Antibody-Secreting Peripheral Blood Lymphocytes Induced by. Immunization with a .... 5 pug of monoclonal anti-IgAl or anti-IgA2 (a gift of J. Radl and J. J. ...
Vol. 58, No. 8
INFECTION AND IMMUNITY, Aug. 1990, p. 2547-2554
0019-9567/90/082547-08$02.00/0 Copyright C 1990, American Society for Microbiology
Antibody-Secreting Peripheral Blood Lymphocytes Induced by Immunization with a Conjugate Consisting of Streptococcus pneumoniae Type 12F Polysaccharide and Diphtheria Toxoid C. LUE,1* S. J. PRINCE,1 A. FATTOM,2 R. SCHNEERSON,2 J. B. ROBBINS,2 AND J. MESTECKY1' Departments of Microbiology' and Medicine,3 University of Alabama at Birmingham, Birmingham, Alabama 35294, and Laboratory of Developmental and Molecular Immunity, National Institute of Child Health and Human Development, Bethesda, Maryland 208922 Received 18 January 1990/Accepted 21 May 1990
Healthy adult volunteers were injected either with one of two conjugates composed of Streptococcus pneumoniae type 12F polysaccharide (Pn12F) covalently coupled to diphtheria toxoid or with Pn12F alone (as a component of Pnu-Imune, a 23-valent pneumococcus vaccine). The conjugates induced Pn12F-specific antibody-secreting cells in peripheral blood with numbers and isotype distribution similar to those induced by Pnu-Imune, with immunoglobulin A (IgA) as the predominant isotype. The conjugates also elicited high numbers of diphtheria toxoid-specific antibody-secreting cells of the IgG class. There was no distinct booster effect, since a second dose of the conjugates induced antibody-secreting cells at significantly lower numbers than after the first dose. In contrast to the cell numbers, the conjugate vaccines induced higher increases of IgAl Pn12F antibodies in serum than did Pnu-Imune. However, neither the conjugates nor Pnu-Imune induced a secretory antibody response. Antibody levels in serum and saliva correlated poorly with the frequency of antigen-specific antibody-secreting cells. Circulating antibody-secreting cells present 7 days postimmunization were probably not responsible for the high increase of antibodies in serum but rather represented a population of in vivo-activated B cells with the ability to disseminate the humoral response from the antigen recognition site to distant locations of antibody production.
Bacterial capsular polysaccharide (CP) vaccines have reduced efficacy in immunocompromised patients and infants (3, 36, 37). Efforts have been undertaken to make polysaccharides more immunogenic by coupling them to protein carriers (1, 7, 10, 35, 36). Conjugates of Haemophilus influenzae type b and Streptococcus pneumoniae type 6B elicited booster responses (a T-cell-dependent property) in infants and children but not in adults (1, 7, 10, 35). We studied the immune responses of healthy adults to a conjugate composed of Pn12F bound to diphtheria toxoid (DT), the chemical characteristics of which have been previously described (12). The Pn12F-DT conjugate elicited higher serum antibody levels in adult volunteers than did Pn12F alone (as a component of the 23-valent pneumococcus vaccine) (11). In this investigation, we studied circulating antibodysecreting cells (AbSC) induced by immunization with Pnl2FDT conjugates or unconjugated Pn12F. We examined whether the numbers of AbSC correlated with the levels of specific antibodies in serum. Earlier studies showed high numbers of antibody-secreting lymphoblastoid B cells (LB) in peripheral blood 5 to 9 days postimmunization with polyvalent pneumococcus vaccines (17, 18, 21, 22). Little is known about the immunoglobulin A (IgA) response to polysaccharide-protein conjugates in serum. Therefore, we analyzed the IgA subclass distribution of antigen-specific antibodies in serum. In addition, we assayed saliva for antibodies to Pn12F to examine whether systemic immunization with either unconjugated Pn12F or Pn12F coupled to DT could induce a secretory antibody response.
*
MATERIALS AND METHODS Immunization. Thirty healthy volunteers 18 to 44 years old, 16 of which were female and 14 of which were male, were recruited with full written consent. The subjects were randomly and evenly distributed into three groups. A preimmunization blood sample was taken, and the subjects were injected twice intramuscularly at a 1-month interval with 0.5 ml of either Pnl2F-40234-006-DT or Pnl2F-812408DT (12). The two lots of the Pnl2F-DT conjugate contained 25 ,ug of Pn12F and about the same weight of DT. Lot 812408 CP was a polydisperse preparation with both high- and low-molecular-mass components, while lot 40234-006 was a homogeneously high-molecular-mass CP. The third group received only one 0.5-ml injection of Pnu-Imune (lot NDC 005-2309-31; Lederle Laboratories, Pearl River, N.Y.). Blood samples were taken at 1, 4, and 8 weeks postimmunization. An additional blood sample was obtained 1 week after the second dose of the conjugates. Side reactions following injection were mild and short-lived (11). Cells. Venous blood was collected into sterile, heparinized syringes. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on a Ficoll-Hypaque (Sigma Chemical Co., St. Louis, Mo.) density gradient. The cells were washed three times in Dulbecco phosphate-buffered saline (PBS) (GIBCO Laboratories, Santa Clara, Calif.) and suspended in RPMI 1640 medium (GIBCO) supplemented with 10% fetal bovine serum (Irvine Scientific, Santa Ana, Calif.)-100 U of penicillin per ml-100 ,ug of streptomycin per ml. The cells were processed in the enzyme-linked immunospot (ELISPOT) assays (see below) immediately after isolation. Body fluids. Serum was obtained by venipuncture. Unstimulated parotid saliva was collected with a Schaefer cup (34) placed over the parotid duct. Unstimulated whole saliva
Corresponding author. 2547
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LUE ET AL.
was obtained from some volunteers by asking them to drool. Saliva and serum samples were immediately frozen at -20°C and thawed only for assay. Enumeration of antigen-specific AbSC. A modification of the ELISPOT technique, with nitrocellulose instead of polystyrene as the solid support, was used (8, 27). Individual wells of a Millititer HA plate (Millipore Corp., Bedford, Mass.) were coated overnight at 4°C with 100 ,ul of Pn12F coupled to poly-L-lysine (14) at 15 ,ug/ml. For detection of DT-specific AbSC, nitrocellulose-bottom wells were sensitized overnight at 4°C with 100 ,ul of DT (kindly provided by Wyeth Laboratories, Marietta, Pa.) at 10 jig/ml. The optimal coating concentrations were determined by checkerboard titration. On the following day, wells were emptied and washed four times with PBS. The remaining binding sites were saturated with 200 j.l of culture medium containing 10% fetal bovine serum for 30 min at 37°C in a humidified atmosphere with 5% CO2. After the blocking solution was discarded, 100 j.l of a PBMC suspension was dispensed into individual wells. PBMC were assayed at 5 x 105, 2.5 X 105, 1.25 x 105, and 6.25 x 104 cells per well. Samples were always run in replicate. Plates were incubated undisturbed for 3 to 4 h at 37°C and 5% CO2. After the cell incubation period, the plate was rinsed three times with PBS and three times with PBS containing 0.05% Tween 20 (Sigma). The plate was immersed in PBS-Tween for 5 min, emptied, and blotted dry with an absorbent paper towel. Biotinylated goat anti-human IgA, IgG, or IgM (Tago, Burlingame, Calif.) diluted 1/750 in PBS-Tween were used to detect specific antibodies of the appropriate isotype secreted by AbSC and bound to the solid phase. The plate was incubated overnight at 4°C. To detect IgA subclasses, wells were incubated with 5 pug of monoclonal anti-IgAl or anti-IgA2 (a gift of J. Radl and J. J. Haaijman, Rijswijk, The Netherlands) per ml for 2 h at 37°C. Incubation with the anti-IgA subclass monoclonal antibodies was followed by overnight incubation with biotinylated goat anti-mouse IgG antibodies (Southern Biotechnology Associates, Birmingham, Ala.) at a 1/750 dilution. After overnight incubation, avidin-alkaline phosphatase (Sigma) was added to the wells at 1.0 jig/ml to bind the secondary antibodies. After incubation at room temperature for 60 min, the plate was washed four times with PBS and left immersed in PBS for 5 min. To prepare the chromogen substrate, 15 mg of 5-bromo-4-chloro-3-indolylphosphate toluidine salt (Bio-Rad Laboratories, Richmond, Calif.) and 30 mg of p-Nitro Blue Tetrazolium chloride (Bio-Rad) were separately dissolved in 1 ml of dimethlylformamide and then added to 100 ml of 0.1 M NaHCO3 plus 1 mM MgCl2, pH 9.8. The substrate solution was passed through a 0.45-,um-poresize filter to remove particulate matter. The nitrocellulosebottom plate was blotted dry, and the wells were exposed to the 5-bromo-4-chloro-3-indolylphosphate toluidine salt-pNitro Blue Tetrazolium chloride substrate solution. Blue spots usually appeared after 10 to 15 min. After the spots reached maximal intensity, the reaction was stopped by thoroughly rinsing the plate with water. The plate was allowed to dry, and spots were enumerated under a stereomicroscope with 40-fold magnification. The specificity of the assays was verified as already described (8, 24). Pn12F (0.1 to 20 ,ug/ml) added to the cell suspension inhibited spot formation in a dose-dependent fashion (data not shown). The specificity of the anti-DT ELISPOT was tested in an analogous fashion (Fig. 1). The spot count was essentially zero in nitrocellulose-bottom wells not sensitized with the antigen but only blocked with fetal bovine serum-containing me-
INFECT. IMMUN.
Anti-DT AbSC/ 60' 10 6 PBMC
50'
40 '
30
-
20 10 0
.01
.1
1
10
100
Soluble DT (jig/ml) FIG. 1. Formation of spots by anti-DT AbSC was inhibited in a dose-dependent fashion by soluble DT. The data shown are means of replicate determinations in a representative experiment.
dium. Addition of soluble Pn12F or DT did not affect this count. Enumeration of immunoglobulin-secreting cels. In this isotype-specific ELISPOT assay, the wells were coated with 2.5 ,ug of goat anti-human IgA antibodies (Pel-Freez, Rogers, Ariz.) per ml, 1.5 ,ug of goat anti-human IgG antibodies (Jackson, West Grove, Pa.) per ml, or 2.5 ,ug of goat anti-human IgM antibodies (Pel-Freez) per ml. After blocking with complete medium, 100 RI of PBMC at various densities was incubated in the wells for 3 to 4 h. Biotinylated goat anti-IgA, anti-IgG, or anti-IgM antibodies (Tago) were used to detect the immunoglobulin secreted. Subsequent development with avidin-alkaline phosphatase and the 5bromo-4-chloro-3-idolylphosphate toluidine salt-p-Nitro Blue Tetrazolium chloride substrate mixture was carried out in the same way as in the antigen-specific assay. ELISA for determination of specific antibody levels. Polystyrene microtiter plates (Dynatech Laboratories, Inc., Alexandria, Va.) were coated with 15 ,ug of Pn12F per ml or 2.5 jig of DT per ml. After being washed with PBS and blocked with 5% fetal bovine serum, serial dilutions of serum or saliva were pipetted into the microwells and incubated for 2 to 3 h at 37°C. After the incubation, the plates were washed with PBS-0.05% Tween 20 (Sigma). Depending on the type of assay, biotinylated anti-human IgA, IgG, or IgM (Tago) at a 1/750 dilution or the first-stage human IgA subclass monoclonal antibodies (5 jig of antibodies per ml) were added. The plates were then incubated overnight at 4°C. Second-stage biotinylated goat anti-mouse IgG antibodies (Southern Biotechnology Associates) were applied in the antigen-specific IgA subclass assay and incubated for 2 h at 37°C. The biotinylated antibodies were detected by incubation with 0.5 ,ug of avidin-horseradish peroxidase (Sigma) per ml for 1 h at room temperature. Wells were then allowed to react with 2.5 mg of the substrate 2,2-azinobis(3-ethylbenzthiazoline sulfonic acid) (Sigma) per ml in citrate buffer (pH 4.2) containing 0.0075% H202. The absorbance was measured in a Vma,, kinetic microplate reader (Molecular Devices Co., Palo Alto, Calif.) at 414 nm. A positive reference serum, which was assigned a value of 100 enzyme-linked immunosorbent assay (ELISA) units of anti-Pn12F and anti-DT antibodies per ml, was used to generate a standard curve. The absorbances of the samples were analyzed and converted into ELISA units per milliliter by using a computer program based on either the four-parameter logistic or weighted logit-log model (kind-
PNEUMOCOCCUS TYPE 12F-DIPHTHERIA TOXOID CONJUGATES
VOL. 58, 1990
2549
TABLE 1. Pn12F-specific AbSC in peripheral blood of immunized individuals Geometric mean no. (SE) of Pnl2F-specific AbSC/106 PBMC (isotype distribution)
Time and immunogen"
Preimmunization Pnl2F-40234006-DT Pnl2F-812408-DT Pnu-Imune 7 Days after dose 1 Pn12F-40234006-DT Pnl2F-812408-DT Pnu-Imune
IgA
0.9 (1.3) 1.6 (1.6) 3.1 (1.9)
150.7 (1.4) (45.5% IgAl, 54.5% IgA2) 208.0 (1.2)b (41.6% IgAl, 58.4% IgA2c) 147.6 (1.1)d (54.4% IgAl, 45.6% IgA2)
IgG
IgM
0.8 (1.4) 0.9 (1.3) 0.5 (1.0)
0.8 (1.3) 1.1 (1.4) 0.7 (1.3)
22.7 (2.2) 31.2 (2.2) 77.1 (1.2)
33.8 (2.2) 57.7 (1.8) 54.8 (1.2) 0.6 (1.3) 0.5 (1.0) 1.1 (1.6)
4 Weeks after dose 1 Pn12F-40234006-DT Pnl2F-812408-DT Pnu-Imune
0.6 (1.1) 0.7 (1.2) 0.8 (1.4)
1.3 (1.6) 0.6 (1.3) 0.8 (1.5)
7 Days after dose 2 Pnl2F-40234006-DT Pn12F-812408-DT
6.3 (1.7)e (54.0% IgAl, 46.0% IgA2) 5.6 (1.8)9 (50.6% IgAl, 49.4% IgA2)
5.9 5.0
(1.7)f (1.8)f
4.1 (1.6)f 3.1 (1.4)9
a Groups of 10 samples were tested. Frequency higher than that of group Pnl2F-812408-DT IgM at day 7 (P < 0.02) and higher than that of group Pnl2F-812408-DT IgG at day 7 (P < 0.05). c Percentage higher than that of IgAl (P < 0.01). d Frequency higher than that of group Pnu-Imune IgM at day 7 (P < 0.01) and higher than that of group Pnl2F-812408-DT IgG at day 7 (P < 0.05). ' Lower response than 7 days after dose 1 (P < 0.002). f Lower response than 7 days after dose 1 (P < 0.05). 8 Lower response than 7 days after dose 1 (P < 0.01). b
ly provided by T. A. Brown, Department of Microbiology, University of Florida, Gainesville). Statistics. Results are presented as geometric means with standard errors (antilogs of the standard errors of the means of the logs). The Kruskal-Wallis test and the Mann-Whitney U test were used to compare the immune responses elicited by the three different vaccine preparations. A significance level of 0.05 was chosen. Differences among isotypes were evaluated with the Wilcoxon paired rank sum test. The Spearman rank correlation coefficient (p) was determined to study the correlation between AbSC and antibody levels in serum or saliva. All calculations were carried out on a Macintosh SE computer (Apple Computer Inc., Cupertino, Calif.) with the software package StatView 512+ (Brain Power, Inc., Calabasas, Calif.). RESULTS AbSC to Pn12F in peripheral blood. Low numbers of Pnl2F-specific AbSC were detectable with no differences between vaccine groups or immunoglobulin classes in preimmune samples (Table 1). At 7 days postimmunization, high numbers of anti-Pn12F AbSC were detected. IgA accounted for most of these AbSC. A predominance of the IgA2 isotype was observed in vaccine group Pnl2F-812408DT (P < 0.01), while the IgA subclasses were evenly distributed in the other groups. At 4 weeks postimmunization, the numbers of anti-Pn12F AbSC were at the preimmunization level. Subjects in the conjugate groups received a second dose of the vaccine. One week later, the frequency of Pn12F-specific AbSC was higher than at 4 weeks but lower than 7 days after the first dose (P < 0.05). There were no significant differences between the two conjugate groups in the frequency and isotype distribution of AbSC. AbSC to DT in peripheral blood. Preimmune numbers of
DT-specific AbSC were low, as observed for anti-Pn12F AbSC (Table 2). Immunization with either Pnl2F-DT conjugate induced a rise of IgG anti-DT AbSC at high numbers (P < 0.002) at 7 days postimmunization. Cells secreting IgG anti-DT outnumbered those secreting IgA and IgM (P < 0.02 for Pnl2F-40234006-DT and Pnl2F-812408-DT). There was a clear preponderance of the IgAl subclass in the IgA anti-DT response. PBMC from five subjects immunized with PnuImune were assayed, and anti-DT AbSC were also detected. In those subjects, the IgG anti-DT response was significantly lower than in the conjugate groups. However, IgM and IgA anti-DT AbSC were found at higher frequencies than in the conjugate groups, although the difference did not reach the significance level of P = 0.05. At week 4 postvaccination, the numbers of anti-DT AbSC returned to preimmunization values. A second dose of the conjugate induced only a moderate increase of AbSC that was lower (P < 0.05) than after the first dose. Again, a predominance of the IgAl subclass was observed. Immunoglobulin-secreting cells. The frequency of total immunoglobulin-secreting PBMC increased after the first injection and to a lesser extent after the second dose of the conjugates. Numbers of immunoglobulin-secreting cells ranged from 72/106 PBMC to 232/106 PBMC preimmunization and from 112/106 PBMC to 1,562/106 PBMC 7 days after the first dose of the vaccine. The increase was accounted for by circulating antigen-specific AbSC. Approximately 50% of immunoglobulin-secreting cells were of the IgA class. There was no significant difference among the three vaccine groups with respect to the total number and the isotype distribution of immunoglobulin-secreting PBMC. IgA subclass antibodies in serum. All three vaccine preparations induced significant increases of IgA anti-Pn12F antibodies of both subclasses (Table 3). Pnl2F-DT, in particular Pnl2F-812408-DT, elicited considerably higher levels of IgAl anti-Pn12F than did Pnu-Imune. IgA2 anti-Pn12F also
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LUE ET AL.
INFECT. IMMUN.
TABLE 2. DT-specific AbSC in peripheral blood of immunized individuals Geometric mean no. (SE) of DT-specific AbSC/106 PBMC (isotype distribution)
Time and immunogen (no. of samples)
Preimmunization Pnl2F-40234006-DT (10) Pnl2F-812408-DT (10) Pnu-Imune (5) 7 Days after dose 1 Pnl2F-40234006-DT (10) Pnl2F-812408-DT (10) Pnu-Imune (5)
IgA 0.5 (1.0) 0.6 (1.2) 1.4 (1.7) 5.4 (1.9) (83.1% IgAl,b 16.9% IgA2) 7.7 (1.5)C (66.3% IgAl,b 33.7% IgA2) 43.5 (1.1)
IgG
IgM
0.6 (1.1) 0.6 (1.2) 0.5 (1.0)
0.7 (1.2) 1.7 (1.5) 1.6 (1.6)
284.1 (1.4)a 12.6 (2.3)
1.6 (1.4) 1.8 (1.4) 14.6 (1.7)
98.2 (1.7)d
4 Weeks after dose 1 Pn12F-40234006-DT (10) Pnl2F-812408-DT (10) Pnu-Imune (5)
0.5 (1.1) 0.6 (1.2) 0.6 (1.1)
0.8 (1.3) 0.6 (1.3) 0.5 (1.0)
0.5 (1.0) 0.5 (1.0) 0.6 (1.1)
7 Days after dose 2 Pnl2F-40234006-DT (10) Pn12F-812408-DT (10)
1.0 (1.3)e (69.9% IgAl,b 30.1% IgA2) 1.0 (1.3)9 (83.3% IgAl,b 16.7% IgA2)
3.3 (1.7/ 2.1 (1.5/
1.3 (1.4) 1.4 (1.3)
a Frequency higher than that of Pnl2F-40234006-DT IgA and IgM on day 7 (P < 0.002) and higher than that of Pnu-Imune IgG on day 7 (P < 0.02). Higher percentage than IgA2 (P < 0.05). c Frequency higher than that of group Pnl2F-812408-DT IgM on day 7 (P < 0.05). d Frequency higher than that of Pnl2F-812408-DT IgA and IgM on day 7 (P < 0.02) and higher than that of Pnu-Imune IgG on day 7 (P < 0.05). e Lower response than 7 days after dose 1 (P < 0.05). f Lower response than 7 days after dose 1 (P < 0.01). g Lower response than 7 days after dose 1 (P < 0.02). b
tended to be higher after immunization with the conjugates, but the difference was not statistically significant. The conjugates induced an IgA response to DT which was exclusively IgAl (Table 4). However, the increase rates were moderate compared with the response to the polysaccharide component. Pnu-Imune did not induce an IgA antiDT response in serum. Antibody levels in saliva. Pn12F antibody activity was detected in pre- and postimmune saliva samples, and IgA was the predominant isotype (Fig. 2). Comparison of parotid and whole-saliva samples showed, in most cases, higher levels of specific antibodies in the latter. Therefore, parotid saliva and whole-saliva results were separately processed in statistical calculations. No significant increase for IgA, IgG, and IgM anti-Pn12F antibody levels was observed after immunization with any of the three vaccine preparations. In the calculations, specific IgA antibody levels were related to the total IgA levels of the respective saliva samples to take into account the dependence of the salivary immunoglobulin concentration on the flow rate. Statistical analysis of those corrected antibody levels showed no significant change of antibody concentrations after immunization (data not shown). Thus, neither Pnl2F-DT conjugates nor the unconjugated Pn12F induced a significant salivary antibody response. Correlations. The correlation coefficient (p) was determined to examine whether there was a correlation between AbSC in the circulation and antibody levels in serum or saliva. The frequency of Pn12F-specific AbSC in blood 7 days postvaccination with either the conjugates or unconjugated Pn12F did not correlate significantly with specific antibody levels in serum. The correlation between IgA anti-Pn12F antibodies in saliva and IgA anti-Pn12F AbSC on day 7 was significant (p = 0.673, P < 0.05) in the Pnu-Imune group. In the same group, IgA2 anti-Pn12F AbSC on day 7 correlated positively with IgA anti-Pn12F antibody levels in saliva at 4 weeks
postimmunization (p = 0.697, P < 0.05). No significant correlations were found in the two conjugate vaccine groups. Antibody levels in saliva and serum correlated poorly in the conjugate groups. In the Pnu-Imune group, there was a positive correlation between IgA2 anti-Pn12F antibodies in serum and Pnl2F-specific IgA in saliva at 8 weeks postvaccination (p = 0.721, P < 0.05). DISCUSSION Previous investigations showed that immunization with polyvalent pneumococcus vaccines induces the transient appearance of antigen-specific AbSC in the circulation (17, 18, 22, 24). The AbSC were phenotypically characterized as LB with an average cell volume of 320 ,um3, with the ability to bind to pneumococcus CP-coated ox erythrocytes through surface IgG or IgM, and a surface marker of activation called 4F2 (23). The antipneumococcus CP response at the singlecell level is dominated by IgA as demonstrated with the hemolytic plaque or ELISPOT assay (18, 24). Analysis of IgA subclasses revealed a predominance of the IgA2 isotype (16, 24). In the present study, subjects responded with high numbers of Pnl2F-specific AbSC 7 days postimmunization with the Pnl2F-DT conjugate or unconjugated Pn12F. IgA was the predominant isotype, with IgG and IgM accounting for lower percentages. There was a predominance of the IgA2 subclass in subjects who received Pnl2F-812408-DT. The Pnl2F-DT conjugates induced a predominant IgG response of AbSC to DT. This is in agreement with the results of previous studies using tetanus toxoid (TT) and DT (33, 39). A modest IgA response to DT was mostly of the IgAl isotype. DT-specific AbSC were also found in five subjects postimmunization with Pnu-Imune. This response is possibly part of the polyclonal B-cell activation which is a known effect of certain T-independent antigens (28) and which has been described previously after immunization with pneumococcus CP (18, 24, 29).
PNEUMOCOCCUS TYPE 12F-DIPHTHERIA TOXOID CONJUGATES
VoL. 58, 1990
TABLE 4. IgAl anti-DT antibody levels in sera of adult volunteers elicited by Pn12F-DT or Pnu-Imunea
05cno
S.,
2551
tIII
I
Immunogen
cHP 9 0
Geometric mean ELISA units/ml (range of percentiles 25-75) at: Preimmunization
4 weeks after dose 1
4 weeks after dose 2
6.3b (3.7-9.1)
5.8b (3.0-6.2)
00
"g - t '"
A
