RESULTS

Altogether 40 adult volunteers and 40 toddlers were vaccinated with one of the study vaccines (Table 2). Few pneumococcal polysaccharide-specific ASCs could be detected in the peripheral blood on day 0 in adults (I, II). After immunisation, their number increased rapidly, so that the peak number of ASCs was seen on day 7 or 9 after immunisation (I). On day 7 after immunisation ASCs were seen in all vaccinees to each of the serotypes (I, II). Thereafter, the number of ASCs decreased, and on day 28 no ASCs could be detected (I). The peak number of ASCs varied by vaccine used. As a whole, the responses in adults were higher after PncT and PncD conjugates than after PncPS and PncOMPC conjugate (p<0.001, except: p=0.034 for serotype 14 and p=0.004 for serotype 19F in the comparison of PncPS with PncD, and p=0.004 for serotype 14 in the comparison of PncPS with PncT) (Fig 4).

In toddlers, the ASC response was studied only on day 7 after immunisation. Pneumococcal polysaccharide-specific IgA, IgG, and IgM ASCs could be detected in all the 40 vaccinees (III). This was true for all the serotypes (for serotype 23F only 29 samples were analyzed), with one exception (one child did not respond to serotype 19F). In toddlers the responses were lower than in adults after PncD and PncT (p<0.001, except for p=0.015 for serotype 14 in comparison with PncT responses), but comparable to those seen in adults after PncPS (Fig 4).

The ASC response consisted mostly of IgA- and IgG-secreting cells, whereas the number of IgM ASCs remained low (2-28 ASC/10⁶ cells) (I-III). The dominant antibody class in the ASC response in most of the cases was IgA (Fig 4). In adults, this was true for all four serotypes after immunisation with PncPS and PncD and for two serotypes after PncOMPC (I, II). In response to PncT, however, the number of IgG ASCs exceeded the number of IgA ASCs (II). The peak number of IgA-ASCs (GM) was higher after vaccination with PncPS than after PncOMPC, but the number of IgG ASCs did not differ between PncPS and PncOMPC (I) (Fig 4). On the other hand, the number of IgA ASCs did not differ between PncD and PncT, but the number of IgG ASCs was 3.0 to 4.4-fold higher after immunisation with PncT than after PncD; the difference was statistically significant for serotypes 6B, 19F, and 23F (p=0.02). In toddlers, the ASC responses to all the serotypes were dominated by IgA (III) (Fig 4).

In the PncD and PncT vaccine groups we also measured ASC response to the carrier proteins, diphtheria toxoid or tetanus protein (II, III). The ASC responses were completely specific: no ASCs to diphtheria toxoid were detected in vaccinees immunised with PncT nor were any ASCs to tetanus protein found in vaccinees immunised with PncD. The ASC responses to the carrier proteins were clearly dominated by IgG in all the vaccinees (Fig 5).

Figure 4. The geometric mean number of IgA- and IgG-ASCs to pneumococcal serotypes 6B, 14, 19F, and 23F on day 7 after parenteral immunisation with the pneumococcal vaccines (with 95% confidence intervals shown).

Figure 5. The geometric mean number of ASCs to the carrier proteins (diphtheria or tetanus toxoid) on day 7 after immunization with the pneumococcal conjugate vaccines PncD and PncT in adults and toddlers (with 95% confidence intervals shown).

ASCs to the capsular PS of the pneumococcal type isolated from the MEF could be detected in all of the 17 patients studied, varying from 7 cells to 1100 ASC/10⁶ cells (all immunoglobulin classes combined). The geometric mean of the ASC response was clearly age-dependent; whereas the overall geometric mean (GM) was 32 ASC/10⁶ cells, it was 63 ASC/10⁶ cells in children older than 24 months and 18 ASC/10⁶ cells in these vaccinees (p = 0.03). Seven (78%) of the nine older children, and three (38%) of the eight younger children had at least 15 ASC/10⁶ cells. The ASC response was independent of gender.

Three children, all younger than 24 months, had their first AOM episode during the study (IV: Table). The causative agents were of serogroups 6 and 19 (two children). These children had only a low or no ASC response, but did not differ significantly from the other five children in this age-group (IV: Table). On the other hand, four older children had had at least 10 previous AOM episodes. Their infection was caused by group/type 9, 14 (two cases), or 19, and they all had an ASC response, although a weaker one than that of each of the other five children in this age-group.

In ten of the children in the study, the dominant antibody class was IgA. IgG-ASC were dominant in one child and IgM-ASC in another; both of these children had had over 10 AOM episodes in their lifetimes.

In order to confirm the specificity of the ASC response in the AOM patients, we enumerated the ASC response to a Pnc-type (6B, 14, 19F, or 23F) different from the type cultured in MEF. Most of the ASCs in the control assays were IgA-producing cells (IV: Table), and IgG or IgM-ASC were detected in only few patients. The number of IgA-ASCs thus detected was less than 10 ASC/10⁶ cells in all the children, supporting the specificity of the assay (Table in IV).

Pneumococcal PS-specific IgA antibodies were already detected in the saliva of most of the adult vaccinees before immunisation, most frequently to serotypes 14 and 19F (I, II). In toddlers, PS-specific IgA antibodies were detected only occasionally before immunisation (III). The increases in the IgA concentrations in adults were modest, but could be seen on day 7 after immunisation (I, II). A total of eight IgA responses (>twofold increase in salivary IgA concentration) was seen in six of the eight vaccinees (25% of the measurements showed a response) who had received PncPS, but only one response was seen in the ten vaccinees who had received PncOMPC (I). The responses were seen with equal distribution among the four serotypes (I). The 22 adults who had received PncD or PncT showed a salivary response in 32% of the measurements (II). The specific IgA concentration on day 28 ranged from <3 ng/ml to 222 ng/ml. The toddlers receiving PncD or PncT showed a more than twofold increase in 35% of the measurements, most frequently to serotype 19F; the specific IgA concentrations in saliva ranging from <3 ng/ml to 52 ng/ml on day 28.

Salivary IgA antibody concentrations showed an excellent correlation with the secretory component (SC) concentrations, indicating that the IgA measured was secretory in nature. This was true both before and after (day 28) immunisation (r=0.93-0.97; I-III).

IgG antibodies were rarely detected in saliva before vaccination. In adults, IgG antibodies could be detected on day 7, and more frequently on day 28. IgG responses were seen in one of the eight volunteers who had received PncPS, in three of the ten who had received PncOMPC, in seven of the twelve who received PncD and in eight of the ten who received PncT. In toddlers, IgG antibodies were not detected in any of the saliva samples before immunisation, and were found in only two samples even after immunisation (day 28).

Pneumococcal PS-specific IgG antibodies were already present in the serum of all the adult vaccinees before immunisation (I, II). The PncPS, the PncD, and the PncT vaccines induced serum antibody responses to all the four PncPS studied. The GM fold increase in IgG concentration varied from 3.5- to 6.0-fold after PncPS (I: Table 2) and from 10- to 22-fold after PncD/PncT (II: Table 3), depending on the serotype. Responses after PncOMPC were lower, less than 3-fold to each of the serotypes (I: Table 2). The IgM responses remained lower in response to each of the vaccines. The IgA concentrations were low before immunisation, below the detection limit in most of the vaccinees. The GM increase in IgA concentration varied from 1.0- to 2.5-fold after PncOMPC (I: Table 2) and from 2.4- to 7.7-fold after the other vaccines (II: Table 3), depending on the serotype.

In toddlers, IgG antibodies were detected in only 25 to 40% of the vaccinees before immunisation, depending on the serotype. IgM antibodies were present in 65 to 83% of the 40 children. After immunisation, increased concentrations of IgM antibodies were detected in virtually all vaccinees, and IgG antibodies were detected in 75 to 95%, depending on serotype. Serotype-specific IgA antibodies were very rarely present in the serum of the toddlers before immunisation (serotype 19F-specific antibodies detected in three of the 40 vaccinees). After immunisation, IgA antibodies were detected in 63 to 68% of the vaccinees on day 7 after immunisation and in 38 to 45% of the vaccinees on day 28 (III: Table 1). Thus the GM IgA antibody concentration was higher on day 7 than on day 28 (III: Fig 4). This was true for each of the vaccines (data not shown) and for each pneumococcal serotype studied. The antibody concentrations in toddlers on day 28 ranged from 0.9 µg/ml to 6.6 µg/ml for IgM and from 0.5 µg/ml to 6.4 µg/ml for IgG (III: Fig 4). On day 7, the GM IgA concentration ranged from 0.2 µg/ml to 1.1 µg/ml, depending on vaccine and on the pneumococcal serotype measured.

Only a little variation in the ASC response was observed between the four serotypes in each of the vaccine groups (Fig 4), whereas serum antibody responses varied by pneumococcal serotypes. Thus, low serum antibody responses were often detected to serotype 6B, (especially after PncPS and PncOMPC). The highest responses, in general, were to serotypes 14 and 19F in adults and to serotype 19F in toddlers. In adults, the secretory IgA antibodies in saliva were mostly to serotypes 14 and 19F before immunisation, but the responses after vaccination occurred equally in all serotypes. In toddlers, IgA antibodies were rarely detected before immunisation, but could be seen most frequently to serotype 19F, although frequently also to other serotypes. Of the two IgG-positive saliva samples detected in children, one was specific for serotype 6B, the other for 19F. The latter was associated with an exceptionally high serum concentration of serotype 19F-specific IgG (223 µg /ml).

In children with AOM, the infection caused by serogroup 19 evoked the highest ASC response, but group 23 was also associated with high ASC responses (Table in IV). The ASC responses to other types were lower, but low responses were also detected in some children to groups 19 and 23. Thus the magnitude of the response showed no clear-cut correlation with the serotype causing the infection.

S. pneumoniae was identified by culture in the nasopharyngeal swabs from four adult vaccinees before immunisation (I, II). One of them was colonised with the vaccine serotype, 23F (I), and three with nonvaccine serogroups 3, 9, and 11 (II). None of the vaccinees was colonised with any pneumococcal serotype on day 28 after immunisation. S. pneumoniae was identified by culture in the nasopharyngeal swabs from 8 children before immunisation (serogroups 6, 14, 15, 18, 19, and 23) and in 11 children (7 new acquisitions) on day 28 after immunisation (serogroups 6, 18, 19, 23) (III). The ASC and antibody concentrations and responses of these culture-positive vaccinees did not differ from those in the other vaccinees

The same serogroup as in MEF was cultured in the NPA sample of each of the 17 patients with pneumococcal AOM. Pneumococcal group 19 was the most common serogroup involved (7 patients). Group 23 was cultured in 4 patients, group 6 in 3 patients, and type 14 in 2. One patient had an AOM caused by group 9.

In adults, the IgA response in saliva was associated with a high number of IgA-ASCs (35/38 of the salivary responses were related to an IgA ASC response of >100 IgA ASC/10⁶ cells to the same serotype). On the other hand, a high ASC response was detected only rarely when no salivary IgA response was seen. In Study I, more than 100 IgA ASC/10⁶ cells were detected in 7 of the 62 cases that showed no salivary IgA response to the same serotype. Furthermore, in most of these cases (5 of 7) antibody concentration was high before immunisation and a 1.2- to 1.9-fold increase in IgA concentration was still seen. In Study II, more than 100 IgA ASC/10⁶ cells were frequently detected even when no salivary IgA response was demonstrated, but very rarely (6 cases) when more than 1000 IgA ASC/10⁶ cells were detected: also in Study II, high salivary antibody concentrations were often already detected before immunisation, but we did not attempt to correlate these cases with those with high ASC responses. In toddlers (III), IgA was rarely detected in saliva before immunisation. After immunisation, a positive correlation was demonstrated between the salivary IgA concentration (on day 28) and the number of IgA ASCs after PncD or PncT vaccination in toddlers (r=0.57, p=0.01; IV: Fig 3).

The ASC responses were also compared to the serum responses to see whether any correlation could be demonstrated. A significant correlation between the fold rise in serum IgA concentration and the number of IgA ASCs appeared in adults in Study II (r=0.64, p<0.01) (III; Fig 3), but in Study I no correlation (Fig 3, r=0.4). The correlation between the responses was stronger after immunisation with PncD (r=0.75, p<0.01) than after PncT (r=0.56, p<0.05). The fold rise in serum IgG concentration showed no correlation with number of IgG ASCs (r=0.3) (I, II). However, in Study II, the serotype-specific IgG concentrations in serum and in saliva correlated on day 28 (r=0.56, p<0.01) (II: Fig 4); (r=0.62 after PncD and r=0.51 after PncT), whereas no correlation appeared between IgA concentrations in serum and in saliva.

In toddlers, IgA antibodies were rarely detected in serum before immunisation. After immunisation, a positive correlation existed between the serum IgA antibody concentration and number of IgA ASCs (r=0.70, p=0.01). The IgA concentrations in serum and saliva did not correlate. A weak positive correlation was found between the fold increase in the serum IgG and the number of IgG ASCs (r=0.60, p=0.01).

Among the AOM patients, all three (Patients # 1, 2, 3) who had more than 100 IgA-ASC/10⁶ cells also had an IgA response in NPA and serum (IV: Table). Each of them also had a serum IgG and/or IgM response. An IgA response in NPA was detected in three additional patients (# 7, 8, 15), whose IgA ASC responses were low. Furthermore, IgA in acute or convalescent NPA could be detected in four other children (# 6, 10, 13, 14) with an IgA ASC response. No IgG responses were detected in NPA.