women receiving either vaccine experienced, at most, only mild redness or swelling at the injection sites. Four subjects, each of whom received conjugate vaccine at the highest dose, experienced grade 2 (13 to .05. d P ! .05, compared with that of II CPS. e P ! .03, compared with that of prevaccination antibody concentrations. f P p .02 for 14.3-mg dose, compared with that of 3.6-mg dose (repeated measures Wilcoxon). g n p 13.

II-TT a,b 57 f 14.3 3.6 II CPS 45

Vaccine, dose in mg

Table 2.

c,d,e

e,g

3.4

(0.1–151; 1.3–9.2)

16.5 (0.7–321; 9.1–30.2) c,d,e 15.7 (1.4–143; 7.9–31.4) e 7.6 (0.5–87.6; 4.2–13.7)

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the specific antibody concentrations measured by RABA or ELISA in sera obtained 8 and 26 weeks after immunization. Before and 4 weeks after vaccination, sera collected to preserve endogenous complement were tested for functional activity in vitro, using an opsonophagocytosis assay for GBS II. There was a good correlation between the concentration of II CPS–specific IgG and opsonophagocytic activity of sera from subjects receiving II-TT or uncoupled II CPS vaccines (r p .57). At a given II CPS–specific IgG concentration, sera from women receiving either GBS vaccine had similar functional activity, affirming that the CPS epitope critical for opsonophagocytic activity is preserved in the CPS of the conjugate vaccine. The mean preimmunization log10 reduction in GBS II cfu was !0.3 and did not differ significantly between the 2 subject groups. Similarly, the mean log10 reduction in cfu of 0.9 or 0.62 for recipients of II-TT and uncoupled CPS 4 weeks after immunization, respectively, did not differ significantly.

Discussion Prevention of perinatal GBS infection has become an attainable goal. Implementation of intrapartum antimicrobial prophylaxis and its attendant significant decrease in the incidence of early-onset GBS infection afford a view of the potential victory over the devastation caused by these infections [1]. The development of a GBS vaccine and its incorporation into routine prenatal care have again been given a level I status by the Institute of Medicine for the development of vaccines in the 21st century [26]. This favorable priority status incorporates an estimate of the feasibility and cost effectiveness of a vaccine prevention method, as well as its favorable impact on societal health. Because recent epidemiological surveys indicate that the disease burden and mortality from GBS disease are greater now among adults than among newborn infants, the potential impact of a pentavalent GBS protein–CPS conjugate vaccine is vast. By comparison with the CPSs of other GBS serotypes, the type II antigen is structurally more complex. It contains the same component monosaccharides as those in the other clinically relevant GBS serotypes. These sugars, however, are in a molar ratio that differs from the other antigens. The II CPS repeating backbone has 2 monosaccharide side chains. One contains only sialic acid and is less susceptible to acid cleavage than sialic acid residues found on types Ia, Ib, III, and V polysaccharides [21]. The other monosaccharide side chain is galactose. In constructing the first II–TT conjugate, controlled periodate oxidation resulted in the conversion of 7% of sialic acid residues to an analogue of sialic acid, 5-acetamido-3, 5dideoxy-D-galactosyloctulosonic acid, and TT was conjugated to free aldehyde groups created on the oxidized sialic acid by reductive amination. This II-TT vaccine, in contrast to uncoupled CPS, which failed to elicit type II–specific antibody, was immunogenic in rabbits [6]. This same II-TT vaccine, as a component of a tetravalent preparation, provided protection

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against homologous serotype challenge in neonatal mice whose dams had received tetravalent immunization [9]. In the present report, studies of GBS II vaccines were undertaken in an effort to design a preparation that would provide optimal immunogenicity in humans and to ensure that antibodies to this GBS serotype would be functional in vitro and protective in vivo. It has been shown for GBS III that the degree of polysaccharide-to-protein cross-linking influences immunogenicity and that opsonic activity in a murine model of infection was greatest in mouse serum raised to a moderately cross-linked CPS [27]. Of the 3 vaccines tested in animals, the II57%-TT was the most immunogenic and protective in the maternal mouse vaccination–neonatal challenge model of GBS II disease, perhaps because of exposure to or stabilization of conformational epitopes or to effects related to B and T cell interactions, as hypothesized for type III conjugate vaccines prepared with different degrees of cross-linking [27]. These experimental results led to the preparation of a II-TT conjugate vaccine, with between 30% and 60% of its sialic acid residues converted to aldehydes for use in the human subject trial. The II-TT vaccine tested clinically had an oxidation of 35% of the total sialic acid residues on II CPS. As reported for Ia-TT, Ib-TT, and III-TT conjugate vaccines, the II-TT conjugate vaccine and the uncoupled CPS were well tolerated by healthy nonpregnant women. Only 1 subject had a systemic reaction, which consisted of symptoms of chills, malaise, and headache, which occurred on the day after vaccination, accompanied by low-grade fever that may have been attributable to the TT component of the vaccine. Such systemic reactions accompany TT immunization in adults. Either no reactions or mild injection site reactions were observed in most subjects. The conjugate was a significantly better immunogen than was the uncoupled polysaccharide. A reverse cumulative distribution plot (figure 2) of II CPS–specific IgG concentrations 8 weeks after vaccination indicated that 97% of women in the conjugate vaccine group had potentially protective IgG concentrations (11 mg/mL) in their sera. The dose-response study indicated the superiority of the 2 higher doses over the 3.6-mg dose, but even the low dose resulted in II CPS–specific IgG concentrations 11 mg/mL in 93% of subjects. The CPS-specific IgG response was rapid and reached its maximum GMC 2 weeks after immunization. Similarly, high GMCs in CPS-specific IgG persisted 2–6 weeks later. This early peak for the II-TT conjugate could relate to a “booster” effect among subjects who had higher preimmunization CPS-specific IgG concentrations in their sera. In concept, the CPS-specific IgG response is similar to that observed when Ia-TT, Ib-TT, and III-TT GBS conjugates were administered to healthy women [10, 11]. The magnitude of the CPS-specific IgG responses from each of the GBS glycoconjugate vaccines tested thus far ensure that adequate CPS-specific IgG would be available for placental transport, if a pentavalent GBS vaccine were administered in the third trimester of pregnancy. In addition,

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15

13

30

a

1.79 (0.03–15.2; 0.70–4.59) 0.13 (0.2–7.0; 0.05–0.33)

a

0.91 (0.2–10.8; 0.39–2.13) a 0.11 (0.01–0.6; 0.05–0.25)

a

0.83 (0.2–4.7; 0.35–1.93) a,b 0.07 (0.02–0.5; 0.03–0.17)

1.01 (0.03–10.4; 0.51–2.01) a,b 0.08 (0.03–0.6; 0.04–0.16)

0

a,b,c,d

a,b,c,d

4

a,b,c,d

8

a,d

5.18 (0.3–151; 1.81–14.85) d 3.91 (0.6–19.7; 1.36–11.2)

a,d

6.26 (0.8–39.4; 3.41–11.49) d 2.28 (0.6–10.9; 1.24–4.18)

a,b,c,d

15.24 (4.6–68.0; 6.94–33.43) a,c,d 8.22 (2.5–113; 3.74–18.03)

a,d

5.00 (0.2–209; 1.78–14.08) d 2.86 (0.7–22.5; 1.02–8.04)

a,d

6.00 (1.2–42.2; 3.28–10.97) b,d 1.04 (0.3–4.0; 0.57–1.90)

a,b,c,d

13.63 (3.6–50.7; 6.01–30.93) a,c,d 3.38 (0.6–43.6; 1.49–7.66)

a,d

4.92 (0.2–204; 1.70–14.24) d 2.51 (0.4–19.4; 0.87–7.27)

a,d

5.50 (1.2–26.6; 2.97–10.18) b,d 0.81 (0.3–2.8; 0.44–1.51)

a,b,c,d

11.77 (3.3–31.5; 5.56–29.94) a,c,d 2.17 (0.3–26.7; 1.02–4.59)

29.62 (0.5–440; 16.34–53.71) 23.35 (0.6–486; 12.84–42.46) 18.28 (1.2–267; 10.03–33.32) a,c,d a,c,d a,c,d 9.25 (0.06–127; 5.10–16.78) 3.82 (0.03–63.6; 2.10–6.95) 2.33 (0.04–42.3; 1.28–4.25)

2

NOTE. GMC, geometric mean concentration; CI, confidence interval; II CPS, type II capsular polysaccharide; II-TT, II CPS–tetanus toxoid conjugate. a Not significant at P > .05. b P ! .05, compared with that of II CPS. c P ! .05, compared with that of 3.56-mg dose of II-TT. d P ! .02, compared with that of prevaccination antibody concentrations. e n p 13.

II-TT 57 IgM IgA 14.3 IgM IgA 3.6 IgM IgA II CPS 45 IgM IgA

GMC (mg/mL) of II CPS–specific IgM and IgA (range; 95% CI) at indicated week after vaccination

Isotype of immune response to type II group B Streptococcus vaccines.

Vaccine, No. of dose in mg recipients

Table 3.

a,d

a,d,e

3.83 (0.1–151; 1.44–10.19) de 1.74 (0.6–14.3; 0.65–4.62)

a,d

3.99 (1.1–20.0; 2.2–7.21) a,c 0.60 (0.2–2.5; 0.33–1.09)

a,c,d

6.68 (2.1–40.1; 3.35–13.33) a,c,d 1.48 (0.3–15.1; 0.74–2.96)

9.53 (0.5–137; 5.22–17.40) a,c,d 1.45 (0.8–2.4; 0.79–2.65)

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Type II GBS Glycoconjugate Vaccine

Figure 3. Geometric mean concentrations (GMCs) of antibodies to type II capsular polysaccharide (CPS) measured by a radioactive antigen-binding assay (RABA) or ELISAs (IgG plus IgM plus IgA) in sera from recipients of II CPS–tetanus toxoid (TT) conjugate vaccine at the 57-mg dose.

our studies demonstrated a significant correlation between CPS-specific IgG elicited in response to the II-TT conjugate or to the uncoupled II CPS vaccine and opsonization, phagocytosis, and killing of an opsono-resistant GBS II strain in vitro. When the isotype of the immune response was examined, both the II-TT and the uncoupled CPS vaccines elicited II CPS-specific IgM and IgA in substantial concentrations. In this regard, GBS II differs from the other GBS serotypes examined to date, in which the response to vaccination is predominantly of the IgG class. The control of the human antibody response to GBS III CPS has been shown to be influenced by T cells and to be optimal for IgM- and IgG-secreting cells when T cells comprised 10%–25% of the total cells in an in vitro culture system [28]. Improvement in antibody responses to polysaccharides is proposed to occur through linkage of the polysaccharide to proteinspecific T helper cells [29]. The specific B cell–T cell interactions responsible for recruitment of T cell help by glycoconjugate vaccines are incompletely elucidated. Adoptive transfer of splenocytes from mice immunized with the III-TT conjugate vaccine, however, confers anti-CPS immunologic memory to naive recipient cells in a murine adoptive lymphocyte transfer model [16]. Furthermore, the expression of specific molecules (B7-1 or B72) on antigen-presenting cells is sufficient for optimal T cell costimulation to a GBS glycoconjugate vaccine [30]. The T cells activated by the vaccine also had a pivotal role in determining the magnitude of the IgM response to the CPS. Finally, it has been shown that the first dose of a Haemophilus influenzae type b conjugate vaccine in an adult reactivated pre-existing memory B cells that had undergone extensive clonal selection, to result in a response primarily confined to IgA antibody-secreting cells [31]. The nature of the antigen also has a profound effect on the immunoglobulin class distribution of the antibody-secreting cell

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response [32]. Parenteral protein vaccines, such as TT, induce an IgG-dominated response, whereas a polysaccharide vaccine, even if administered parenterally, may elicit an IgA-dominated response. CPS-specific IgG, IgM, and IgA are produced in response to pneumococcal vaccination and infection. Johnson et al. [33] characterized the molecular forms of serum IgA to 3 types of pneumococcal CPS after vaccination and found that it remained primarily polymeric up to 1 year after immunization. In contrast, the IgA produced, in response either to the purified polyribosyl-ribitol-phosphate (PRP) of H. influenzae type b or to PRP conjugated to diphtheria toxoid, was predominantly monomeric. The persistence of polymeric IgA in response to vaccination with pneumococcal CPS was proposed to facilitate binding and clearance of pneumococci from the systemic circulation or to reflect limited maturation of the immune response to pneumococcal CPS. The immunological explanation(s) for the different distribution of isotype-specific responses to the II-TT, compared with that of the Ia-TT, Ib-TT, or III-TT vaccines, is of interest and requires further study. The National Vaccine Advisory Committee has examined the research and development pathways of several vaccines, such as the H. influenzae type b conjugate, that reached licensure expeditiously and some that were licensed only after considerable delay [34]. Some common themes that emerged included the expediting influence of a strong scientific background, the need for firm quantitation of disease burden and clear identification of target population, the availability of reliable animal models, high-quality reagents, and standardized assays to measure immune response. In addition, a critical role was noted for individuals or teams who acted as “champions” to overcome the inevitable obstacles encountered on the path to licensure. Taking its current stage of testing into account, the program for the development of GBS glycoconjugate vaccines satisfies each of the conditions associated with expeditious licensure that were voiced by the National Vaccine Advisory Committee. As with the former phase I and II testing of Ia-TT, Ib-TT, and III-TT vaccines, the II-TT conjugate vaccines are well tolerated and suitably immunogenic. They evoke specific antibodies to the CPSs within 2 weeks of vaccination, are functional in vitro, reach concentrations 11 mg/mL in almost all subjects, and are durable. It is becoming apparent that conjugates of a design similar to that in this and our previous studies [10, 11] have a high likelihood of preventing perinatal GBS disease. Acknowledgments We thank Pamela McInnes (National Institute for Allergy and Infectious Diseases) for advice and assistance; April Blodgett and Julianne Pinel (Channing Laboratory) for invaluable technical assistance in the production of vaccines, and Robin Schroeder for help in preparation of the manuscript; Judith Campbell, Claire Skeeter, and Sally Mason for assistance in recruitment and enrollment of subjects, collection of specimens, and performance of serological assays; and

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Morven S. Edwards (Baylor College of Medicine) for thoughtful review of the manuscript.

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