Med Microbiol Immunol (1992) 181:35-42

�9 Springer-Verlag 1992

Induction of anti-pneumococcal cell wall polysaccharide antibodies by type 4 pneumococcal polysaccharide-protein conjugates Carla Peeters 1,2, Anne-Marie Tenbergen-Meekes 1 , Jan Poolmann 2, Ben Zegers 1 , and Ger Rijkers 1

1 Department of Immunology, Univeristy Hospital for Children and Youth "Het Wilhelmina Kinderziekenhuis', State University, School of Medicine, Utrecht, The Netherlands 2 Unit for Bacterial Vaccine Development and Pathogenesis Research, National Institute of Public Health and Environmental Protection, Antonie van Leeuwenhoeklaan 9, P.O. Box 1, NL-3720 BA Bilthoven, The Netherlands

Received October 7, 1991

Abstract. We have prepared polysaccharide-protein conjugates consisting of type 4 pneumococcal capsular polysaccharide (PS4) coupled to tetanus toxoid. The PS4 preparation used contained 2.5% pneumococcal cell wall polysaccharide (CPs). During the conjugation process, in addition to PS4-protein conjugates, CPs- protein conjugates were also formed. After immunization with PS4-protein conjugates, CPs-protein conjugates that are present as a contaminant induce IgG anti-CPs antibodies in mice. Pneumococcal oligosaccharides, prepared by perio- date oxidation of PS4, did not contain detectable amounts of CPs; hence, oligosaccharide-protein conjugates did not induce anti-CPs antibodies.

Introduction

Antibodies againts the type-specific capsular polysaccharides of Streptococcus pneumoniae confer clinical protection against invasive diseases caused by these bacteria. Capsular polysaccharides are classified, in both mice [15] and humans [2], as T cell-independent type 2 (TI-2) antigens. TI-2 antigens are characterized by a number of features which distinguish them from T cell-dependent (TD) antigens: responsiveness appears relatively late in o ntogeny [5, 6]; immunological memory is not induced [4]; and IgM and IgG2 (in man)/IgG3 (in mice) are the predominant isotypes [9, 20, 23,]. Consequently, pneumococcal polysaccharide vaccines are ineffective in children below 2 years of age, a group which, because of the prevalence of invasive pneumococcal disease, would most benefit from an effective vaccine [8, 22].

Recently, it became evident that immunization with polyvalent pneumococcal vaccine not only induces type-specific anti-capsular polysaccharide antibodies but also antibodies to the pneumococcal cell wall polysaccharide (CPs). This CPs, which is a common contaminant of most capsular polysaccharide preparations

Offprint requests to: C. Peeters 2

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[24, 25], is a complex teichoic acid polymer containing phosphorylchol ine [ 10]. For that reason, the anti-capsular polysaccharide ant ibody responses in both man [12, 17] and mice [7, 14] have recently been re-evaluated to analyze possible interference of anti-CPs antibodies. These studies show that, for example, the response to type 6A and 19F pneumococca l polysaccharide may be misinterpreted since it consists to a large degree of anti-CPs antibodies. The TI-2 nature of polysaccharide antigens can be changed into that of a TD antigen by conjugat ion to a protein carrier. This strategy has succesfully been used to generate effective vaccines against Haemophilus influenzae type b [1, 21]. Currently, similar approaches are being used in the development of pneumococca l polysaccharide conjugate vaccines. We have prepared experimental polysaccharide-protein conju- gates, consisting of type 4 pneumococca l polysaccharide (PS4) coupled to tetanus toxoid (TT) [19]. In the present study we show that, during the conjugat ion process, CPs-protein conjugates are also formed in addit ion to PS4-protein conjugates. These CPs-protein conjugates are able to induce IgG anti-CPs antibodies in mice. Pneumococca l oligosaccharides, prepared by periodate oxidat ion of polysaccharides, did not contain CPs, hence, ol igosaccharide-protein conjugates did not induce anti-CPs antibodies.

Methods

Mice

Random outbred NIH/RIVM mice were bred and maintained at the animal facility of the National Institute of Public Health and Environmental Protection, Bilthoven. Female mice, 8-12 weeks old, were used in all experiments.

Antigens and immunization

PS4 was obtained from the American Type Culture Collection (ATCC, Rockville, Md). Type 4 pneumococcal oligosaccharide (OS4; mean chain length of 12 repeating units) was prepared by periodate oxidation of PS4 as described earlier [19]. CPs was kindly donated by Dr. U. Sorensen (Streptococcal Department, State Serum Institute, Copenhagen, Denmark) and was prepared as described elsewhere [18]. PS4- and OS4-protein conjugates were prepared as described earlier [19]. Briefly, polysaccharides and oligosaccharides were activated with cyanogen bromide and coupled to 6-aminohexanoic acid. Next, the derivatized poly- and oligosaccharides were coupled to TT using 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide. Conjugates were purified by gel filtration on Sepharose CL4B.

Mice were immunized subcutaneously with 2.5 lag native or conjugated poly- or oligosaccha- ride. Three immunizations were given; the interval between the first and second immunization was 4 weeks, and 6 weeks between the second and third immunization. Blood samples were taken from the tail vein at regular intervals; sera were stored at --20~

Determination of lgM and IgG anti-PS4 and anti-CPs antibodies

Individual wells of 96-well microtiter plates (Flow, Irvine, UK) were coated with appropriate diluted rabbit anti-PS4 antibodies (State Serum Insitute) in 0.5 M Na2Co3, pH 6.9. Plates were incubated at 37~ for 3h, washed, and incubated overnight at 4~ with 1 lag/ml PS4 in 0.9% NaCI. Next, plates were washed and incubated for 3 h at 37~ with serum samples, serially

37

diluted in PBS, 0.05 % (v/v) Tween-20, 1% BSA. Plates were washed and incubated for 13 h at 37~ with alkaline phosphatase-conjugated goat antisera to mouse IgG or IgM (Southern Biotechnology, Birmingham, Ala.). Plates were washed and incubated with the phosphatase substrate (p-nitrophenylphosphate(1 mg/ml; Sigma, St Louis Mo.) in 10% diethanolamine buffer, pH 9.8. Plates were incubated for 30-60 min, the reaction was stopped by addition of 50 lal 2.4 NaOH, and absorbance at 405 nm was determined using a Titertek Multiscan enzyme-linked immunosorbent assay (ELISA) Reader (Flow). All antibody titers are expressed as percentages of a pooled hyperimmune serum.

Anti-CPs antibodies were determined, using a similar method, on plates coated overnight at room temperature with CPs covalently linked to phenyl at a concentration of 5gg/ml in 0.1 M NaHCO3, pH 8.2.

ELISA inhibition

CPs-coated plates were washed and incubated for 3h at 37~ with immune serum of a Pneumovax-vaccinated donor, prediluted to a concentration yielding an absorbance of approximately 1.0. Native or conjugated poly- or oligosaccharides were mixed in varying concentrations (ranging from 0.01 to 100 gg/ml) with the immune serum. Plates were washed and incubated with peroxidase-conjugated goat antisera to human IgG (Dako Immunoglobulin, Copenhagen, Denmark). Next, plates were washed and incubated for 10-30min at room temperature with the peroxidase substrate 3,Y,5,5'-tetramethylbenzidine (0.1 mg/ml; Sigma), 0.01% H202 in 0.11 M sodium acetate buffer, pH 5.5. The reaction was stopped by addition of 100 gl 0.5 M H2SO4 and absorbance at 450 nm was determined.

Double sandwich ELISA to detect conjugated CPs

Microtiter plates were coated with goat antibodies to TT. Plates were washed and incubated with native or conjugated poly- or oligosaccharides. Next, plates were washed and incubated with murine monoclonal anti-CPs antibodies (clone HASP8, kindly provided by Dr U. Sorensen [25]. Plates were washed and incubated with alkaline phosphatase-conjugated goat antibodies to mouse IgM and processed further as described above.

Results

PS4 appears to be contaminated with approximately 2.5% (w/w) CPs, as determined by inhibition ELISA (Fig. 1). Type 14 pneumococcal polysaccharide contained a similar amount of CPs, while type 3 pneumococcal polysaccharide was virtually free of CPs (Fig. 1, left panel). The oligosaccharide preparation of PS4 (OS4) did not inhibit an anti-CPs ELISA, suggesting that the procedure used to generate oligosaccharides (i.e. periodate oxidation) leads to destruction of CPs. Polysaccharide-protein conjugates (PS4TT) displayed an inhibition profile similar to that of the native PS4, while oligosaccharide-protein conjugates (OS4TT) were not inhibitory, similar to native OS4 (Fig. 1, right panel).

Since the native PS4 preparation contained CPs, it is possible that during the process of conjugation CPs-protein conjugates may be produced in addition to the PS4-protein conjugates. Poly- and oligosaccharide conjugates were, therefore, analyzed in a double-sandwich ELISA for the presence of CPs-protein conjugates. Figure 2 shows that the PS4TT conjugates did indeed contain conjugated CPs. As expected, OS4TT conjugates were devoid of CPs conjugates.

Immunization of mice with 2.5 ~tg PS4, which, as shown above, contains approximately 0.06 gg Cps, induced IgM anti-CPs antibodies. Immunization with

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38

inhibitor (pg/rnl)

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Fig. 1. Presence of cell wall polysaccharide (CPs) in native and conjugated type 4 pneumococcal capsular polysaccharide (PS). CPs-coated plates were incubated with human immune serum, mixed with: variable concentration CPs (I); type 3 (PS3; A). type 4 (PS4; A), and type 14 (PS14; [~) pneumococcal capsular polysaccharide; PS4-tetanus toxoid conjugate (@); type 4 oligosac- charide-tetanus toxoid conjugate (V); or BSA (O). Following incubation, plates were developed with peroxidase-conjugated goat anti-human IgG. The CPs content of native or conjugated capsular polysaccharide was estimated by comparing, relative to CPs, the amount of polysaccha- ride required to obtain 59% inhibition of absorbance at 405 nm

rel. absorbance 0 0.1 0.2 0.3 0.4 I , I u I , I , I

PS4xTT (a) Iiii~;~i~i~i{~;{~!i!i~:ii~i!i~iii!i~i~i!i~i~!!~!i!!~!i!i!i~i!i~i!i;i~i~i{~!~i~!~i!;:ii!ii!;~iii!{~i~i!ii~!i!iii~!i~!!!i~i~ii!~

OS4xTT

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0.5 Fig. 2. Type pneumococcal poly- saccharide (PS4)-tetanus toxoid (TT) conjugates contain CPs. Anti-TT-coated plates were incu- bated with serial dilutions of two different batches (a) and (b) PS4TT conjugates, type 4 oligo- saccharide (OS4)-TT conjugate, native PS4, native OS4, or TT. Plates were developed with mouse anti-CPs and alkaline phospha- tase-conjugated goat anti-mouse IgM. Shown is the relative absor- bance (405 nm) at equivalent sac- charide concentrations

PS4TT induced approximate ly five fold higher IgM anti-CPs an t ibody titers (Fig. 3). Secondary and tertiairy immuniza t ion (which were given 4 and 10 weeks following pr imary immunizat ion, respectively) did not result in an increase in IgM anti-CPs titers. IgM anti-CPs antibodies were never detected in mice immunized with OS4TT conjugates. Pr imary immunizat ion with PS4TT (a conjugate which also contains conjugates CPs) induced IgG anti-CPs antibodies (Fig. 3). Repeated immunizat ion led to a clear-cut booster response. IgG anti-CPs antibodies could not be induced by immuniza t ion with native PS4 or OS4. These data indicated that CPs, when conjugated to a protein carrier, is capable to induce IgG antibodies.

39

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anti-PS4 antibody titer (- CPs)

Fig. 3. Induction of anti-CPs antibodies by native or conjugated PS4. Groups of ten mice were immunized three times with type 4 polysaccharide-TT conjugate (PS4TT; II), type 4 oligosaccha- ride-TT conjugate (OS4TT; 4,) or native PS4 (0). Anti-CPs IgM antibodies were determined in pooled serum samples obtained before (pre) and at day 7-8 following each immunization; anti- CPs IgG antibodies were determined before (pre) and at day 28 following each immunization. Antibody titers are expressed as percentage of pooled hyperimmune serum

Fig. 4. Specificity of IgM and IgG anti-PS4 antibodies induced by native or conjugated PS4. Mice were immunized with native PS4 (circles) or PS4-TT conjugates (triangles) as described in legend to Fig. 3. Anti-PS4 IgM (closed symbols) and anti-PS4IgG antibodies (open symbols) were determined in pooled serum samples in the presence or absence of 250/.tg/ml soluble CPs. Antibody titers are expressed as percentage of pooled hyperimmune serum. Pre-immune sera contained less than 5 % anti-PS4 IgM and anti-PS4 IgG antibodies. The number of immuniza- tions is indicated with roman numbers. The broken line and drawn lines connecting the data points are indicated for visual reference only

The fine specifity of anti-CPs antibodies, generated by immunization with native or conjugated PS4, was studied by an inhibition E L I S A using CPs and phosphorylcholine (PC), the immunodominant moiety of CPs [11]. While the binding of IgM antibodies to CPs could be completely inhibited by PC, binding of IgG antibodies to CPs was not affected by PC in concentrations up to 500 ~tg/ml (Table 1).

Having established that immunization with polysaccharide-protein conju- gates induces both IgM and IgG anti-CPs antibodies, we subsequently studied the potential interference of anti-CPs antibodies on the determination of anti-capsular polysaccharide antibodies. The binding of IgM antibodies, induced by immuniza- tion with native PS4, to plates coated with PS4 can be completely inhibited by excess (250 ~tg/ml) of soluble CPs (Fig. 4). This finding confirms earlier observa- tions with other pneumococcal capsular polysaccharides, namely that the apparent IgM anti-polysaccharide response basically reflects an anti-CPs response [7, 14]. IgM antibodies induced by (repeated) immunization with conjugated polysaccha- rides, however, are specific for PS4 (Fig. 4). The binding of IgG antibodies to PS4- coated plates could not be inhibited by soluble CPs. This also holds true for IgG antibodies induced by immunization with native PS4 (Fig. 4).

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Table 1. Fine specificity of anti-cell wall polysaccharide (CPs) antibodies

Inhibitor Concentration % Inhibition (~tg/ml)

IgM antibodies IgG antibodies

CPs 25 63 42 CPs 100 68 61 CPs 500 76 80 PC 25 >99 <1 PC 500 >99 <1

CPs-coated plates were incubated with pooled sera of mice immunized with type 4 pneumococcal polysaccharide-tetanus toxoid conjugate. Serum incubation was performed in the presence of variable amounts soluble CPs or phospholycholine (PC). Plates were developed using goat anti- mouse IgM or IgG reagents. Results are expressed relative to unhibited anti-CPs antibody titers

Discussion

The data presented in this report on the induction of anti-CPs IgM antibodies following immunization with polysaccharide-protein conjugates confirm recent data of Milligan et al. [14]. These authors, however, did not find anti-CPs IgG antibodies following immunization with type 6 PS or type 19 PS coupled to chicken erythrocytes (CRBC) [14]. Moreover, all IgG secreting hybridomas, obtained from PS19- or PS6-CRBC secondary response fusions secreted PS-specific antibody [13].

CPs-specific IgG could, however, be induced by immunization with CPs- CRBC conjugates [14]. We have no adequate explanation as to why anti-CPs IgG is readily induced by PS4TT conjugates and not by PS6-CRBC and PS 19-CRBC. The amount of CPs in the different PS preparations is unlikely to have caused this difference, because PS6 and PS 19 contain similar (or even higher) amounts of CPs [24, 25]. The differences in the ability to induce anti-CPs IgG antibodies could potentially be due to differences in coupling reagent used (CrC13 for coupling to CRBC, carbodiimide for coupling to protein) or to the differences in carrier (corpuscular CRBC versus protein TT).

The observation that in mouse sera, which contain anti-PS4 and anti-CPs antibodies of both IgM and IgG isotype, soluble CPs can significantly inhibit binding of IgM antibodies to PS4-coated plates, but is not capable of inhibiting the binding of IgG antibodies, seems intruiging. Because anti-PS4 as well as anti-CPs antibodies were measured by ELISA in the absence of a quantitative standard, their levels cannot be compared directly. As far as IgM antibodies are concerned, our data demonstrate that after primary immunization, most, if not all IgM appears to be anti-CPs. The binding of IgM antibodies to PS4-coated plates in sera from mice which were immunized repeatedly with PS4TT, however, could not be inhibited by CPs. Because ist has been well demonstrated that, even during anamnestic anti-polysaccharide antibody responses, no affinity maturation oc- curs, we can conclude that in the latter situation the anti-PS4 IgM titer by far exceeds the anti-CPs IgM titer. This conclusion is supported by the finding that repeated immunization with PS4TT does not increase anti-CPs IgM antibodies (Fig. 2). With regard to IgG antibodies, it can be argued that, due to the 40-fold

41

difference in antigen dose, anti-PS4 IgG antibody levels are far higher than anti- CPs antibody levels. In this case, an effective prevention of binding ofanti-CPs IgG by soluble CPs would remain unnoticed in an anti-PS4 IgG ELISA.

Wether induction of anti-CPs IgG antibodies is a favorable or unwanted side effect of vaccination depends on the role of anti-CPs antibodies in invasive pneumococcal disease. However, the level of protection against pneumococcal infections conferred by anti-CPs antibodies still is controversial. Antibodies to the PC determinant of CPs have been shown to protect mice against a lethal challenge with several pneumococcal serotypes [3, 27, 29]. In contrast, rabbit antibodies to CPs elicited by the CPs-BSA conjugate failed to protect mice against intraperito- neal challenge with a strain of type 3 or type 6A pneumococci [28]. In man, naturally present anti-CPs IgG does not protect against invasive pneumococcal infection {10, 16].

Our data indicate that protection conjugates of pneumococcal polysaccha- rides may contain conjugated CPs and, therefore induce anti-CPs IgG antibodies. Because of the potential interference of anti-CPs IgG antibodies in routinely used assays for determination of anti-capsular polysaccharide antibodies, this caveat should be realised in the evaluation of pneumococcal polysaccharide conjugate vaccines.

Large scale production of pneumococcal capsular polysaccharides which are free of CPs may be difficult to accomplish, which is reflected by the fact that most, if not all, commercially available capsular polysaccharides contain a certain level of CPs ([24, 25] and this report). The reason may be that CPs is probably convalently linked to capsular polysaccharides [26]. Oligosaccharides, prepared by periodate oxidation of capsular polysaccharides, no longer contain detectable amounts of CPs. In this respect, oligosaccharides would be the saccharide moiety to be preferred in synthesis of future pneumococcal conjugate vaccines.

Acknowledgements. We thank Mr. B. van Hilten for his invaluable biotechnical assistance and Dr. U. Sorensen (State Serum Insitute, Copenhagen, Denmark) for kindly supplying phenyl-CPs, CPs and the monoclonal antibody HASP8 which is directed against CPs. This study was supported in part by a grant from the Prevention Fund (grant #24-1246).

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