Immunogenicity of Streptococcus pneumoniae type 6B and 14 polysaccharide-tetanus toxoid conjugates and the effect of uncoupled polysaccharide on the antigen-specific immune response

PII: SO264-410X(98)00129-7

Vaccine, Vol. 16, No. 20, pp. 1941-1949, 1998 0 1998 Elsevier Science Ltd. All riahts reserved Printed in-Great Britain 0264-410X/98 $lQ+O.OO

ELSEVIER

Immunogenicity of Streptococcus pneumoniae type 6B and I4 polysaccharide-tetanus toxoid conjugates and the effect of uncoupled polysaccharide on the antigen-specific immune response Maria E. Rodriguez:“-& Germie P.J.M. van den Dobbelsteen”, Lukas A. Oomen”, Odo de Weers”, Leo van Buren*, Michel Beurret”, Jan T. Poolman*+ and Peter Hoogerhout* The immunogenic@ of two types of Streptococcus pneumoniae capsular polysacchatide-tetanus toxoid conjugates (PS6BTT and PS14TT) was evaluated in mice. Both conjugates induced high titres of high avidity type-specific anti-PS IgG, which include all IgG isotypes except IgG2a. Repeated immunization resulted in booster responses in both cases. The antibodies induced exhibited opsonic activity, as measured in an in vitro opsonophagocytosis assay, using the mouse macrophage cell line RAW-264. Furthermore, the influence of spiking PS6BTT with fi-ee PS6B of either 1000 kDa (native) or 37 kDa was investigated. The results indicate that not only the amount but also the molecular weight of the free PS6B present in the conjugate vaccine affect the anti-PS6B immune response. Large amounts of free PS6B of both molecular weights decrease each anti-PS6B IgG isotype response. Howevel; unlike admixture of the low molecular weight PS6B, addition of the high molecular weight PS6B leads to a rather persistent state of unresponsiveness. 0 1998 Elsevier Science Ltd. All rights reserved Keywords:

Streptococcus pneurnoniae;

Conjugate

vaccine: Immunogenicity

Streptococcus pneumaniae is an important human pathogen, being a principal cause of bacterial pneumonia, meningitis and bacteraemialm3. Host defence against S. pneumoniae depends on opsonic activity of anticapsular polysaccharide antibodies, complement and phagocytosis by macrophages. The existing polyvalent antipneumococcal vaccine, formulated from 23 capsular polysaccharides (PS) of the over *Laboratory of Vaccine Development and Immune Mechanisms, National Institute of Public Health and the Environment, Antonie van Leeuwenhoeklaan 9, 3720 BA Bilthoven, The Netherlands. Wentro de Investigation y Desarrollo de Fermentaciones lndustriales (CINDEFI), Facultad de Ciencias Exactas, Universidad National de La Plata, 47 y 115, (1900) La Plata, Argentina. SPresent address: SmithKline Beechman Biologicals, Rue de I’lnstitut 89, B-1330, Rixensart, Belgium. 3Author to whom all correspondence shoukl be addressed. Tel.: +54 (0) 21 33794; fax: +54 (0) 21 254533; e-mail: [email protected]. unlp.edu.ar (Received 1 October 1997; revised version received 23 January 1998; accepted 20 March 1998)

90 serologically distinct types currently recognized4, has been shown to induce antibody responses and increased opsonization of pneumococcr . However, revaccination does not result in anamnestic response and the vaccine is poorly immunogenic in population groups that are at risk for pneumococcal diseasebm9, namely, people with frequent respiratory tract infections, asplenic patients, HIV-infected individuals, elderly people and young children. Some of the limitations of the efficacy of the current vaccine are due to the T independent type 2 (TI-2) character of PS antigen?‘. This problem is being addressed by the conjugation of the most widely spread pneumococcal PS types to protein carriers”-“. The application of conjugate vaccines has been shown to be successful in the prevention of Haemophilus influenzae type b infection in infants”. The covalent coupling of H. influenzae type b capsular polysaccharide with proteins resulted in an enhanced immunogenicity, anamnestic response and prevalence of anticapsular polysaccharide IgG antibodies, which

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are properties characteristic for a T-cell dependent immune response. In this study, two different types of pneumococcal polysaccharide, 6B and 14, of high epidemiological importance all over the world”’ and of recognized low immunogenicity”, were conjugated with tetanus toxoid (TT). The immunogenicity of these two conjugates was analysed. Furthermore, PS6BTT was employed to gain further insight into the reported” immune suppression induced by the presence of free polysaccharide in the conjugate vaccine formulation. MATERIALS AND METHODS Antigens

Tetanus toxoid was obtained from the Laboratory of Vaccine Production of RIVM (Bilthoven, The Netherlands). The material meets the requirements of the World Health Organisation for human administration. Purified Streptococcus pneumoniae type 6B and 14 capsular PS (PS6B and PS14, respectively) were purchased from the American Type Culture Collection (Rockville, MD, USA) or obtained from the Laboratory of Product and Process Development of RIVM (Bilthoven, The Netherlands). The polysaccharides meet the requirements of the European Pharmacopoeia. pneumococcal In this study two different polysaccharide-tetanus toxoid conjugates were used. They were prepared by a coupling procedure described elsewhere’“. In brief, the PS were partially depolymerized by sonication to an average molecular weight (m.w.) of 37 kDa (54 repeating units (RU)) or 46 kDa (67 RU) for PS6B, and 37 kDa (54 RU) for PS14. The 46 kDa PS6B or 37 kDa PS 14 were activated with cyanogen bromide in a sodium carbonate buffer. Cystamine was then coupled to the cyanogen bromideactivated PS, the pH being periodically adjusted with NaOH. Thereafter, the disulphide bond of the linker was reduced by dithioerythritol and the resulting thiolcontaining PS was conjugated to bromoacetylated tetanus toxoid. The conjugates were purified by gel permeation chromatography over Sephacryl” S-400 HR (Pharmacia, Uppsala, Sweden) and fractions were analysed for sugar- and protein content. Selected fractions, essentially devoid of non-conjugated PS, were pooled, sterile-filtered, and stored at approx. 4°C. PS6B-IT and PS14-TT showed a polysaccharidel protein ratio (mol/mol) of 3.2 and 2.2, respectively. Mice and immunization

seven 8-12 week-old female (a) Groups of NIH/RIVM mice (random outbred strain) bred and kept at RIVM, were immunized subcutaneously (s.c.) with 0.1 pg of PS6B (average molecular weight: 46 kDa) or PS14 (average molecular weight: 37 kDa) conjugated with TT and absorbed in 0.1% (w/v) AlPO, suspension. The animals were immunized on days 0, 28, and 56. The serum samples were collected on days 0, 14, 28, 42, 56, and 70 and stored at -20°C until use. (b) Groups of seven 8-12 week-old female NIHiRIVM mice were immunized subcutaneously (s.c.) with PS6BTT containing 0.1 pg of conjugated

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PS6B and variable amounts of free PS6B of either 1000 kDa (native) or 37 kDa. The immunization and sampling scheme was as described above. Some of these groups of mice were further immunized with PS6BTT, containing 0.1 /lg of conjugated PS6B, without free PS, on days 98 and 126. Serum samples were taken on days 98, 112, 126, and 140. Antibody titres

Antibody titres against PS6B and PS14 were determined by enzyme-linked immunoabsorbent assay. PolySorp microtitre plates (Nunc-Immune Plates, Inter Med) were coated with PS6B or PS14 from Streptococcus przeumoniae (American Type Culture Collection) dissolved in 0.01 M PBS (pH 7.2), at a concentration of 10 and 1 @ml, respectively. After incubation overnight at room temperature, plates were washed and incubated for 2 h at room temperature with serial dilutions of serum samples in PBS, containing 0.1% (w/v) Tween-80. Previous experiments had shown that blocking agents for non-specific binding (such as fetal bovine serum or albumin) were not necessary. After incubation with serum dilutions, the plates were washed again and incubated for 2 h at room temperature with isotype-specific peroxidase conjugated goat anti-mouse immunoglobulin (Ig) IgG, IgGl, IgG2a, IgG2b or IgG3 antibodies (Southern Biotechnology Associates, Birmingham, AL). Plates were washed and incubated with the peroxidase substrate tetramethylbenzidine (0.1 mg/ml; Sigma) O.Ol%(v/v) HIOz in 0.11 M sodium acetate buffer (pH 5.5). After 10 min of incubation at room temperature, the reaction was stopped by the addition of 100 ~11of 2 M HZSO, and the &so was read using an ELISA Titretek Multiscan spectrophotometer. Antibody titres were measured for individual sera. A linear fit was made for optical density values of eight dilutions and the antibody level was calculated in arbitrary units, namely the reciprocal dilutions that gave 50% of the maximum absorbance. To exclude interference by antipneumococcal cell wall polysaccharide antibodies in the determination of type-specific (anti-PS6B or anti-PS14) IgG antibodies, all sera were absorbed with soluble cell wall polysaccharide (CPS; Statens Seruminstitut). To that end, each sample was pre-incubated with soluble CPS at a concentration of 120 pg/ml (which proved to be sufficient to neutralize anti-CPS antibodies present in these serum samples), at 37°C for 30 min. before serum incubation. IgG avidity

In this assay NaSCN was used as a chaotropic agent as described elsewhereZ4 with minor changes. Briefly, the basic method of ELISA as described above was followed with one exception. Before the detection antibody was introduced, 100 ~1 of 0.5 M NaSCN in PBS (0.1 M, pH 6) or 100/d of PBS (0.1 M, pH 6; control) was added to the wells for 15 min at room temperature. The wells were then washed four times and the ELISA was completed as described above. The results were expressed as avidity indices, i.e. titre (with NaSCN)/titre (without NaSCN) x 100.

Pneumococcal conjugate vaccine: M. E. Rodriguez et al. Opsonophagocytosis

assay

The opsonic activity of mouse antisera was determined in an opsonophagocytosis assay according to the method described by Velasco et ~1.~’ with minor changes. Briefly, fluorescein-labelled S. pneumoniae serotype 6B or 14 were opsonized with several dilutions (O-20%) of the respective heat-inactivated (30 min at 56°C) mouse antisera, with or without a fixed amount of baby rabbit serum (2%) as a source of complement. Pre-immune sera were tested at 20% of the serum concentration. Anti-CPS antibodies present in the sera were neutralized before performing the assay as described above. The mouse macrophage cell line RAW-26416 was employed for phagocytosis. Cells were grown in a 5% CO2 humidified atmosphere in RPM1 1640 medium supplemented with 10% inactivated fetal bovine serum (GiBCO), penicillin/streptomycin (10 @ml) and glutamine (4 mM). For the phagocytosis assay, cells (viability > 90%) were washed once with Hank’s balanced salt solution 0.1% gelatine (HBBS-G) and then resuspended in HBBS-G at a concentration of 10’ cells/ml. After phagocytosis by RAW-264 cells (bacteria/macrophage ratio: 10/l), samples were washed and analysed in a flow cytometer (FACScan; Becton Dickinson, Mountain View, CA). The FITC-fluorescence intensity of 10000 cells was quantitated in each sample. The mean of the FITCfluorescence intensity of the cells in each sample was used to estimate the opsonic capacity of the antiserum. Statistical

methods

Before statistical analysis, antibody titres were loglo converted, which normalized their distributions. Results are expressed as, the mean of logarithmic titres of n independent observations f standard deviations (S.D.). Analysis of Variance (ANOVA) was used for statistical data evaluation. The significance of the differences between the mean values of each condition was determined with the Least Significant Difference (LSD) test at a confidence level of 95%.

using increasing concentrations of thiocyanate ranging from 0 to 6 M, according to the method described by Pullen et ~1.” (d a t a not shown). This affinity maturation could be detected in animals immunized with PS6BTT, as well as in those immunized with PS14TT. The third immunization exerted more influence on the affinity maturation of anti-PS6B IgG antibodies, as compared with the anti-PS14 IgG response. The analysis of individual sera showed no correlation between the avidity index and the antibody titre, suggesting that the determination of the avidity was not affected by the level of antibodies. To gain further insight into the T-dependence (TD) of the immune response, type-specific IgG subclass distribution induced by each conjugate was investigated. IgG isotype distribution of anti-PS6B and antiPS14 2 weeks after the second and the third immunization are shown in Figure 3A and 3B, respectively. The isotype pattern of type-specific IgG

(4 5-

432-

‘-4 02

0

28

56 day

(W

RESULTS Immunogenicity

of PS6EITT and PS14TT conjugates

Groups of seven mice were immunized three times within 8 weeks with PSGBTT or PS14TI’ in a dose of 0.1 pg of conjugated polysaccharide per immunization in combination with 0.1% (w/v) AlPOh. This dose proved to be optimal in previous studies (data not shown). Repeated immunizations with both conjugates led to a booster response of 1gG anti-PS antibodies (Figure I). The third immunization yielded a further small increase of the immune response induced by the second immunization. The avidity of type-specific IgG anti-PS was analysed after each immunization by an ELISA and expressed as the percentage of antibody still bound to the antigen after incubation with a chaotropic agent (SCN ion). The antibodies produced during the secondary response showed higher average affinity than those produced in the primary response. After the third immunization, the avidity index of the sera showed a further increase (Figure 2). Similar results were found

0

28

56

Figure 1 Time course of type-specific anti-PS IgG antibody response after priming and booster immunizations with PSGBlT and PS14TT. Groups of seven mice were immunized on days 0, 28, and 56 (indicated by arrows) with PS6BTT (0) or PSl4lT (m). Serum samples were obtained on days 0, 28, 42, 56, and 70. Antibody responses (means i S.D.) were determined in PSGB and PS14 ELISA, respectively.

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+-anti-PSGB

antibodies

Groups of seven mice were immunized with 0.1 pg of PS6BTT mixed with 0 ;lg (group l), 0.1 pg (group 2) and 1 pg (group 3) of free native PS6B, as was described in Materials and Methods. As shown in Figure 5A, all anti-PS6B IgG isotypes were affected by the presence of free native polysaccharide during the immunization. The level of each anti-PS IgG isotype achieved after immunization decreased when increasing quantities of free PS6B were added. However, anti-PS6B IgG2b seemed to be the isotype most depleted. At the highest amount of free native PS6B added to the conjugate vaccine, only a low antiPS6B IgG2b titre could be detected, even after three immunizations (Figure _5Az).

75

50

25

lgG1

(4 !__I

28

k

56

5

1

lgG2b

mlgG3

days Figure 2 Avidity index of type-specific anti-PS IgG antibodies after priming and booster immunizations with PS6BTT and PS14lT. Groups of seven mice were immunized three times on days 0, 28, and 56 (indicated by arrows) with PSGBTT (0) or PS14TT (m). Serum samples were obtained on days 0, 28, 42, 56, and 70. Avidity indexes are expressed as the mean & S.D.

antibodies did not change after the successive immunizations. Both conjugates induced high titres of all antiPS IgG subclasses except IgG2a, which remained at the pre-immune level. Both conjugates induced anti-TT IgG antibodies distribution characterized by with a subclass IgGl gIgG2b. Anti-T-I IgG2a antibodies stayed at pre-immune levels. Small amounts of anti-TT IgG3 could be detected in some individual sera (data not shown). The opsonic activity of type-specific anticapsular antibodies was measured by complement-dependent of fluorescein-labelled pneumococci phagocytosis serotype 6B or 14, using RAW-264 as effector cells. To exclude the effects of serum factors other than pneumoPS-specific antibodies, fluorescein-labelled cocci opsonized with pre-immune serum were included in each assay. A negative control, fluorescein-labelled pneumococci incubated with complement, but without sera, was also included. As depicted in Figure 4, pre-immune sera did not show higher opsonic activity than observed in the absence of sera (0% of serum concentration). Both conjugates induced sera with opsonic activity as compared with the pre-immune sera. More than one serum concentration was employed to determine the opsonic activity induced after three immunizations. The opsonophagocytosis assay appeared to be type specific, since phagocytosis was only achieved in the presence of the homologous antiserum for both serotypes tested in the present study (data not shown). Opsonic activity was proportional to the serum concentration up to 5% of anti-PS6B antiserum and 10% of anti-PS14 antiserum.

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three Number of immunizations

IgGl

tW0

I

lgG2b

--c

lgG3 IgG

three Number of immunizations

Figure 3 Subclass distribution of type-specific anti-IgG antibodies upon a second and a third immunization with PSGBTT or PS14TT. Groups of seven mice were immunized three times on days 0, 28, and 56 with PSGBTT (A) or PS14lT (B). Serum samples were obtained on days 0, 28, 42, 56, and 70. The individual titre of each isotype of the respective type-specific anti-PS IgG was determined by ELISA. The figure shows mean titres, in which the bars represent the standard errors.

Pneumococcal conjugate vaccine: M. E. Rodriguez et al.

+

anti-PSI 4 antisera

+-anti-PSGB

antisera

13 pre-Immune sera + pneumococo serotype 14 0

0

0,5

1

2

5

10

pre-Immune sera + pneumococo serotype 6B

20

Serum concentration (%) Figure 4 Serum concentration dependence of the opsonic activity towards S. pneumoniae serotype 6B and serotype 14. Two groups of seven mice were immunized .three times within 8 weeks with PS6BlT (0) or PS14lT (M). After the third immunization (day 70) the sera from mice belonging to the same group were pooled and tested in different concentrations as a source of opsonins in an in vitro phagocytic test with fluorescein-labelled S. pneumoniae of the respective serotype and RAW-264 cells. S. pneumoniae of both serotypes were also opsonized in the absence of mouse serum (0% of serum concentration) or with pre-immune mouse serum (20%). Ten thousand cells of each sample were analysed by flow cytometry to determine the mean FITC-fluorescence concentrations of the pooled ‘sera from each group of mice were tested in triplicate.

The presence of anti-PS IgM was tested in each group of mice after the third immunization. The same pattern of immunosupre,jsion at the IgM level could be detected. Although anti-PS6B IgM antibodies were induced in group 1 (mean ,,Jog titre: 1.8f0.2), antiPS6B IgM antibodies were undetectable in the sera of mice in groups 2 and 3. Groups of seven mice, randomly chosen among the mice initially belonging to groups 2 and 3, were immunized twice more within 4 weeks with PS6BTT plus adjuvant or saline solution plus adjuvant (to be used as control). Figure 5B shows that, in comparison with the control group, mice re-immunized with PS6BTT showed some type-specific antibody induction. However, the antibody level induced upon two re-immunizations with PS6BTT were not higher than those achieved initially in group 2 (Figure 5A). The influence of the chain length of PS6B on the persistence of the state of unresponsiveness was studied by inoculating mice with PS6BTT admixed with different amounts of free PS6B of reduced chain length (average molecular weight: 37 kDa). Groups of seven mice were immunized with 0.1 pg of PS6BT”T mixed with 0, 0.01, 0.03, 0.1 or 1 /lg of free PS6B. Table 1 shows that low amounts of PS6B were unable to suppress the IgG anti-P:3 antibody response. A significant fp~O.05) decrease in anti-PS IgG2b production was detected when the amount of free PS6B was 0.03 pg per dose. However, a significant (p ~0.05) decrease in all the other anti-PS6B IgG isotypes’ antibody levels was detected only when 0.1 or 1 /lg of free sonicated PS was simultaneously administered with the conjugate vaccine. Remarkably, no significant differences were detected in the anti-PS6B IgG antibody response induced in the group of mice inoculated with conjugate admixed with 0.1 pg of free sonicated polysaccharide, as compared with the group

intensity of the cells in each case. The different

immunized with conjugate plus 1 pg of free sonicated polysaccharide (Table I). Since no significant differences were observed between the two latter groups of mice, they were selected to gain further insight into the state of unresponsiveness. One group was inoculated with PS6BTT plus adjuvant and the other with saline plus adjuvant (to be used as control). After the first re-immunization with PS6BTT, the level of anti-PS6B IgG antibody (all isotypes) induced showed no statistical differences (p ~0.05) with levels observed in mice immunized three times with PS6BTT alone (Table I). This indicates that the responsiveness was completely recovered in these mice. No further significant increase was detected after the second re-immunization. The presence of free polysaccharide at the concentrations and chain lengths employed in this study showed no influence on the immune response raised against the protein carrier. Finally, the admixture of PS6BTT with high amounts (more than 10 times the concentration of the conjugated polysaccharide) of free pneumococcal polysaccharide of types different from PS6B (PS14, PS19F and PS23F) did not affect the immune response against PS6B induced by PS6BTT (data not shown). DISCUSSION The covalent attachment of carbohydrate epitopes to a carrier protein is expected to increase the immunogenic@ by converting the saccharide from a TI to a TD immunogen. The importance of the carrier protein for the b&yten. is its ability to stimulate the Th cell efficacy of H. response . Since the demonstrated infruenzae type b conjugate vaccine in young children, research on pneumoccocal vaccine has also been directed towards the development of a capsular

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--c

0 ug PSGB

-u-o.1

“T

0

No)

et al.

-A-l

+ PSGBTT -O-control

ug PSGB ug PSGB

56

28

140 days

days

(Al

WI

1 T

0 28

I

140

70

56

days

days

6

I40

6

6T

28

(83)

56

Figure 5 Effect of different quantities of native PSGB on the immunogenicity of PSsBTT.(A) Groups of seven times on days 0, 28, and 56 with PSGBlT admixed with 0 (group I, l), 0.1 (group 2, q ) or 1 (group 3, A) pg of seven mice randomly chosen among those mice initially belonging to groups 2 and 3 were immunized two 126 with PSGBlT (0) or saline (0). Individual mice sera were analysed 2 weeks after each immunization. The isotype was determined by ELISA and expressed as mean f S.D.

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days

140

days

mice were immunized three of native PSGB. (B) Groups more times on days 98 and titre of each anti-PSGB IgG

Pneumococcal conjugate vaccine: ALE Rodriguezet al. Table 1

Influence of the presence of free PSGB of 37 kDa (mean molecular weight) on the immunogenicity Mean of “log IgG”

+ + + +

anti-PSGB titre (No. responders/No.

vaccine

in group)

IgG2b”

lgGld

lgG3’

After three immunizations

Immunization” PSGBTT PSGBTT PS6Bl-r PSGBTT PSGBTT

of PSGBlT conjugate

4.9 4.9 4.5 3.7 3.7

0.01 pg PS6B 0.03 /1g PSGB 0.1 ,1g PSGB 1 1’9 PSGB

4.1 4.1 4.0 3.3 3.3

(7/7) (7/7) (7/7) (7/7) (7/7)

(7/7) (7/7) (7/7) (7/7) (7/7)

3.0 3.1 2.1 1.7 1.5

(7/7) (7/7) (6/7) (6/7) (6/7)

2.9 3.2 2.9 2.0 1.9

(7/7) (7/7) (6/7) (6/7) (6/7)

After first re-immunization

Re-immunization* 4.5 (7/7) 3.7 (717)

PS6Bl-r Saline

4.3 (7/7) 3.4 (717)

2.9 (7/7) 1.6 (617)

3.2 (7/7) 2.0 (6/7)

After second re-immunization 4.6 (7/7) 3.7 (7/7)

PS6Bl-r Saline

4.5 (7/7) 3.5 (7/7)

3.1 (7/7) 1.7 (6/7)

3.2 (7/7) 2.1 (6/7)

“Groups of seven mice were immunized three times within 8 weeks (days 0, 28 and 56) with PSGBTT admixed with different amounts of free PSGB. Individual mice sera were analysed 2 weeks after the third immunization. The titre of each anti-PSGB IgG isotype was determined by ELISA and expressed as means which were compared by LSD test with a confidence level of 95%. The number of responder mice/size of the experimental group are indicated. bThe two groups previously immunized three times with 0.1 and 1 Llg of free PSGB admixed with PSGBTT were immunized twice more within 4 weeks with PSGBTT and saline, respectively. Individual mice sera were analysed 2 weeks after the first and the second re-immunization. The titre of each anti-PSGB IgCi isotype was determined by ELISA and expressed as a mean. The means were compared by LSD test with a confidence level of 95%. Number of responder mice/size of the experimental group are indicated. CLSD0.05= 0.8. “LSD -07 “LSD’ O5I 0’9’ oos‘LSDo,os= 0.8. polysaccharide

conjugate

vaccine

capable

of

inducing

in infants. In this study, we investigated the immunogenicity displayed by two of the least immunogenic, and widely spread types of capsular polysaccharide of Streptococcus pneumoniae, 6B and 14, covalently coupled to tetanus toxoid. This toxoid proved to be safe for human use and a good carrier when conjugated to other polysaccharides”.““. The size of both polysaccharides was reduced by sonication to a mean molecular weight of 46 and 37 kDa for PS6B and PS14, respectively, before conjugation. Both conjugates induced anti-TT IgG antibodies with a subclass distribution characterized by IgGl$ IgG2b. Only small amounts of anti-TT IgG3 were detected and anti-TT IgG2a remained at its pre-immune level. On the other hand, both PS6BTT and PS14TT induced a type-specific anti-PS IgG antibody response which could be boosted by repeated immunization. After the second immunization a marked increase in anti-PS IgG production rate was observed. A further increase, but to a lesser extent, was induced by a third immunization with both conjugates. Antipolysaccharide antibodies induced by booster vaccination with PS6BTT or PS14TI’ showed significant higher avidity for their respective carbohydrate with the avidity of the antigens, as compared antibodies induced upon priming immunization. This indicates that antibody affinity maturation was induced. Both conjugates induced all anti-PS IgG subclasses, except for IgG2a, which remained at its pre-immune level. The third immunization with both conjugates led to an increase in the respective type-specific anti-PS antibody levels but no significant changes were detected in the anti-PS IgG subclass distribution compared with that induced upon the second immunization. protection

The results obtained indicate that conjugation of the capsular polysaccharide of pneumococcal types PS6B and PS14 actually stimulates Th co-operation in the anti-PS antibody response, which was in many aspects comparable with the TD response to proteins. The conjugates induced high levels of anti-PS IgG antibodies in a secondary response. Antibody affinity maturation was detected and the isotype restriction characteristic of TI-2 antigens3’ was not observed. However, high titres of type-specific anti-PS antibodies do not guarantee biological activity of antibodies. The avidity of the antisera is a good indicator of the quality of the antibodies induced but the biological functionality of these antibodies must be assessed. The results obtained indicate that both conjugates are able to induce functional antibodies capable of acting as opsonins towards pneumococcal phagocytosis in vitro, despite the lack of type-specific IgG2a antibody. The latter observation is in agreement with previous studies in which the opsonic activity of mouse serum was mainly correlated with IgG isotypes other than IgG2a3’. The efficacy of a conjugate vaccine could be diminished by the presence of free polysaccharide in the vaccine formulationz2. The admixture of free pneumococcus capsular polysaccharide type 4 (PS4) of 1.6-120 kDa with a PS4TT vaccine resulted in typespecific immune suppression when the amount of free polysaccharide was higher than 10% of the conjugated polysaccharide. An inappropriate ligation of the antigen receptor on B cells effected by the poly- or oligosaccharide was suggested to be the cause of the tolerance induction. In our hands, a similar pattern of unresponsiveness towards PS6B was induced by the presence of high doses of native PS6B in the conjugate vaccine Vaccine 1998 Volume 16 Number 20

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(PS6BTT). Booster injections of conjugate admixed with free polysaccharide did not result in the rise of anti-PS6B IgG antibody levels comparable with those observed in mice injected with conjugate solely. AntiPS6B IgG2b was the isotype most affected; its production was almost totally suppressed at the highest dose (1 pg) of free polysaccharide. On the other hand, antiPS6B IgG3 was less affected by the presence of free polysaccharide, which could suggest that free PS6B competes with the conjugate and activates B cells in a TI form. However, the finding that anti-PS6B IgM production was also suppressed by the presence of free polysaccharide did not support this possibility. Interestingly, free PS6B of shorter chain length (37 kDa) was shown to be less effective than native polysaccharide in suppressing antigen-specific immune response. Moreover, further immunization with conjugate alone reversed the state of unresponsiveness in those mice previously immunized with conjugate and free PS6B of lower molecular weight. Only one re-immunization with pure conjugate was able to induce high titres of each anti-PS6B IgG subclass. The titres induced showed no significant (p ~0.05) differences with those achieved in the group of mice immunized three times with conjugate solely. Even the level of anti-PS6B IgG2b, which was again the isotype most deprived by the presence of free polysaccharide, was recovered after one immunization with conjugate. In contrast, further immunizations with conjugate failed to induce significant anti-PS6B IgG antibody response in those mice previously immunized with conjugate mixed with free native polysaccharide. The above data seem to indicate that the relative incapacity of the immune system to respond to a secondary injection when high doses of free PS6B were present in the conjugate vaccine could have been caused by persistence of PS in the organism. The reduction in chain length of PS6B could allow a faster clearance from the organism as compared with native PS. In general, antigens with repetitive epitopes are poorly degradable in vivo, which is a prerequisite to stimulate B cells to TI antibody production’3. This might also be the cause of tolerance induction”“-“6, as was demonstrated when the immune unresponsiveness induced by serotype 3 of pneumococcal polysaccharide was restored by injecting tolerant animals with hydrolytic enzymes37.38.The difference between the state of unresponsiveness induced by free PS6B (mean m.w.: 37 kDa) and that reported for PS4”, despite the fact that they were comparable in terms of chain lengths, could be explained by the fact that PS6B, being a phosphorylated polysaccharide, is more easily degradable in viva. The presence of native or sonicated free polysaccharide in the conjugate vaccine formulation was shown to have no influence on the immune response raised against the protein carrier. Moreover, the B-cell tolerance induction showed to be typespecific since the presence of other pneumococcal polysaccharide serotypes, even in high amounts (1 pg), did not affect the anti-PS6B antibody response. In conclusion, the two pneumococcal polysaccharide conjugates presented in this paper induce a high antiPS IgG antibody response with good biological activity. which is a requirement for a good vaccine candidate.

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Furthermore, this study shows that the immunogenic@ of a PS-protein conjugate vaccine is decreased by the presence of uncoupled PS. However, the magnitude of this effect seems to be PS type dependent. ACKNOWLEDGEMENTS The research for this publication was partially financed by the Netherlands ‘Ministry for Development Co-operation’ and the European Union. Dr Maria Eugenia Rodriguez was supported by an External Fellowship from the Argentinian Postdoctoral Research Council (CONICET). REFERENCES 1

2

3

4 5

6

7

8

9

10

11

12

13

14

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Austrian, Ft., 1984. Pneumococcal infections. In: Germanier, R. (Ed.), Bacterial Vaccines. Academic Press, New York, pp. 257-285. Garcia-Leoni, M.E., Cercenado, E., Rodeno, P., Bernaldo de Quirks, J.C.L., Martinez-Hernandez, D. and Bouza, E. Susceptibility of Streptococcus pneumoniae to penicillin: a prospective microbial and clinical study. J. Infect Dis. 1992, 14(Z), 427-435. Musher, D.M. Infections caused by Streptococcus pneumoniae: clinical spectrum, pathogenesis, immunity, and treatment. C/in. Infect. Dis. 1992, 14(4), 801-809. Henrichsen, J. Six newly recognised types of Streptococcus pneumoniae. J. C/in. Microbial. 1995, 33(10), 2759-2762. Bardardottir, E., Jonsson, S., Jonsdottir, I., Sigfusson, A. and Valdimarsson, H. IgG subclass response and opsonization of Streptococcus pneumoniae after vaccination of healthy adults. J. infect Dis. 1990, 162(Z), 482-488. Sanders, E.A.M., Rijkers, G.T. and Kuis, W. et al. Defective antipneumococcal polysaccharide antibody response in children with recurrent respiratory tract infections. J. AIIergy C/in. Immunol. 1993, 91(1 pt l), 110-l 19. Musher, D.M., Chapman, A.J., Goree, A., Jonsson, S., Briles, D. and Baughn, R.E. Natural and vaccine-related immunity to Streptococcus pneumoniae. J. Infect. Dis. 1986, 154(Z), 2455256. Forrester, H.L., Jahnigen, D.W. and LaForce, F.M. Inefficacy of pneumococcal vaccine in a high-risk population. Am. J. Med. 1987, 83(3), 425-430. Shapiro, E.D., Berg, A.T. and Austrian, R. et a/. The protective efficacy of polyvalent pneumococcal polysaccharide vaccine. N. Engl. J. Med. 1991, 325(21), 1453-1460. Stein, K.E. Network regulation of the immune response to bacterial polysaccharide antigens. Curr. Top. Microbiol. Immunol. 1985, 119,57-74. Stein, ES. Thymus-independent and thymus-dependent responses to polysaccharide antigens. J. Infect. Dis. 1992, 165(suppl l), S49-52. Alonso de Velasco, E., Merkus, D. and Snippe, H. et al. Synthetic peptides representing T-cell epitopes act as carriers in pneumococcal polysaccharide conjugate vaccines. fnfect. Immun. 1995, 63(3), 961-968. Lee, C.-J., Lock, R.A., Andrew, P.W., Timothy, J.M., Hansman, D. and Paton, J.C. Protection of infant mice from challenge with Streptococcus pneumoniae type 19F by immunization with a type 19F polysaccharide-pneumolysoid conjugate. Vaccine 1994, 12(10), 875-878. Schneerson, R., Levi, L., Robbins, J.B., Bryla, D.M., Schiffman, G. and Lagergard, T. Synthesis of a conjugate vaccine composed of pneumococcus type 14 capsular polysaccharide bound to pertussis toxin. infect Immun. 1992, 60(g), 3528-2532. Kayhty, H., Ahman, H., Ronnberg, P-H., Tillikainen, R. and Eskola, J.J. Pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine is immunogenic in infants and children. J. infect. Dis. 1995, 172, 1273- 1278. Sigurdardottir, ST., Vidarsson, G. and Gudnason, T. et a/. Immune response of infants vaccinated with serotype 6B pneumococcal polysaccharide conjugated with tetanus toxoid. Pediatr. infect. Dis. J. 1997, 16(7), 667-674.

Pneumococcal conjugate vaccine: M. E. Rodriguez et al. 17

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19

20

21

22

23

24

25

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Dagan, Ft., Melamed, Ft., Zamir, 0. and Leroy, 0. Safety and immunogenicity of tetravalent pneumococcal vaccines containing 6B, 14, 19F and 23F polysaccharides conjugated to either tetanus toxoid or diphtheria toxoid in young infants and their boosterability by native polysaccharjde antigens. Pediatr. Infect. Dis. J. 1997. 16(11). 1053-1059. Ahman, H., Kayhty, H., Tamminen, P., Vuorela, A., Malinoski, F. and Eskola, J. Pentavalent pneumococcal oligosaccharide conjugate vaccine PncCRM is well-tolerated and able to induce an antibody response in infants. Pediatr. Infect. Dis. J. 1996, 15(2), 134-139. Insel, R.A. and Ander:son, P.W. Oligosaccharide-protein conjugate vaccines induce and prime for oligoclonal IgG antibody responses to the Haemophilus influenzae type b capsular polysaccharide in human infants. J. Exp. Med. 1986, 163(2), 262-269. Sniadack, D.H., Schwartz, B. and Lipman, H. et al. Potential interventions for prevention of childhood pneumonia: geographic and temporal differences in serotype and serogroup distribution c,f sterile site pneumococcal isolates from children-implications for vaccine strategies. Pediafr. Infect. Dis. J. 1995, 14(6), 503-510. van Dam, J.E.G., Feer, A. and Snippe, H. lmmunogenicity and immunochemistry of Streptococcus pneumoniae capsular polysaccharides. Anton& Leeuwenhoek 1990, 56, l-47. Peeters, C.A.M., Tenbergen-Meekes, A-M.J., Poolman, J.T., Zegers, B.J.M. and Rijkers, G.T. lmmunogenicity of Sfreptococcus pneumoniae type 4 polysaccharide-protein conjugate vaccine is decreased by admixture of high doses of free saccharide. Vaccine 1992, 10(12), 833-840. de Weers, O., Beurret, M., van Buren, L., Oomen, L. A., Poolman, J.T., Hoogerhout, P., 1998. Application of cystamine and N,N’-bis[glycyl]cystamine as linkers in polysaccharide-protein conjugation. Bioconjugates Chemistry 1998, 9,309-315. Anttila, M., Eskola, J., Allman, H., Kayhty, H., 1998. Avidity of IgG for Streptococcus pneumoniae type 6B and 23F polysaccharide in infants primed with pneumococcal conjugates and boostered with polysaccharide or conjugate vaccines. J. Infect. Dis. 1998, 177, 1614-1620. Alonso de Velasco, E., Dekker, B.A.T. and Antal, P. et al. The Quil A improves protection in mice and enhances the opsonic capacity of antisera induced by pneumoccocal polysaccharide conjugates. Vac,sine 1994, 12(15), 1419-1422. Raschke, WC., Baird, S., Ralph, P. and Nakoinz, I. Functional

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28

29

30

31

32

33

34

35

36

37

38

macrophage cell line transformed by Abelson leukaemia virus. Cell 1978, 15(l), 261-267. Pullen. G.R.. Fitzaerald. M.G. and Hoskina. C.S. Antibodv avidity’ determinat&n by ELISA using thiocianate elution. j. Immunol. Meth. 1986, 86(l), 83-87. Avery, O.T. and Goebel, W.F. Chemo-immunological studies on conjugated carbohydrate-proteins II. Immunological specificity of synthetic sugar protein antigens. J. fxp. Med 1929,50,533-550. Bixler, G.S. and Pillai, S. The cellular basis of the immune response by conjugate vaccines. Contrib. Microbial. Immunol. 1989,10,18-47. Laferriere, CA., Sood, R.K., de Muys, J-M., Michon, F. and Jennings, H.J. The synthesis of Streptococcus pneumoniae polysaccharide-tetanus toxoid conjugates and the effect of chain length on immunogenicity. Vaccine 1997, 15(2), 179-l 86. Perlmutter, R.M., Hansburg, D., Briles, D.E., Nicoletti, R.A. and Davie, J.M. Subclass restriction of murine anti-carbohydrate antibodies. J. Immunol. 1978, 121(2), 566-572. Alonso de Velasco, E., Dekker, B.A.T., Verheul, A.F.M., Feldman, R.G., Verhoef, J. and Snippe, H. Anti-polysaccharide immunoglobulin isotype levels and opsonic activity of antisera: relationships with protection against Streptococcus pneumoniae infection in mice. J. Infect. 0s. 1995, 172(2), 562-565. Sela, M., Mozes, E. and Shearer, G.M. Thymus independence of slowly metabolised immunogens. Proc. Nat/. Acad. Sci. USA 1972,69(g), 2696-2700. Moreno, C., 1987. In: Bell, R., Torrigiani, G. (Eds.), Towards Better Carbohydrate Vaccines. J. Wiley, Chichester, UK, pp. 263-277. Howard, J.G., Christie, G.H. and Courtenay, B.M. Treadmill neutralisation of antibody and central inhibition: separate components of pneumococcal polysaccharide paralysis. Transplantation 1970, 1 O(4), 251-253. Dint%, R.Z., Middleton, M.H. and Dintzis, H.M. Studies on the immunogenicity and tolerogenicity of T-independent antigens. J. Immunol. 1983, 131(5), 2196-2203. Avery, O.T. and Dubos, R. The protective action of a specific enzyme against type Ill pneumococcus infection in mice. J. Exp. Med. 1931, 54, 73-90. Brooke, M.S. Breaking of immunological paralysis by injection of specific depolymerase. Nature 1964, 204, 1319-l 320.

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