Abstract
The presence of immunoglobulin G (IgG) antibodies against group B streptococcus (GBS) type III polysaccharide (PS) has been correlated with protection against GBS disease. The GBS type III PS is structurally similar to the pneumococcal type 14 PS, differing only in the presence of sialic acid residues. Four different preparations of GBS type III PS were evaluated for their specificity in enzyme-linked immunosorbent assay (ELISA): free PS, free PS mixed with methylated human serum albumin (mHSA), PS conjugated to biotin and PS conjugated to human serum albumin. Three groups of human sera were used to evaluate these PS preparations: sera from recipients of a GBS PS vaccine, sera from women receiving a GBS type III PS-tetanus toxoid conjugate vaccine, and sera from nonimmunized healthy women of childbearing age. Estimated antibody concentrations were different depending on the PS preparation used. Using any of the four preparations, we were able to measure ≤0.05 μg of IgG antibody to the GBS type III PS per ml. The specificity of the assay was determined by competitive inhibition with homologous and heterologous PS. The pneumococcal type 14 PS did not inhibit binding of antibody to the native GBS type III PS in sera from adults receiving the GBS PS vaccine or in sera from nonimmunized adults (except serum G9). The pneumococcal type 14 PS inhibited 50% in sera from recipients of GBS type III conjugate vaccine and in serum G9 when GBS type III PS conjugated to biotin or to HSA was used as antigen in ELISA. These data show that free GBS type III PS or PS mixed with mHSA is a sensitive and specific antigen for ELISA and that conjugation can alter the antigenic specificity of a PS.
Group B streptococci (GBS) are the leading cause of neonatal sepsis and meningitis (3, 13). The virulence of GBS is due to the presence of the type-specific polysaccharide (PS) capsule (28). The GBS PS induces type-specific antibodies that are opsonophagocytic and protective against GBS disease in human infants and animals (4, 12). Maternal immunoglobulin G (IgG) antibodies to the GBS PS protect the neonate from invasive GBS disease (6). There is a correlation between the risk for development of symptomatic GBS disease and low concentrations of maternal serum PS antibodies (7, 19). Nine different GBS serotypes have been isolated from humans (types Ia, Ib, II, III, IV, V, VI, VII, and VIII). Types Ia, III, and V are most prevalent in early-onset disease (5, 32). All GBS have a common group B cell wall antigen, composed of rhamnose, galactose, and N-acetylglucosamine (23, 27), but antibodies to this antigen are not protective against GBS disease (1, 24, 26). The PSs of all GBS types contain glucose, galactose, N-acetylglucosamine, and a side chain terminating in sialic acid (21). Although the PSs have structural similarities, they exhibit immunological specificity (13). Jennings et al. (17, 18) demonstrated that when the sialic acid of GBS type III PS is removed by treatment with neuraminidase, the structure of the PS becomes identical to that of the pneumococcal type 14 (PN-14) capsular PS. The structures of the GBS type III and PN-14 PSs are shown in Fig. 1. Antisera against PN-14 PS generally do not cross-react with the GBS type III PS and do not protect against GBS type III disease. Similarly, rabbit antisera against the type III GBS PS do not react with the PN-14 PS (19).
FIG. 1.
Structures of GBS type III PS and PN-14 PS repeating units.
The objective of the present study was to evaluate free GBS type III PS, PS mixed with methylated human serum albumin (mHSA), and PS conjugated to biotin or to HSA for their specificities and sensitivities for estimating concentrations of IgG antibody to the GBS type III-specific PS. This work was done in preparation for determining protective levels of maternal antibody in ongoing neonatal studies. Biotinylated and HSA-conjugated PSs have been used to estimate GBS type-specific antibody (8, 16), while free PS and PS mixed with mHSA have been used in enzyme-linked immunosorbent assay (ELISA) to measure antibodies to other bacterial PSs (2). The chemistry of conjugation for HSA and that for biotin are different. HSA is covalently coupled to C-7 or C-8 of sialic acid by reductive amination with sodium cyanoborohydride after the GBS type III PS is oxidized with sodium periodate (16). In contrast, biotin is randomly attached to the hydroxyl groups of the PS, following activation with cyanogen bromide and derivatization with adipic acid dihydrazide (8).
We show that free GBS type III PS or PS mixed with mHSA is sufficient as a sensitive and specific coating antigen for ELISA. We also show that following conjugation of the PS to either HSA or biotin, the resulting antigens bind both GBS type III-specific antibodies and PN-14-reactive antibodies. Therefore, these findings have broader implications because various PSs are commonly chemically modified by addition of proteins or peptides for use as ELISA antigens or for preparation of conjugate vaccines.
(This research was presented in part at the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 28 September to 1 October 1997.)
MATERIALS AND METHODS
Bacteria and sera.
The nontypeable GBS strain CDC 1073 was obtained from the Centers for Disease Control and Prevention, Atlanta, Ga. Reference serum 19, a pool of sera from adults immunized with a tetravalent PS vaccine (GBS types Ia, Ib, II, and III), was provided by North American Biologics Inc., Rockville, Md. (22). Reference serum SHRS-III, a pool of sera from five adults immunized with GBS type III PS conjugated to tetanus toxoid (TT), was provided by Dennis Kasper, Channing Laboratory, Harvard Medical School, Boston, Mass. (16). Immune globulin intravenous (IGIV) 004 and 006 were provided by Gerald Fischer, Department of Pediatrics, Uniformed Services University of the Health Services, Bethesda, Md., and were obtained from plasma of adults immunized with a tetravalent PS vaccine (GBS types Ia, Ib, II, and III) (14). Sera G1 through G12 were obtained from unimmunized healthy women of childbearing age. Polyclonal rabbit typing antiserum to PN-14 was obtained from the New York State Health Department, Albany.
Antigens used for ELISA.
PN-14 and PN-23F PSs were obtained from the American Type Culture Collection, Rockville, Md.; mHSA was provided by George Carlone, Centers for Disease Control and Prevention; Haemophilus influenzae type b (Hib) PS was obtained from Wyeth-Lederle Vaccines and Pediatrics, Rochester, N.Y.; GBS type Ia, Ib, II, and III PSs and GBS type III PS conjugated to biotin were obtained from North American Biologics Inc.; (8); and GBS type III PS conjugated to HSA was obtained from North American Vaccine Inc., Beltsville, Md., and from Dennis Kasper, Channing Laboratory, Harvard Medical School (16).
ELISA.
Four preparations of GBS type III PS were used as coating antigens: (i) free GBS type III PS, (ii) GBS type III PS mixed with mHSA, (iii) GBS type III PS conjugated to biotin (8), and (iv) GBS type III PS conjugated to HSA (16). Initial experiments for the PS mixed with mHSA indicated that 5 μg of GBS type III PS per ml and 0.5 μg of mHSA per ml were optimal for binding of immune and nonimmune sera. Increasing the concentration of mHSA was found to inhibit binding.
PS preparations were used to coat Immulon 4 plates in phosphate-buffered saline (PBS) (pH 7.4) and incubated overnight at 28°C. The plates were washed six times (with PBS–0.05% Tween 20) in an EL404 automated microplate washer (Bio-Tek Instruments, Winooski, Vt.). Reference and test sera were serially diluted twofold in triplicate. Dilution of sera was done in serum conjugate incubation buffer (PBS containing 0.1% Brij 35, 5% newborn calf serum, and 0.05% NaN3). The plates were incubated overnight at 4°C. An optimal dilution of anti-human IgG conjugated to alkaline phosphatase (Sigma, St. Louis, Mo.) was added, and the mixture was incubated for 2 h at 37°C. Then 100 μl of 1-mg/ml p-nitrophenyl phosphate (Sigma 104) in 1 M Tris–0.3 mM MgCl2 (pH 9.8) was added. Absorbance at 405 nm was determined 30 to 40 min after addition of substrate in a Bio-Tek 900C microtiter plate reader.
PN-14 PS antibodies were measured by using 2 μg of PN-14 PS per ml mixed with 0.5 μg of mHSA per ml as coating antigen. Pneumococcal C PS (1 μg/ml) was added to the serum conjugate incubation buffer to block C PS antibodies (11). Hib PS antibody was measured by using 5 μg of Hib PS per ml mixed with 5 μg of mHSA per ml as coating antigen (2).
Endotoxin-free water (Biofluids, Inc., Rockville, Md.) was used for all buffers to minimize nonspecific binding. Reference and test sera were assayed in triplicate. The absorbance was read 30 to 40 min after the addition of the substrate (p-nitrophenyl phosphate), and the absorbance values were extrapolated to 100 min for comparison between experiments. Reference serum 19 was used as a standard for calculating antibody concentrations in test sera. A weighted Log-Logit computer program was used to calculate the IgG concentrations (11).
Competitive inhibition assay.
A dilution of sera was selected from the upper half of the linear range of the dilution curve, mixed with various concentrations (0.04 to 5 μg/ml) of PS, and incubated at 37°C for 2 h. Then 100 μl of the mixture was added to the antigen-coated microtiter plates and incubated overnight at 4°C. Plates were washed and anti-human IgG enzyme conjugate and substrate were added as described above. Percent inhibition was determined by comparison of the absorbances with and without inhibitor.
Group PS.
Group B PS antigen was extracted from the nontypeable GBS strain CDC 1073 with nitrous acid as described previously (29), with slight modifications. Casein hydrolysate medium (0.5%) (10) was inoculated with GBS and incubated with shaking overnight at 35°C. The bacteria were centrifuged, washed twice with 0.15 M NaCl, and resuspended in 40 ml of water. Five milliliters of 4 M NaNO3 and 5 ml of acetic acid were stirred for 15 min. The cell-free supernatant was dialyzed extensively against water, concentrated with Centriprep (Amicon Corp., Danrivers, Mass.), and lyophilized. Nuclear magnetic resonance (NMR) spectroscopy was performed in the Laboratory of Biophysics, Center for Biologics Evaluation and Research (CBER), Bethesda, Md., to detect rhamnose, confirming the presence of group B PS.
Group B antigen (50 μg/ml) was incubated with sera for 2 h at 37°C and then added to the microtiter plates and incubated overnight at 4°C. ELISA was performed to compare absorbed and unabsorbed sera.
RESULTS
Relative assay sensitivity.
Standard reference serum 19 (8) and serum SHRS-III (16) with previously assigned GBS type III PS antibody concentrations were used to compare the sensitivities of four different PS preparations in ELISA for measuring less than 0.1 μg of antibody per ml. The slopes of absorbance versus antibody concentration were parallel for all methods (Fig. 2). PS conjugated to HSA or to biotin was slightly more sensitive than free PS or PS mixed with mHSA as coating antigen. When an antibody concentration of 80 ng/ml was used with reference serum 19, there was no difference in binding between any of the four different antigen preparations (Fig. 2A). When SHRS-III serum was used at the same antibody concentration, marked differences between antibody binding to conjugated PS preparations and antibody binding to free PS or PS mixed with mHSA were seen (Fig. 2B). Although the apparent sensitivity was dependent on the serum used, all four PS preparations measured at least 0.05 μg of IgG antibody to GBS type III PS per ml.
FIG. 2.
Relative sensitivities of ELISAs with four GBS type III PS preparations as antigens. Symbols: ⧫, free GBS type III PS; ■, GBS type III PS mixed with mHSA; ▴, GBS type III PS conjugated to HSA; ×, GBS type III PS conjugated to biotin. The assays were performed with reference serum 19 (A) and SHRS-III serum (B) based on their previously assigned IgG antibody concentrations.
Estimation of GBS type III IgG antibody in sera from immunized and nonimmunized adults.
Antibody concentrations calculated in micrograms per milliliter for IGIV 004 and type 006 (14) hyperimmune IGIV fall within a 10% range with the four different antigen preparations (Table 1). IgG concentrations for reference serum 19 were also comparable with those for all four antigens when IGIV 004 was used as the standard (data not shown). When PS alone or PS mixed with mHSA was used as the ELISA coating antigen, the GBS type III antibody concentration estimated in SHRS-III was less than half (33 μg/ml) of that estimated when PS conjugated to biotin or HSA (81 to 83 μg/ml) was used as coating antigen. The concentration of GBS type III antibody in sera from 12 adult female volunteers was also calculated with the four antigen preparations against reference serum 19 (Table 2). Antibody values obtained with free PS or PS mixed with mHSA as antigen in ELISA were similar for all sera. Similarly, the biotin- or HSA-conjugated PS also gave comparable IgG values, but IgG antibody concentrations were generally much lower than those measured by the free PS and PS mixed with mHSA. The geometric means of antibody concentrations were 5.7 μg/ml with free GBS PS as coating antigen and 0.7 μg/ml for the HSA-conjugated PS (P < 0.002 by Student’s t test). This suggests that the native PS has a conformation-dependent epitope whose expression is reduced following conjugation.
TABLE 1.
Estimation of anti-GBS type III and PN-14 PS IgG antibody concentrations in sera from GBS type III-immunized adults with different coating antigens in ELISA
TABLE 2.
Estimation of GBS type III and PN-14 IgG antibody concentrations in sera from nonimmunized adult female volunteers
| Serum | Concn of anti-GBS type III IgG by ELISA
(μg/ml)a
|
Concn of antibody to PN-14 (μg/ml) | |||
|---|---|---|---|---|---|
| Free PS | PS with mHSA | PS-biotin conjugate | PS-HSA conjugate | ||
| G1 | 6.0 | 7.9 | 0.7 | 0.1 | 1.0 |
| G2 | 12.0 | 14.0 | 6.3 | 5.9 | 0.6 |
| G3 | 3.0 | 4.5 | 0.7 | 0.1 | 1.6 |
| G4 | 24.3 | 31.0 | 11.9 | 18.4 | 2.8 |
| G5 | 2.5 | 3.3 | 0.7 | 0.3 | 2.5 |
| G6 | 4.0 | 5.1 | 1.7 | 1.2 | 1.8 |
| G7 | 12.1 | 8.0 | 2.9 | 3.9 | 3.3 |
| G8 | 3.0 | 4.0 | 0.7 | 0.1 | 0.6 |
| G9 | 4.1 | 3.4 | 2.8 | 5.2 | 15.0 |
| G10 | 4.6 | 3.0 | 1.2 | 1.2 | 1.3 |
| G11 | 6.3 | 6.4 | 0.9 | 0.2 | 0.3 |
| G12 | 4.3 | 5.1 | 0.4 | 0.1 | <0.1 |
Arithmetic mean of two to four assays.
Competitive inhibition showing assay specificity.
Competitive inhibition assays were performed with reference serum 19 (Fig. 3). With 0.04 μg of GBS type III PS, 90% inhibition was achieved when PS alone or PS mixed with mHSA was used as coating antigen. To achieve 90% inhibition with PS conjugated to biotin or HSA, 5 μg of GBS type III PS was necessary. Heterologous GBS type Ia, Ib, II, and V PSs, the GBS group B antigen, and PN-14 PS did not inhibit serum 19 in ELISA with any of the four antigen preparations. Competitive inhibition of other immune sera (IGIV 004 and 006) with GBS type III PS resulted in 90 to 100% inhibition with the homologous PS for all four PS preparations (data not shown).
FIG. 3.
Competitive inhibition with reference serum 19. Fivefold-increased PS concentrations from 0.04 to 5 μg/ml were used. Percent inhibition was determined by comparing the absorbances in the presence and absence of the PS inhibitor. III, II, Ia, Ib, and V, PSs from GBS types III, II, Ia, Ib, and V; B, GBS group B antigen; 14, PN-14 PS.
Competitive inhibition assays were also performed to evaluate the GBS type III specificity in sera from nonimmunized adult women (G1 to G12). With 5 μg of GBS type III PS, approximately 70 to 90% inhibition was achieved for all assays (data not shown). No cross-reactivity was seen with any heterologous inhibitors in any serum except G9. The results for G9 are described below.
Measurement of concentration of antibody to the PN-14 PS-reactive epitopes.
The structures of PN-14 and GBS type III PSs are identical except for the presence of terminal side chain sialic acid residues on the GBS type III PS (Fig. 1). We examined the degree to which the different assays measured antibodies to the PN-14-reactive epitope. One serum, G9, from a nonimmunized volunteer had a high (17-μg/ml) concentration of antibodies to the PN-14 PS, while the other sera from nonimmunized volunteers had a <3.5-μg/ml concentration of antibodies to the PN-14 PS (Table 2).
The SHRS-III immune-serum pool had a high concentration of antibodies (41 μg/ml) against the PN-14-reactive determinant as measured in the PN-14 ELISA (Table 1). In a competitive inhibition assay, 100% of this antibody was inhibitable in both SHRS-III and G9 with 1 μg of homologous PN-14 PS per ml.
Inhibition of SHRS-III and G9 sera with GBS type III and PN-14 PSs.
Competitive inhibition studies using SHRS-III were performed with all four PS preparations. We show the data at a single concentration because of the number of inhibitions involved (inhibitions were done with several concentrations of PS inhibitor, similar to the assays whose results are shown in Fig. 3). The homologous GBS type III PS inhibited SHRS-III 90 to 100% with all four PS preparations (Fig. 4). When GBS type III PS alone or mixed with mHSA was used as coating antigen, there was no inhibition with PN-14 PS. However, 50 to 55% inhibition with PN-14 PS was observed when the GBS type III PS antigen preparations conjugated to HSA or biotin were used to coat the ELISA plates. Similar results were observed with serum G9. Inhibition with GBS-III PS was approximately 80% for all methods in which G9 serum was used. Inhibition of SHRS-III or G9 with PN-14 PS was insignificant when free GBS III PS or PS mixed with mHSA was used as coating antigen but approximately 50% when conjugated PS was used as coating antigen. These data indicate that the GBS type III PS, when conjugated to a protein carrier, displays both PN-14- and GBS type III-specific epitopes.
FIG. 4.
Competitive inhibition with G9 and SHRS-III sera. A serum dilution corresponding to the upper portion of the linear range of a dilution curve was mixed with 5 μg of GBS type III PS per ml (A) or 5 μg of PN-14 PS per ml (B) and then preincubated at 37°C for 2 h. The rest of the GBS type III ELISA was performed as described in the text. Symbols: ■, free GBS type III PS; ▧, GBS type III PS mixed with mHSA; □, GBS type III PS conjugated to HSA; ░⃞, GBS type III PS conjugated to biotin.
Reactivity with rabbit PN-14 antiserum.
To confirm the above results, we examined the reactivity of a hyperimmune rabbit PN-14 typing serum with all four antigen preparations (Fig. 5). There was high reactivity even at a 1:64,000 serum dilution with the GBS type III PS conjugated-antigen preparations, but the cross-reactivity was observed only at 1:2,000 dilution with free PS or PS mixed with mHSA, similar to the results obtained with the heterologous Hib PS. The rabbit serum also reacted at high dilution against the native PN-14 PS (data not shown). This confirms that when GBS PS is conjugated to either biotin or HSA and used as a coating antigen, antibodies to both PN-14-reactive epitopes and GBS type III-specific epitopes are detectable.
FIG. 5.
Results of ELISA with a rabbit PN-14 typing antiserum. Free and conjugate GBS type III PSs and Hib PS were used for coating microtiter plates. The ELISA was performed by using twofold dilutions of the PN-14 antiserum, and the absorbance was extrapolated to 100 min. Symbols: ⧫, free GBS type III PS; ■, GBS type III PS mixed with mHSA; ▴, GBS type III PS conjugated to HSA; ×, GBS type III PS conjugated to biotin; ✻ Hib PS.
Absorption by group B antigen.
To analyze whether antibodies specific for the group B antigen could account for the differences in the estimated antibody concentrations among the methods, sera from nonimmunized individuals were absorbed with purified group B antigen and assayed by using GBS type III PS mixed with mHSA. For sera G1 through G12, there was no difference in antibody concentrations with and without preabsorption with B antigen (data not shown). To confirm this, a competitive inhibition assay was performed with B antigen and PN-23F PS. PN-23F PS contains terminal rhamnose residues, and antiserum against PN-23F PS cross-reacts with the B antigen (27). No inhibition was observed with either PS. To further rule out contamination of type-specific PS with group B PS, NMR proton spectrum analysis was performed. No rhamnose was detected in the GBS type III PS or in the HSA-conjugated PS. Thus, antibodies to the B antigen do not appear to account for the differences in estimations of antibody in nonimmunized sera.
Inhibition of serum binding with mHSA.
Competitive inhibition was used to determine if mHSA used for binding GBS type III PS interfered with the measurement of antibody. The mHSA did not interfere because no differences in inhibition were seen with sera from immunized and nonimmunized adults when GBS type III PS was used as a competitive inhibitor, alone or mixed with 0.5 μg of mHSA per ml (data not shown).
DISCUSSION
This is the first study to systematically compare several different PS preparations as coating antigens for their sensitivities and specificities in the measurement of GBS type III IgG antibodies. We found that conjugation of a protein carrier to PS can alter the PS conformation, exposing new epitopes. The sensitivities of all four antigen preparations in the ELISA are dependent on the serum used. All PS preparations measured IgG antibody concentrations as low as 0.05 μg/ml. Antibody concentrations estimated in different sera varied greatly depending on the PS antigen preparation used. The relative avidity of the antibodies measured by the different assays (data not shown) cannot account for the differences in the antibody concentrations estimated by different methods (15, 25). Competitive inhibition data suggest that conjugation of biotin or HSA to the PS resulted in expression of a PN-14-reactive epitope in addition to GBS type III-specific epitopes. When used as coating antigens, free GBS type III PS and PS mixed with mHSA do not display the PN-14-reactive epitopes. This was confirmed by the high reactivity of a hyperimmune anti-rabbit PN-14 serum only against GBS type III PS conjugated to either biotin or HSA. Thus, conjugation of the GBS type III PS to biotin or HSA may alter the GBS type III PS conformation to expose regions of the PS reactive with PN-14 antibodies.
ELISA with biotin- and HSA-conjugated PSs measured lower antibody concentrations in most nonimmune sera than ELISA with free PS or PS mixed with mHSA as coating antigen. We show that attachment of a protein to the GBS type III PS following conjugation causes a conformational change in the PS. The existence of a conformational epitope was reported in 1990 by Wessels and coworkers (30). It can be inferred that the conformational change in the PS that results in the PN-14-reactive epitope is in place of an alternative GBS type III-specific epitope. Evidence for this is seen in some sera from unimmunized healthy women which had higher antibody concentrations when free PS and PS mixed with mHSA were used as coating antigens than when conjugated PS was used (Table 2). Furthermore, the GBS type III conjugate vaccine induces antibodies to both GBS type III and PN-14 epitopes in humans (Table 1 and reference 8a).
Kasper et al. (19) showed that rabbit antiserum to PN-14 did not react with GBS type III PS in agar gel diffusion, while partially desialyated core PS reacted well with the antiserum. Inhibition of binding was observed for reference serum 19 only with the homologous GBS type III PS. Estimations of antibody concentrations in serum 19 were similar for all four PS preparations. In SHRS-III serum, higher GBS type III antibody concentrations were measured by ELISA with conjugated PS as antigen than with free PS. Antibodies in SHRS-III measured by using conjugated PSs as antigens were inhibitable with both GBS type III PS and PN-14 PS. In contrast, antibodies in SHRS-III estimated by using free PS were inhibitable only by the GBS type III PS. Furthermore, rabbit antiserum against PN-14 reacted strongly in ELISA when conjugated PS was used but not when PS alone or PS mixed with mHSA was used. Therefore, GBS type III PS conjugate preparations measure both GBS type III-specific antibodies and PN-14-reactive antibodies. Kasper and coworkers (19) also showed that antibody to PN-14 PS does not protect against GBS disease. They demonstrated that development of opsonic antibody and natural immunity to group B streptococcus was correlated with native GBS antibody rather than with antibody to the core PS.
Previous studies showed that the terminal sialic acid is not the immunodeterminant per se but exerts conformational control over the immunodominant epitope (9, 17, 18). Serology and NMR studies suggested that the carboxylate group of the sialic acid residues and the hydroxyl groups of the glucosamine residues are involved in intramolecular hydrogen bonding (31). This interaction of sialic acid with the hydroxyl groups was found to be important in defining the conformational GBS type III PS epitope. Jennings et al. (17) showed that antibody to the native GBS type III PS did not react with the PS if the carboxylate groups were reduced to hydroxymethyl groups. Oxidized PS, obtained by periodate oxidation of the sialic acid residues to heptulosonic acid, still reacts with antisera to native GBS type III PS.
NMR studies on the sialic acid-induced conformational change to the GBS type III core PS indicate that the sialic acid residues control the torsion angles of the glycosidic bond between the galactose and glucose residues (17). It is possible that the conjugation process interferes sufficiently with a portion of the hydrogen bonding to alter some of the antigenic specificity of the GBS type III PS so that it resembles the PN-14 PS specificity. The sialic acid-dependent epitopes do not appear to be distorted by mixing the PS with mHSA or by direct PS attachment.
Studies on the immune response in humans (20) and animals (30) to GBS type III PS conjugated to TT showed that GBS type III PS-TT induces antibodies to the native GBS type III PS. However, our data show that the SHRS-III reference serum pool prepared from sera from five individuals who had received the GBS type III PS-TT conjugate also contains high levels of antibody to a PN-14-reactive epitope. We found that SHRS-III had 33.0 μg of antibody to the native GBS type III PS per ml and 41.0 μg of antibody to the PN-14 PS per ml. Furthermore, we found that SHRS-III is opsonic for PN-14. This implies that the existing conjugate vaccines induce antibodies to a PN-14-reactive epitope in addition to inducing antibodies to GBS type III-specific epitopes.
Sialic acid is essential for the formation of the native GBS type III-specific antibodies, and it is the sialic acid-dependent epitope to which protective antibodies are directed (18). To determine the level of maternal capsular PS IgG antibodies sufficient to protect the neonate against GBS disease, an assay that measures the native GBS-specific antibodies and that correlates the antibodies with a known protective epitope should be used.
ACKNOWLEDGMENTS
We thank Ali Fattom and V. Pavliak for reference serum 19, GBS type III PS, and GBS type III PS-biotin and Dennis Kasper for the GBS type III PS-HSA conjugate and the SHRS-III immune serum. We also thank Milan Blake for the GBS type III PS-HSA conjugate, William Eagan for performing the NMR, and the women at CBER who donated sera. We thank John Robbins, Prem Seth, Kimi Lin, and Nelly Concepcion for their suggestions and discussion and also for critically reviewing the manuscript.
This work was supported by the Division of Epidemiology, Statistics and Preventive Research at the National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md., and by the Postgraduate Research Participation Program at CBER administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration.
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