Abstract
Neisseria meningitidis serogroup C bactericidal titers and class-specific enzyme-linked immunosorbent assay (ELISA) antibody concentrations were measured in sera from 173 children (1 to 5 years old) before and 6 weeks and 7 months following vaccination with a quadrivalent (A/C/Y/W-135) polysaccharide vaccine. The immune responses of the children were compared with those of 40 adults 6 weeks postvaccination. Both bactericidal titers and ELISA antibody concentrations were significantly higher in the adults than in the children (P < 0.05). In addition, the ratio of immunoglobulin G (IgG) to IgM was higher in the children than in the adults. With an ELISA total antibody concentration of ≥2 μg/ml used as a measure of seroconversion, ≥84% of the individuals from each age group responded to the serogroup C polysaccharide. However, with a ≥4-fold-increase in bactericidal titer used, only 18% of 1-year-olds, 32% of 2-year-olds, and 50 to 60% of 3-, 4-, and 5-year-olds seroconverted. The ELISA results suggest that >50% of all children retained ≥2 μg of total antibody per ml at 7 months postimmunization. However, the bactericidal titers suggest that <10% of children <4 years old retained a ≥4-fold increase at 7 months following vaccination. Of particular note, 59 of 79 sera (75%) from the 1- and 2-year-olds had high ELISA antibody concentrations (2 to 20 μg/ml) with no associated bactericidal titer (<1:8). Discordant results between bactericidal titers and ELISA antibody concentrations were not explained by the presence of IgA blocking antibody or relative levels of IgG and IgM. The bactericidal results show age-dependent differences in the production and retention of antibody in young children immunized with serogroup C polysaccharide; these differences are not evident with the ELISA data.
There are approximately 1,200 cases of disease caused by Neisseria meningitidis serogroup C in the United States each year, and serogroup C outbreaks in the United States appear to be increasing (15). Although a higher proportion of community outbreak cases occur among persons 5 to 24 years of age, sporadic serogroup C cases occur mainly in young children.
The prevalence of meningococcal disease is inversely proportional to the presence of polysaccharide-specific bactericidal antibody in serum. Meningococcal disease rates in the United States are highest for those between 6 and 24 months of age, when naturally acquired serum bactericidal activity is lowest (10). Immunization with serogroup C polysaccharide vaccine produces bactericidal antibodies in both children (3 to 16 years of age) (5) and adults (38, 40). Recently, production of serogroup C bactericidal antibodies in response to polysaccharide vaccine was shown to be age dependent (17, 24). This corresponds with the observation of age-dependent efficacy of serogroup C polysaccharide vaccine (36).
Although some investigators have described the immunogenicities of meningococcal vaccines in terms of the presence of polysaccharide-binding antibody (measured by radioimmunoassay, radio-antigen binding assay [RABA], enzyme-linked immunosorbent assay [ELISA], or hemagglutination [HA]) (4, 7–9, 16–19, 23, 24, 26, 40), only bactericidal titers measured with the serum bactericidal assay (SBA) were shown to be associated with protection from meningococcal disease (10). In general, serogroup A bactericidal titers, in a limited number of sera from immunized adults, were shown to correlate with both RABA and HA assay results, although some individual samples gave discordant results when tested by different methods (16). In addition, a positive correlation was observed between ELISA total serogroup C antibody and bactericidal titers in sera from vaccinated military personnel (r = 0.7; P < 0.0001) (40). Recently, a positive correlation (r = 0.8) was observed between bactericidal titers and total ELISA antibody concentrations in sera from children 2 to 19 years old immunized with serogroup C polysaccharide vaccine (24). However, no such correlation was observed with sera from children <18 months old (r = 0.06; P = 0.6) (17).
Little is known about the class-specific antibody produced in response to meningococcal serogroup C polysaccharide vaccine. Gotschlich et al. (11) found bactericidal activity only in the immunoglobulin G (IgG) fraction of the serum from an adult volunteer immunized with serogroup A meningococcal polysaccharide; both the IgG- and the IgM- and IgA-containing fractions had HA activity. Skevakis et al. (34) showed that both IgG and IgM antibodies produced in response to serogroup C polysaccharide vaccine were bactericidal. Other investigators have studied the influence of class-specific antibodies on the bactericidal activity of polysaccharide-specific antibody produced in adults during infection. Käyhty (16), measuring serogroup A class-specific antibody in convalescent-phase sera by RABA, found that bactericidal activity correlated with the level of IgG. However, IgM was found to be the most lytic antibody in another study (12). Both of these studies demonstrated that IgA produced in individuals with meningococcal infections may block bactericidal activity.
Until recently (17, 24), little was known about the production of bactericidal antibody in young children immunized with serogroup C polysaccharide vaccine. A vaccination campaign initiated in response to an outbreak of serogroup C disease in Montana provided the opportunity to evaluate the immune response of young children to meningococcal polysaccharide. In 1992, seven cases of serogroup C meningococcal disease were identified in Butte-Silver Bow County, Mont., giving an incidence rate of 20.5/100,000 for the population (>20 times the expected rate) (15). Four of the seven cases were in children under 2 years old, giving an incidence rate of 440/100,000 for this population (>140 times the expected rate for this age group). In response to the outbreak, vaccination with a single dose of quadrivalent (serogroup A/C/Y/W-135) meningococcal polysaccharide vaccine was offered to all children and adolescents 1 to 17 years old living in Butte-Silver Bow County. No cases of meningococcal serogroup C disease were reported in the region following the vaccination campaign.
ELISA class-specific antibody concentrations and serum bactericidal titers were measured in paired (pre- and 6-week-postimmunization) sera from 173 children (1 to 5 years old) and 40 adults who received the quadrivalent meningococcal polysaccharide vaccine. The correlations between these antibody concentrations and serum bactericidal titers, as well as age-dependent differences in the response to polysaccharide vaccine, were evaluated. In addition, children’s sera from 7 months and 1 year postimmunization were analyzed for retention of both serogroup C ELISA antibody and bactericidal titers.
MATERIALS AND METHODS
Participants.
Healthy children (n = 173), all residing in Butte, Mont., and adult military personnel (n = 40) from different geographic locations (40) were immunized with an N. meningitidis quadrivalent (A/C/Y/W-135) polysaccharide vaccine (Menamune; Connaught Laboratories Inc., Swiftwater, Pa.). Sera were obtained after informed consent from either the individual or the legal guardian. Paired pre- and 6-week-postimmunization sera were obtained from 55 children 12 to 23 months old (1-year-olds), 31 children 24 to 35 months old (2-year-olds), 36 children 36 to 47 months old (3-year-olds), 32 children 48 to 59 months old (4-year-olds), 19 children 60 to 71 months old (5-year-olds), and 40 adults (20 to 30 years old). All children from Montana were asked to return at 7 months postimmunization; however, some failed to return. Seven-month-postimmunization sera were obtained from 42 (76%) of the 1-year-olds, 15 (48%) of the 2-year-olds, 24 (67%) of the 3-year-olds, 20 (63%) of the 4-year-olds, and 14 (74%) of the 5-year-olds. Only 20 Montana children who still had measurable bactericidal titers (≥1:8) at 7 months were requested to return 1 year following immunization. At that time, sera were obtained from two of the 1-year-olds, three of the 2-year-olds, five of the 4-year-olds, and three of the 5-year-olds. All sera were stored at −70°C until tested for serogroup C antibody.
ELISA.
N. meningitidis serogroup C polysaccharide-specific antibodies were measured by a standardized ELISA (8). The serogroup C polysaccharide used to coat the microtiter plate wells was provided by Connaught Laboratories. The antibody concentrations in each serum sample were measured relative to a standard reference serum, CDC1992 (14), and data were analyzed by the method of Plikaytis et al. (27). The class-specific antibody concentration assignments for CDC1992 were as follows: IgG, 24.1 μg/ml; IgM, 2.3 μg/ml; and IgA, 5.9 μg/ml. Polysaccharide-specific IgG, IgM, or IgA antibodies in each unknown serum sample were measured by using murine monoclonal antibodies (anti-human IgG HP6043, anti-human IgM HP6083, and anti-human IgA HP6123) conjugated with alkaline phosphatase (American Qualex International, Inc., La Mirada, Calif.). Total antibody was determined by the addition of the individual measurements of IgG, IgM, and IgA polysaccharide-specific antibodies. All other conditions were as described previously (8).
SBA.
N. meningitidis serogroup C bactericidal titers were determined as described earlier (22), except that the target strain (G8050) was recently isolated from an outbreak in the United States (15). The target strain was grown to log phase in sufficient volume to complete all tests with a single lot. The strain was serotyped (C:2a,P1.2:L3,7,9) as described previously (22). The complement source was pooled sera from 3- to 4-week-old rabbits (Pel-Freeze Biologicals, Brown Deer, Wis.). All of the adult sera and 28 of the postimmunization child sera were also tested for bactericidal titers by using the target strain C11 (the strain that is recommended for the standardized SBA [22] and is the vaccine type strain [C:NT,P1.1:L3,7,9]). All titers were within ±1 dilution from the titers obtained with G8050. A strain isolated from a patient in Montana (G7880; C:2b,P1.2:L3,7,9) was unavailable when the testing of the sera began. Subsequent testing of Centers for Disease Control and Prevention quality control sera with this strain gave titers similar (±1 dilution) to the titers obtained with G8050 and C11 (22).
Immunoadsorption with monoclonal antibody.
IgA was removed from sera (selected based on high ELISA antibody concentration and low bactericidal titer) by immunoadsorption. An anti-human IgA monoclonal antibody, purified from ascites (HP6123) (28), was immobilized on cyanogen bromide-activated Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden) (21). Each serum sample was incubated with the monoclonal antibody-bound Sepharose for 2 h at room temperature with constant rotation. The suspension was centrifuged for 15 s at 1,600 rpm in a microcentrifuge. The supernatant was removed and assayed for bactericidal titer and for IgA and IgG ELISA antibody.
Data analyses.
Geometric mean antibody concentrations and bactericidal titers were determined from the log2-transformed concentrations and reciprocal bactericidal titers, respectively. Bactericidal titers of <1:8 were assigned a titer of 4, and antibody concentrations of <0.02 μg/ml were assigned a concentration of 0.01 μg/ml for analyses. The differences between pre- and postimmunization (6-week or 7-month) antibody levels and between 6-week and 7-month-postimmunization antibody levels were measured by the Wilcoxon signed rank sum test. Differences in response between age groups were measured by Kruskal-Wallis one-way analysis of variance on ranks; between age groups, pairwise comparisons were done by Dunn’s method (significance was tested at P < 0.05). Association of ELISA antibody concentrations with bactericidal titers was measured by Spearman correlation analyses. All comparisons were considered significant at P < 0.05. Seroconversion was defined and reported as a ≥4-fold rise in bactericidal titer (based on recommendations for meningococcal vaccine licensure [39]), a postimmunization bactericidal titer of ≥1:8 (for comparison to earlier studies [7, 9, 17, 19, 23, 24]), or a postimmunization ELISA total antibody concentration of ≥2 μg/ml (the previously reported protective level [9]).
RESULTS
Total and class-specific antibody concentrations.
The antibody concentrations in the pre- and 6-week-postimmunization sera measured by ELISA are shown in Table 1. There was a significant increase in total antibody concentrations in each age group following immunization (P < 0.01 for each age group). IgG accounted for most of the serogroup C postimmunization antibody in each of the child sera; IgM levels were <1 μg/ml. Because of the wide range of antibody responses within each age group, there were no detectable age-related differences in antibody concentrations (total, IgG, IgM, or IgA) measured by ELISA in children from 1 to 5 years old (P > 0.05 between each age group). Total, IgG, IgM, and IgA antibody concentrations in the adult sera were significantly higher than those in any other age group (P < 0.05 for adults compared with each other age group). The median fold increases between pre- and 6-week-postimmunization total antibody levels were 7.8, 11.6, 26.3, 19.2, 14.0, and 31.0 for the 1-, 2-, 3-, 4-, and 5-year-olds and adults, respectively. In contrast to the differences between the adult and child antibody levels at 6 weeks postimmunization, the fold increases for the adults were significantly different (P < 0.05) only from those for the 1-year-olds. The fold increases in the child sera were similar (P > 0.05), except those for the 1-year-olds were significantly different (P < 0.05) from those for the 3- and 4-year-olds.
TABLE 1.
Age-dependent meningococcal serogroup C total and class-specific ELISA antibody concentrations
| Immunization status | Antibody class | Antibody concn (μg/ml)a in:
|
|||||
|---|---|---|---|---|---|---|---|
| 1-yr-olds (n = 55) | 2-yr-olds (n = 31) | 3-yr-olds (n = 36) | 4-yr-olds (n = 32) | 5-yr-olds (n = 19) | Adults (n = 40) | ||
| Preimmunization | Total | 0.43 (0.30–0.61) | 0.32 (0.18–0.57) | 0.37 (0.25–0.55) | 0.38 (0.26–0.56) | 0.41 (0.27–0.62) | 1.01 (0.68–1.48) |
| Postimmunization (6 wk) | Total | 4.61 (3.80–5.61) | 6.19 (4.64–8.25) | 8.90 (6.04–13.13) | 8.47 (6.25–11.48) | 6.24 (3.85–10.11) | 33.24 (23.55–46.90) |
| IgG | 3.64 (2.96–4.49) | 4.58 (3.52–5.97) | 6.62 (4.37–10.04) | 5.67 (3.98–8.08) | 4.16 (2.36–7.32) | 17.74 (11.54–27.26) | |
| IgM | 0.27 (0.23–0.31) | 0.29 (0.21–0.41) | 0.42 (0.30–0.59) | 0.67 (0.46–0.96) | 0.54 (0.37–0.78) | 3.45 (2.56–4.65) | |
| IgA | 0.39 (0.28–0.54) | 0.57 (0.27–1.20) | 1.09 (0.61–1.94) | 1.32 (0.94–1.85) | 1.19 (0.77–1.86) | 6.23 (4.25–9.12) | |
Geometric mean log2 antibody concentrations, with 95% confidence limits in parentheses.
Bactericidal titers.
The pre- and 6-week-postimmunization bactericidal titers for each age group are shown in Table 2. There was a significant rise in bactericidal titer in each age group following immunization (P < 0.01 for each age group). As with ELISA results, there was a significant difference in the 6-week-postimmunization bactericidal titers between the adult group and each age group of children (P < 0.05 for each comparison). In contrast to the ELISA results, the 6-week-postimmunization bactericidal titers measured in the sera from the 1-year-olds were also significantly lower than the titers measured in the sera from the 3-, 4-, and 5-year-olds (P < 0.05 for each comparison). The bactericidal titers in the sera from the 1-year-olds were similar to the bactericidal titers measured in the sera from the 2-year-olds (P > 0.05). There was no significant difference in the titers between the 3-, 4-, and 5-year-olds (P > 0.05 for each age group comparison).
TABLE 2.
Age-dependent meningococcal serogroup C bactericidal titers
| Immunization status | Titera in:
|
|||||
|---|---|---|---|---|---|---|
| 1-yr-olds (n = 55) | 2-yr-olds (n = 31) | 3-yr-olds (n = 36) | 4-yr-olds (n = 32) | 5-yr-olds (n = 19) | Adults (n = 40) | |
| Preimmunization | 4.0 (—b) | 4.3 (3.9–4.7) | 4.1 (3.9–4.2) | 4.0 (—) | 4.2 (3.9–4.5) | 9.0 (5.22–15.62) |
| Postimmunization (6 wk) | 7.2 (5.0–10.4) | 11.7 (6.0–20.5) | 38.1 (17.5–83.0) | 72.9 (34.1–155.9) | 44.4 (14.6–135.6) | 2,521.4 (1,450.5–4,383.0) |
Geometric mean log2 reciprocal titers, with 95% confidence intervals in parentheses.
—, no variability about the mean; all were <1:8.
Correlation between bactericidal titers and ELISA antibody concentrations.
The correlation between bactericidal titers and total antibody concentrations in 6-week-postimmunization sera of all age groups (n = 213) was significant (r = 0.74; P < 0.0001). With few exceptions, in each age group there was a significant correlation (P < 0.01) between bactericidal titers and total or class-specific antibody concentrations (Table 3). The Spearman correlation coefficient (r) increased with increasing age (from 1- to 3-year-olds) at the time of immunization. The correlations between bactericidal titers and either total or IgG antibody concentrations were similar within each age group.
TABLE 3.
Correlation between 6-week-postimmunization bactericidal titers and ELISA antibody concentrations
| Antibody class | Correlationa for:
|
|||||
|---|---|---|---|---|---|---|
| 1-yr-olds (n = 55) | 2-yr-olds (n = 31) | 3-yr-olds (n = 36) | 4-yr-olds (n = 32) | 5-yr-olds (n = 19) | Adults (n = 40) | |
| Total | 0.33 (0.01) | 0.46 (0.009) | 0.74 (0.0001) | 0.40 (0.02) | 0.76 (0.0002) | 0.66 (0.0001) |
| IgG | 0.34 (0.01) | 0.41 (0.02) | 0.72 (0.0001) | 0.37 (0.04) | 0.76 (0.0002) | 0.58 (0.0001) |
| IgM | 0.13 (0.35) | 0.49 (0.006) | 0.49 (0.003) | 0.48 (0.006) | 0.73 (0.0004) | 0.65 (0.0001) |
| IgA | 0.30 (0.03) | 0.50 (0.004) | 0.69 (0.001) | 0.34 (0.05) | 0.58 (0.01) | 0.51 (0.0008) |
Spearman correlation coefficient (r); P values are in parentheses.
Although positive correlations were obtained with all age groups, discordant results between ELISA antibody concentration and bactericidal titer at 6 weeks postimmunization were observed with some sera. Of 51 of the sera from the 1-year-olds (n = 55) with ELISA total antibody concentrations of ≥2 μg/ml, 41 (80%) had no measurable bactericidal titer (<1:8); 6 (15%) of these 41 sera had total antibody concentrations of >10 μg/ml. Twenty-eight of the sera from the 2-year-olds (n = 31) had antibody concentrations of ≥2 μg/ml. Eighteen (64%) of these sera had no measurable bactericidal titers; 2 (11%) of these 18 sera had total antibody concentrations of >10 μg/ml. Bactericidal activity was not observed in the sera from the 1- and 2-year-olds until the ELISA antibody concentration was >7 μg/ml. However, bactericidal activity was present with antibody concentrations as low as 3 μg/ml in sera from the 3-, 4-, and 5-year-olds. In all age groups except the 3-year-olds, at least one serum had no bactericidal titer even though the total antibody concentration was ≥20 μg/ml. Bactericidal activity was observed in all 6-week-postimmunization adult sera; none of these sera had total antibody concentrations of <4 μg/ml. Discrepancies between bactericidal titers and ELISA antibody concentrations could not be explained by relative differences in class-specific antibody concentrations. Sera from different individuals (even within a given age group) with equivalent concentrations of IgG, IgM, and IgA had different bactericidal titers.
Percent seroconversion at 6 weeks postimmunization.
The proportions of individuals in each age group who seroconverted are given in Table 4. Over 80% of the individuals in each age group had an ELISA total antibody response of ≥2 μg/ml. However, the production of bactericidal antibody was age dependent. The proportions of individuals who seroconverted increased with increasing age. Only two of the children had a preimmunization bactericidal titer of >4; only three had a preimmunization ELISA antibody concentration of ≥2 μg/ml. Eight adults had a preimmunization bactericidal titer of >4, and nine had a preimmunization ELISA antibody concentration of ≥2 μg/ml.
TABLE 4.
Percentage of seroconverted individuals in each age group at 6 weeks postimmunization
| Test criterion | % of seroconverted:
|
|||||
|---|---|---|---|---|---|---|
| 1-yr-olds | 2-yr-olds | 3-yr-olds | 4-yr-olds | 5-yr-olds | Adults | |
| ELISA antibody concn of ≥2 μg/ml | 93 | 94 | 92 | 94 | 84 | 100 |
| ≥4-fold rise in SBA titer | 18 | 32 | 53 | 66 | 58 | 98 |
| Titer of ≥1:8 | 18 | 35 | 56 | 75 | 68 | 100 |
Immunoadsorption with monoclonal antibodies.
Selected sera were tested for the presence of IgA antibody, which may block bactericidal activity. The sera were selected based on having a relatively high IgA ELISA concentration and low bactericidal titer. IgA antibody was removed from five sera from children of each age group by immunoadsorption with an anti-human IgA monoclonal antibody bound to Sepharose beads. The IgG and IgA antibody concentrations were measured before and after adsorption. IgA serogroup C meningococcal antibody was reduced by >95% in all sera; >90% of the IgG was recovered. There was no increase in bactericidal titer in any of the sera following removal of IgA.
In vivo decay of bactericidal and ELISA antibody levels.
Total antibody concentrations and bactericidal titers in serial (pre-, 6-week-, and 7-month-postimmunization) sera from 1- to 5-year-olds are shown in Table 5. In all age groups, the ELISA antibody concentration at 7 months was significantly higher (P < 0.002) than the preimmunization concentration; however, the concentration at 7 months was significantly lower (P < 0.0001) than the concentration at 6 weeks. For all age groups, the average decline between 6 weeks and 7 months postimmunization was ∼50%. Although the serial specimens from each individual differed, in general the IgG, IgM, and IgA concentrations at 7 months showed a decline of the same rate (one-half to one-third the level at 6 weeks). In contrast to the ELISA antibody concentrations, the bactericidal titers in the 7-month-postimmunization sera could not be distinguished (P > 0.1) from preimmunization titers, except for the 4-year-old group (P = 0.03). There was a significant decline (P < 0.03) in titer between 6 weeks and 7 months postimmunization for all age groups except the 1-year-olds (P = 0.07). The percentages of individuals in each group that still retained antibody levels reflecting seroconversion, as determined by ELISA antibody concentration or bactericidal titer at 7 months postimmunization, are given in Table 6.
TABLE 5.
Meningococcal serogroup C total ELISA antibody concentrations and bactericidal titers preimmunization and at 6 weeks and 7 months postimmunizationa
| Test | Immunization status | Concn (μg/ml) or titerb in:
|
||||
|---|---|---|---|---|---|---|
| 1-yr-olds (n = 43) | 2-yr-olds (n = 15) | 3-yr-olds (n = 24) | 4-yr-olds (n = 20) | 5-yr-olds (n = 14) | ||
| ELISA | Preimmunization | 0.37 (0.24–0.57) | 0.44 (0.25–0.78) | 0.32 (0.18–0.55) | 0.35 (0.19–0.62) | 0.37 (0.21–0.64) |
| Postimmunization | ||||||
| 6 wk | 4.61 (3.72–5.71) | 5.59 (3.79–8.27) | 8.43 (5.11–13.88) | 8.46 (6.08–11.79) | 6.35 (3.41–11.84) | |
| 7 mo | 2.34 (1.86–2.95) | 2.59 (1.64–4.10) | 3.31 (1.89–5.79) | 3.84 (2.69–5.49) | 2.89 (1.47–5.68) | |
| SBA | Preimmunization | 4.0 (—c) | 4.6 (3.8–5.6) | 4.1 (3.9–4.4) | 4.0 (—) | 4.2 (3.8–4.6) |
| Postimmunization | ||||||
| 6 wk | 6.1 (4.4–8.4) | 10.6 (5.3–21.0) | 32.0 (12.4–82.8) | 66.3 (22.5–195.5) | 43.1 (10.2–181.1) | |
| 7 mo | 4.8 (3.8–6.0) | 4.0 (—) | 5.5 (3.7–8.2) | 14.9 (5.8–38.4) | 13.1 (4.3–40.1) | |
Only those individuals who returned at 7 months are included.
For ELISA, geometric mean log2 antibody concentrations (with 95% confidence limits in parentheses) are shown; for SBA, geometric mean log2 reciprocal titers (with 95% confidence intervals in parentheses) are shown.
—, no variability about the mean; all were <1:8.
TABLE 6.
Percentage of seroconverted individuals in each age group at 7 months postimmunization
| Test criterion | % of seroconverted:
|
||||
|---|---|---|---|---|---|
| 1-yr-olds | 2-yr-olds | 3-yr-olds | 4-yr-olds | 5-yr-olds | |
| ELISA antibody concn of ≥2 μg/ml | 54 | 60 | 67 | 80 | 71 |
| ≥4-fold rise in SBA titer | 5 | 0 | 8 | 30 | 29 |
| Titer of ≥1:8 | 7 | 0 | 12 | 30 | 29 |
The number of 1-year-postimmunization sera (n = 13) available for analyses was limited, since only 20 individuals had measurable bactericidal titers (≥1:8) at 7 months postimmunization. However, none of the sera from the 13 individuals who returned at 1 year had measurable bactericidal titers (all titers were <1:8) (6-week titer range, 1:8 to 1:4,096; 7-month titer range, 1:8 to 1:2,048). In contrast, 10 of the 13 had ELISA total antibody concentrations of ≥2 μg/ml. One serum had 73 μg of total ELISA antibody per ml with no measurable bactericidal titer (<1:8). Eleven of the sera had little or no reduction (difference of <0.2 μg/ml) in IgM levels between the 7-month- and the 1-year-postimmunization sera. The reduction in total ELISA antibody concentration between 7 months and 1 year postimmunization ranged from 22 to 75%, with a mean of 40%.
DISCUSSION
Age-related differences in response to various polysaccharide vaccines have been clearly documented (6). The age-related development of cross-linking receptors (CR2) has been implicated as a cause of the lower immune response of infants to polysaccharide antigens (29). Since polysaccharides are not processed in major histocompatibility complex class II molecules and infants have fewer receptors for cross-linking carbohydrate moieties, the class response in infants is usually restricted and of low affinity. Studies with mice have shown that the IgG-to-IgM ratio produced in response to polysaccharide antigens is age dependent but that the ratio produced in response to oligosaccharide-protein conjugates is not age dependent (35). Human vaccine trials have focused on development of oligosaccharide- or polysaccharide-protein conjugates because of the observed differences in response to polysaccharide antigens between young infants and adults (6). However, the polysaccharide vaccine is the only currently licensed product available for protection against meningococcal disease. Recent studies (17, 23, 24) showed discrepancies between antibody binding in ELISA and bactericidal activity after immunization with the licensed meningococcal vaccine. These results prompted an evaluation of the class-specific response of children to meningococcal serogroup C polysaccharide.
Protective efficacy of serogroup A and C polysaccharide vaccines has been shown for adults (3, 37), and that of serogroup A polysaccharide vaccine has been shown for infants (≥3 months) and children (26). Protective efficacy of serogroup C polysaccharide vaccine has not been shown for infants <24 months of age (36). These observations suggest that infants <24 months of age produce antibody to serogroup C polysaccharide that is quantitatively and/or qualitatively different from the antibody produced by those ≥24 months of age. Our data show both quantitative (measured by ELISA) and qualitative (measured by SBA) differences in the serogroup C response between adults and children <5 years old. In addition, there are qualitative differences in the response between those ≤2 and those ≥3 years old. These results are similar to those obtained by Mitchell et al. (24), in which significant differences in bactericidal titers were observed between children 2 to 6 years old and children 9 to 19 years old (P < 0.05); quantitative differences were also observed between these age groups. A comparison of their data with this study suggests that 9- to 19-year-olds have a response intermediate between those of young children (<6 years old) and adults.
We compared the serogroup C class-specific responses in children and adults. The IgG and IgM antibody concentrations in sera from children (1- to 5-year-olds) were similar but were lower than those in adult sera. The geometric mean IgM antibody concentrations in the children’s sera were relatively low (<0.7 μg/ml). Both IgM and IgA concentrations were much higher in the adult sera than in the child sera. The IgA concentration in the adult sera was 15 times higher than that in the sera from the 1-year-olds; the IgG concentration was only 5 times higher in the adult sera. The significance of these age-related differences for protection from meningococcal disease is unknown.
Gold et al. (9) reported a significant decline in the serogroup C geometric mean antibody concentrations 7 months following immunization of infants. Our data also show a significant decline in ELISA antibody 7 months following immunization; however, bactericidal activity appeared to decline at a higher rate in the children <4 years old. King et al. (17) and Mitchell et al. (24) also observed a significant decline in both ELISA and bactericidal antibody 1 year following immunization of infants and children with polysaccharide vaccine. In our study, only 13 of 20 children with a bactericidal titer of ≥1:8 at 7 months had sera available at 1 year postimmunization. None of the sera from these children had measurable bactericidal activity after 1 year. Ten of the 13 sera had total antibody levels of ≥2 μg/ml, suggesting retention of meningococcal serogroup C antibody. Although the number of sera available at 1 year was small, the reduction in bactericidal titer appeared to be due to a reduction in IgG, not IgM, antibody. Thus, the absence of bactericidal activity was probably not due to a loss of IgM antibody. Our study did not include immunogenicity data for the adults beyond 6 weeks postimmunization; however, earlier studies (4, 40) showed that antibody levels (HA, ELISA binding, and bactericidal) in adults persist, although at reduced levels, for >5 years.
Of special note is that immunization with one serogroup C oligosaccharide-protein conjugate vaccine did not appear to provide bactericidal antibody in infants that was retained longer than that observed in our study (7). Although infants immunized with the conjugate vaccine responded with ELISA antibody concentrations (38.5 μg/ml) and bactericidal levels (geometric mean titer = 3,082) similar to the response to polysaccharide vaccine observed in our adult volunteers, the infants’ antibody levels declined significantly by 10 months postvaccination. The geometric mean antibody titer in sera from these infants dropped to 10, while 52% of the infants still had ≥2 μg of serogroup C antibody per ml (geometric mean concentration = 2.2 μg/ml). It is unknown at this time whether other meningococcal conjugate vaccines will produce antibody with similar characteristics. However, a significant advantage to the conjugate vaccine is the induction of immunologic memory (18). The long-term retention of antibody levels would not be as important as the generation of polysaccharide-specific memory B cells ready to respond to antigenic epitopes.
There was a positive correlation between ELISA total antibody concentrations and serum bactericidal activity (r = 0.74, P < 0.0001) that was similar to that observed in other studies (17, 22, 24, 40). However, except for the sera from the adults, some sera with high antibody concentrations (2 to 20 μg/ml) had no measurable bactericidal activity. The highest proportion of sera with these discordant results was from the 1- and 2-year-olds. The number of sera (n = 59) from the 1- and 2-year-olds that had an ELISA total antibody level of ≥2 μg/ml and yet had a bactericidal titer of <1:8 was high (75%). The reasons for the discordant results are unknown. Earlier studies with serogroup C convalescent-phase sera showed that IgA antibody blocked the bactericidal activity of IgG and IgM (12, 16). Although the presence of IgA blocking antibody cannot be totally dismissed in this study, removal of IgA from selected sera did not result in an increase in bactericidal titer. In addition, the sera from the 1-year-olds were more likely to have discordant ELISA and SBA results, and these sera had the lowest level of IgA. Discordant results also did not appear to be related to the relative IgG and IgM concentrations. Different sera with similar concentrations of IgG and IgM gave very different bactericidal titers.
Differences in IgG subclass production may result in differences in bactericidal titer, since IgG subclasses differ in their ability to activate complement (31). The ability to produce IgG2 has been shown to correlate with the ability to produce antibody to polysaccharides (32, 33). Milagres et al. (23) observed a relative increase in serogroup C IgG2 levels in older children (10 to 14 years) compared with younger children. Although the reduced ability of young children to form IgG2 antibodies may explain the lower level of antibody produced in 1- and 2-year-olds compared to all other age groups, this does not explain the high number of sera from the 1- and 2-year-olds which had relatively high antibody concentrations (2 to 20 μg/ml) but no bactericidal titer. IgG1 antibody, directed against Haemophilus influenzae type b (Hib) capsular polysaccharide, has been shown to be more efficient than IgG2 in eliciting bactericidal or opsonophagocytic activity (2). Perhaps differences in IgG1/IgG2 ratios are responsible for differences in bactericidal activity. Currently no reference standards are available to quantify meningococcal serogroup C IgG1 and IgG2 levels.
It is likely that differences in serogroup C bactericidal activity for a given antibody concentration are associated with antibody avidity. Differences in antibody avidity have been observed in sera from infants immunized with different Hib-protein conjugate vaccines (1, 20). In addition, Hetherington and Lepow (13) observed a positive correlation between Hib capsular polysaccharide-specific antibody affinity and bactericidal activity (r = 0.76; P < 0.04). Sera with low affinity had no bactericidal activity. Schlesinger et al. (30) observed an association between antibody avidity and bactericidal activity. The standardized meningococcal serogroup C ELISA possibly binds low-avidity antibody, which is not detectable in the bactericidal assay. To date, there are no studies which show the influence of low-avidity antibodies on protection from meningococcal disease.
Goldschneider et al. (10) demonstrated an inverse correlation between the presence of meningococcal bactericidal antibodies and the incidence of disease. Bactericidal activity appears to be critical for host defense against meningococcal disease (25). In addition, the demonstration of a fourfold rise in bactericidal titer following immunization is a requirement for production and release of a meningococcal polysaccharide vaccine (39). Our data suggest that the serogroup C meningococcal ELISA cannot be used as a substitute for the SBA in the evaluation of the immunogenicities of serogroup C meningococcal polysaccharide vaccines in young children. Until efficacy studies demonstrate the influence of ELISA antibody on protection from serogroup C meningococcal disease, the measurement of bactericidal titers must be used to evaluate the immunogenicities of current and developing serogroup C vaccines.
In summary, we report the class-specific antibody concentrations produced in response to serogroup C polysaccharide vaccine. This information will help in the evaluation of new vaccines to ensure a more effective serogroup C immune response. Our study shows both quantitative and qualitative differences between the antibodies produced in young children and adults in response to serogroup C polysaccharide vaccine. In general, for each age group, there was a positive correlation between ELISA antibody concentrations and bactericidal titers. However, many sera, particularly from the children <4 years old, had high concentrations of ELISA antibody with no measurable bactericidal titers. Serogroup C meningococcal ELISA may overestimate protective antibody levels produced by young children because of measurement of low-avidity antibodies. Alternatively, the bactericidal assay may underestimate protective antibody levels, since low-avidity antibodies probably are not measured and antibodies with opsonophagocytic activity (or other functional activity) may not be detected. Vaccine efficacy studies in conjunction with immunogenicity studies are needed to establish the serologic correlate to protection from serogroup C meningococcal disease.
ACKNOWLEDGMENTS
We thank Connaught Laboratories, Inc., for providing purified polysaccharide and American Qualex International for alkaline phosphatase-labeled monoclonal antibodies.
Portions of this research were funded by the National Vaccine Program Office.
REFERENCES
- 1.Agbarakwe A E, Griffiths H, Begg N, Chapel H M. Avidity of specific IgG antibodies elicited by immunization against Haemophilus influenzae type b. J Clin Pathol. 1995;48:206–209. doi: 10.1136/jcp.48.3.206. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Amir J, Scott M G, Nahm M H, Granoff D M. Bactericidal and opsonic activity of IgG1 and IgG2 anticapsular antibodies to Haemophilus influenzae type b. J Infect Dis. 1990;162:163–171. doi: 10.1093/infdis/162.1.163. [DOI] [PubMed] [Google Scholar]
- 3.Artenstein M S, Gold R, Zimmerly J G, Wyle F A, Schneider H, Harkins C. Prevention of meningococcal disease by group C polysaccharide vaccine. N Engl J Med. 1970;282:417–420. doi: 10.1056/NEJM197002192820803. [DOI] [PubMed] [Google Scholar]
- 4.Brandt B L, Artenstein M S. Duration of antibody responses after vaccination with group C Neisseria meningitidis polysaccharide. J Infect Dis. 1975;131S:S69–S72. doi: 10.1093/infdis/131.supplement.s69. [DOI] [PubMed] [Google Scholar]
- 5.Cadoz M, Armand J, Arminjon F, Gire R, Lafaix C. Tetravalent (A,C.Y, W 135) meningococcal vaccine in children: immunogenicity and safety. Vaccine. 1985;3:340–342. doi: 10.1016/s0264-410x(85)90266-x. [DOI] [PubMed] [Google Scholar]
- 6.Carlone G M, Wenger J D, Perkins B A, Maslanka S E, Popovic T. Haemophilus influenzae type b, Neisseria meningitidis, Streptococcus pneumoniae, and Corynebacterium diphtheriae vaccines. In: Rose N R, Conway de Macario E, Folds J D, Lane H C, Nakamura R M, editors. Manual of clinical laboratory immunology. 5th ed. Washington, D.C: ASM Press; 1997. pp. 458–469. [Google Scholar]
- 7.Fairley C K, Begg N, Borrow R, Fox A J, Jones D M, Cartwright K. Conjugate meningococcal serogroup A and C vaccine: reactogenicity and immunogenicity in United Kingdom infants. J Infect Dis. 1996;174:1360–1363. doi: 10.1093/infdis/174.6.1360. [DOI] [PubMed] [Google Scholar]
- 8.Gheesling L L, Carlone G M, Pais L B, Holder P F, Maslanka S E, Plikaytis B D, Achtman M, Densen P, Frasch C E, Käyhty H, Mays J P, Nencioni L, Peeters C, Phipps D C, Poolman J T, Rosenqvist E, Siber G R, Thiesen B, Tai J, Thompson C M, Vella P P, Wenger J D. Multicenter comparison of Neisseria meningitidis serogroup C anticapsular polysaccharide antibody levels measured by a standardized enzyme-linked immunosorbent assay. J Clin Microbiol. 1994;32:1475–1482. doi: 10.1128/jcm.32.6.1475-1482.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Gold R, Lepow M L, Goldschneider I, Draper T F, Gotschlich E C. Kinetics of antibody production to group A and group C meningococcal polysaccharide vaccines administered during the first six years of life: prospects for routine immunization of infants and children. J Infect Dis. 1979;140:690–697. doi: 10.1093/infdis/140.5.690. [DOI] [PubMed] [Google Scholar]
- 10.Goldschneider I, Gotschlich E C, Artenstein M S. Human immunity to the meningococcus. I. The role of humoral antibodies. J Exp Med. 1969;129:1307–1326. doi: 10.1084/jem.129.6.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Gotschlich E C, Goldschneider I, Artenstein M S. Human immunity to the meningococcus. IV. Immunogenicity of group A and group C meningococcal polysaccharides in human volunteers. J Exp Med. 1969;129:1367–1384. doi: 10.1084/jem.129.6.1367. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Griffis J M, Bertram M A. Immunoepidemiology of meningococcal disease in military recruits. II. Blocking of serum bactericidal activity by circulating IgA early in the course of invasive disease. J Infect Dis. 1977;136:733–739. doi: 10.1093/infdis/136.6.733. [DOI] [PubMed] [Google Scholar]
- 13.Hetherington S V, Lepow M L. Correlation between antibody affinity and serum bactericidal activity in infants. J Infect Dis. 1992;165:753–756. doi: 10.1093/infdis/165.4.753. [DOI] [PubMed] [Google Scholar]
- 14.Holder P K, Maslanka S E, Pais L B, Dykes J, Plikaytis B D, Carlone G M. Assignment of Neisseria meningitidis serogroup A and C class-specific anticapsular antibody concentrations to the new standard reference serum CDC1992. Clin Diagn Lab Immunol. 1995;2:132–137. doi: 10.1128/cdli.2.2.132-137.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Jackson L A, Schuchat A, Reeves M W, Wenger J D. Serogroup C meningococcal outbreaks in the United States. An emerging threat. JAMA. 1995;273:383–389. [PubMed] [Google Scholar]
- 16.Käyhty H. Comparison of passive hemagglutination, bactericidal activity, and radioimmunological methods in measuring antibody responses to Neisseria meningitidis group A capsular polysaccharide vaccine. J Clin Microbiol. 1980;12:256–263. doi: 10.1128/jcm.12.2.256-263.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.King W J, MacDonald N E, Wells G, Huang J, Allen U, Chan F, Ferris W, Diaz-Mitoma F, Ashton F. Total and functional antibody response to a quadrivalent meningococcal polysaccharide vaccine among children. J Pediatr. 1996;128:196–202. doi: 10.1016/s0022-3476(96)70389-x. [DOI] [PubMed] [Google Scholar]
- 18.Leach A, Twumasi P A, Kumah S, Banya W S, Jaffar S, Forrest B D, Granoff D M, LiButti D E, Carlone G M, Pais L B, Broome C V, Greenwood B M. Induction of immunologic memory in Gambian children by vaccination in infancy with a group A plus group C meningococcal polysaccharide-protein conjugate vaccine. J Infect Dis. 1997;175:200–204. doi: 10.1093/infdis/175.1.200. [DOI] [PubMed] [Google Scholar]
- 19.Lepow M L, Beeler J, Randolph M, Samuelson J S, Hankins W A. Reactogenicity and immunogenicity of a quadrivalent combined meningococcal polysaccharide vaccine in children. J Infect Dis. 1986;154:1033–1036. doi: 10.1093/infdis/154.6.1033. [DOI] [PubMed] [Google Scholar]
- 20.Lucas A H, Granoff D M. Functional differences in idiotypically defined IgG1 anti-polysaccharide antibodies elicited by vaccination with Haemophilus influenzae type B polysaccharide-protein conjugates. J Immunol. 1995;154:4195–4202. [PubMed] [Google Scholar]
- 21.Mannik M, Stage D E. Antibody-agarose immunoadsorbents: complete removal of classes of immunoglobulins from serum. J Immunol. 1971;106:1670–1672. [PubMed] [Google Scholar]
- 22.Maslanka S E, Gheesling L L, Libutti D E, Donaldson K B J, Harakeh H S, Dykes J K, Arhin F F, Devi S J N, Frasch C E, Huang J C, Kriz-Kuzemenska P, Lemmon R D, Lorange M, Peeters C C A M, Quataert S, Tai J Y, Carlone G M The Multilaboratory Study Group. Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. Clin Diagn Lab Immunol. 1997;4:156–167. doi: 10.1128/cdli.4.2.156-167.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Milagres L G, Lemos A P S, Meles C E A, Silva E L, Ferreira L H M L, Souza J A M, Carlone G M. Antibody response after immunization of Brazilian children with serogroup C meningococcal polysaccharide noncovalently complexed with outer membrane proteins. Braz J Med Biol Res. 1995;28:981–989. [PubMed] [Google Scholar]
- 24.Mitchell L A, Ochnio J J, Glover C, Lee A Y, Ho M K K, Bell A. Analysis of meningococcal serogroup C specific antibody levels in British Columbian children and adolescents. J Infect Dis. 1996;173:1009–1013. doi: 10.1093/infdis/173.4.1009. [DOI] [PubMed] [Google Scholar]
- 25.Nicholson A, Lepow I H. Host defense against Neisseria meningitidis requires a complement-dependent bactericidal activity. Science. 1979;205:298–299. doi: 10.1126/science.451601. [DOI] [PubMed] [Google Scholar]
- 26.Peltola H, Mäkelä P H, Käyhty H, Jousimies H, Herva E, Hällström K, Sivonen A, Renkonen O, Pettay O, Karankp V, Ahvonen P, Sarna S. Clinical efficacy of meningococcus group A capsular polysaccharide vaccine in children three months to five years of age. N Engl J Med. 1977;297:686–691. doi: 10.1056/NEJM197709292971302. [DOI] [PubMed] [Google Scholar]
- 27.Plikaytis B D, Holder P F, Pais L B, Maslanka S E, Gheesling L L, Carlone G M. Determination of parallelism and nonparallelism in bioassay dilution curves. J Clin Microbiol. 1994;32:2441–2447. doi: 10.1128/jcm.32.10.2441-2447.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Reimer C B, Phillips D J, Aloisio C H, Moore D D, Galland G G, Wells T W, Black C M, McDougal J S. Evaluation of thirty-one mouse monoclonal antibodies to human Ig epitopes. Hybridoma. 1984;3:263–275. doi: 10.1089/hyb.1984.3.263. [DOI] [PubMed] [Google Scholar]
- 29.Rijkers G R, Sanders L A M, Zegers B J M. Anti-capsular polysaccharide antibody deficiency states. Immunodeficiency. 1993;5:1–21. [PubMed] [Google Scholar]
- 30.Schlesinger Y, Granoff D M the Vaccine Study Group. Avidity and bactericidal activity of antibody elicited by different Haemophilus influenzae type b conjugate vaccines. JAMA. 1992;267:1489–1494. [PubMed] [Google Scholar]
- 31.Schumaker V N, Calcott M A, Spiegelberg H L, Müller-Eberhard H J. Ultracentrifuge studies of the binding of IgG of different subclasses to the C1q subunit of the first component of complement. Biochemistry. 1976;15:5175–5181. doi: 10.1021/bi00668a035. [DOI] [PubMed] [Google Scholar]
- 32.Shackelford P G, Granoff D M, Nahm M H, Scott M G, Suarez B, Nelson S J. Correlation of serum immunoglobulin subclass concentrations with antibody responses of children to immunization with Haemophilus influenzae type b polysaccharide-pertussis vaccine. J Clin Immunol. 1985;5:390–395. doi: 10.1007/BF00915336. [DOI] [PubMed] [Google Scholar]
- 33.Siber G R, Schur P H, Aisenberg A C, Weitzman S A, Schiffman G. Correlation between serum IgG-2 concentrations and the antibody response to bacterial polysaccharide antigens. N Engl J Med. 1980;303:178–182. doi: 10.1056/NEJM198007243030402. [DOI] [PubMed] [Google Scholar]
- 34.Skevakis L, Frasch C E, Zahradnik J M, Dolin R. Class-specific human bactericidal antibodies to capsular and noncapsular surface antigens of Neisseria meningitidis. J Infect Dis. 1984;149:387–396. doi: 10.1093/infdis/149.3.387. [DOI] [PubMed] [Google Scholar]
- 35.Stein K E. Thymus-independent and thymus-dependent responses to polysaccharide antigens. J Infect Dis. 1992;165:S49–S52. doi: 10.1093/infdis/165-supplement_1-s49. [DOI] [PubMed] [Google Scholar]
- 36.Taunay A, Galvao P A, de Morais J S, Gotschlich E C, Feldman R A. Disease prevention by meningococcal serogroup C polysaccharide vaccine in preschool children: results after eleven months in Sao Paulo, Brazil. Pediatr Res. 1974;8:429A. [Google Scholar]
- 37.Wahdan M H, Sallam A A, Hassan M N, Abdel Gawad A, Rakha A S, Sippel J E, Hablas R, Sanborn W R, Kassem N M, Riad S M, Cvjetanovic B. A second controlled field trial of a serogroup A meningococcal polysaccharide vaccine in Alexandria. Bull W H O. 1977;55:645–651. [PMC free article] [PubMed] [Google Scholar]
- 38.Weibel R E, Villarejos V M, Vella P P, Woodhour A F, Mclean A A, Hilleman M R. Clinical and laboratory investigations of monovalent and combined meningococcal polysaccharide vaccine, groups A and C. Proc Soc Exp Biol Med. 1976;153:436–440. doi: 10.3181/00379727-153-39563. [DOI] [PubMed] [Google Scholar]
- 39.World Health Organization. Requirements for meningococcal polysaccharide vaccine (requirements for biological substances no 23) WHO Tech Rep Ser. 1976;594:72–73. [Google Scholar]
- 40.Zangwill K M, Stout R W, Carlone G M, Pais L, Harakeh H, Mitchell S, Wolfe W H, Blackwood V, Plikaytis B D, Wenger J E. Duration of antibody response after meningococcal polysaccharide vaccination in US Air Force personnel. J Infect Dis. 1994;169:847–852. doi: 10.1093/infdis/169.4.847. [DOI] [PubMed] [Google Scholar]