Pneumococcal conjugate vaccines

Immunology Letters, 30 (1991) 2 6 7 - 274 Elsevier IMLET 01699

Pneumococcal conjugate vaccines Carla C. A. M. Peeters l, 2, Anne-Marie Tenbergen-Meekes l, Bart H a a g m a n s 1, D o l f Evenberg 2, Jan T. P o o l m a n 2, Ben J. M. Zegers I and Ger T. Rijkers 1 IDepartment of Immunology, University Hospital for Children and Youth "'Het Wilhelmina Kinderziekenhuis", Utrecht, The Netherlands; and 2Unit of Bacterial VaccineDevelopment and Pathogenesis Research, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands (Received 8 July 1991; accepted 24 July 1991)

1. Summary

We have prepared conjugates of pneumococcal type 4 polysaccharides (PS4) or oligosaccharides to tetanus toxoid using the carbodiimide method. The use of a spacer, 6-aminohexanoic acid, resulted in higher incorporation of carrier protein. Conjugates contained up to 10°70 free polysaccharide, but no free protein. In general, polysaccharide conjugates induced higher anti-PS4 IgG antibody titers than oligosaccharide conjugates. Conjugates with the highest amount of incorporated protein were the most immunogenic. The response to conjugated PS4 does show characteristics of a T cell-dependent antibody response, in terms of both isotype distribution and induction of immunological memory. Repeated immunization with high doses of PS4TT conjugate resulted in a virtually negative anti-PS4 IgG response, suggestive of the induction of high dose tolerance. 2. Introduction

Antibodies against the capsular polysaccharides of bacteria like Streptococcus pneumoniae, Haernophilus influenzae type b, and Neisseria meningitidis, confer protection against invasive diseases caused by corresponding bacteria. Vaccines Key words: Pneumococcal polysaccharide; Conjugate vaccine; Antibody synthesis; Tolerance Correspondence to: Carla C. A. M. Peeters, Dept. of Immunology, Her Wilhelmina Kinderziekenhuis, P.O. Box 18009, Utrecht, The Netherlands.

based on capsular polysaccharides can induce protective antibody levels, but high risk groups, such as children under two years of age and the elderly, do not respond satisfactorily to these vaccines [1, 2]. The poor antibody response of the former group is most probably associated with the fact that capsular polysaccharides belong to the so-called T cellindependent type 2 (TI-2) antigens [3, 4]. TI-2 antigens can activate B cells without the need for direct participation of T cells, although the magnitude of an anti-TI-2 antibody response appears to be regulated by amplifier and suppressor T cells [3, 5, 6]. The antibody response to TI-2 antigens also differs in other aspects from that to T cell-dependent (TD) antigens: the inability to generate memory cells, the restriction of produced antibodies to # and 3,2 isotypes, and the relatively late onset of responsiveness in ontogeny [7]. As mentioned above, anti-TI2 antibody responses are in general not detectable until 1 8 - 2 4 months of age, while TD antibody responses can be induced from birth onwards [ 8 - 10]. Since the pioneering work of Avery and Goebel [11], it has become clear that the immunogenicity of polysaccharides can be greatly improved by chemical coupling to protein carriers. This principle has been applied to a number of polysaccharides, and the first so-called conjugate vaccines have now been introduced [12- 15]. The efficacy of conjugate vaccines in early childhood is due to the fact that conjugation to proteins changes the nature o f the anti-polysaccharide antibody response into that of a TD response [16]. From initial studies with polysaccharide protein

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conjugates, it has become clear that the immunogenicity of a given conjugate is determined by several variables, such as the molecular size of the saccharide, the immunogenicity of the carrier protein, the method of coupling including the use of spacer molecules, the saccharide to protein ratio, and the conformation of the conjugate. We have prepared conjugates of type 4 S. pneumoniae polysaccharide (PS4) using tetanus toxoid (TT) as carrier protein. We chose this particular polysaccharide because it is the pneumococcal serotype most frequently isolated from blood cultures world-wide [17]. Moreover, the TI-2 nature of PS4 has been demonstrated in man [18]. Here, we describe the preparation of PS4TT conjugates and the effects of variation in saccharide size, the use of a spacer, and the saccharide-to-protein ratio on the immunogenicity of these conjugates in mice. 3. Materials

and Methods

3.1. Antigens Type 4 pneumococcal polysaccharide (PS4) [19] was obtained from ATCC, Rockville, MD. Tetanus toxoid (TT) was supplied by Dr. J. Nagel (RIVM) as bulk material that fulfilled the W H O requirements for vaccine production. T T was fractionated on Sephadex G200 before use.

6-aminohexanoic acid (Merck, Amsterdam, The Netherlands) was added and incubated for 90 min at room temperature. The resulting product was dialyzed, filter-sterilized, and stored at 4 °C until use. 3.4. Conjugation o f polysaccharide and oligosac-

charide to tetanus toxoid Conjugation of PS4 or OS4 to T T was performed by slowly adding N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) to equal amounts of activated poly/oligosaccharide (10mg/ml) and protein (10 mg/ml) until a polysaccharide/EDC ratio of 10:0.75, 1.5, 3, or 6 (mg/ml) was reached. During this process the pH was kept constant at 4.9. The mixture was incubated for 2 h at room temperature and the reaction was stopped by increasing the pH to 10. The formed conjugates were dialysed overnight at 4 °C against PBS. Large aggregates were removed by centrifugation for 20 min at 2 0 0 0 0 x g . The supernatant was purified on Sepharose CI-4B. The sugar and protein contents [22] of the eluted fractions were determined; those fractions with a detectable protein content and a positive outcome of a double sandwich ELISA, detecting TT and PS4 (see below) were pooled and used as conjugate.

3.5. Mice and immunization 3.2. Generation o f oligosaccharide fragments PS4 was degraded into smaller fragments by periodate oxidation (35 mM 104 ; 75 h at 4 °C). OIigosaccharides were purified by chromatography on Sephadex G100 and Sepharose C1-4B columns. Eluted fractions were analyzed for total and reducing sugar content [20, 21]. The mean oligosaccharide size was calculated from the gel filtration profiles. Fractions with a mean size of 12 repeating units (OSdpl2) were pooled.

3.3. Coupling o f a 6-aminohexanoic acid spacer to poly- and oligosaccharides

In all experiments, adult (8 - 12 week old) female random outbred N I H / R I V M mice were used. In experiments assessing the immunogenicity of conjugates prepared with or without a 6-aminohexanoic acid spacer, mice were immunized intraperitoneally with A 1 P O 4 adsorbed PS4TT conjugates. Blood samples were obtained by retro-orbital puncture. In other experiments, mice were immunized subcutaneously and blood was obtained from the tail vein. Blood was allowed to clot at room temperature; the serum was withdrawn and stored at - 20 °C until use. 3.6. ELISA procedures

Cyanogen bromide ( 2 m g / m l final concentration) was added to a 5 mg/ml PS4 solution (pH 10.5) [14]. This mixture was incubated for 6 min at 4 °C. The pH was then adjusted to 7.2 and 5 m g / m l 268

The assays used for detection of anti-PS4 and anti-TT antibodies have been described in detail previously [23]. In order to exclude the interference

of antibodies against cell wall polysaccharides (CPs) in the detection of anti-PS4 antibodies, 250/~g/ml CPs was added to the serum samples. A double sandwich ELISA was used to detect the presence of both TT and PS4 in the conjugates. To that end, ELISA plates (Flow, Irvine, U.K.) were coated with 10/~g/ml sheep anti-TT in 0.9% NaC1 [24]. Plates were washed and incubated with column fractions. Plates were washed again and incubated sequentially with rabbit anti-PS4 and peroxidaselabeled sheep anti-rabbit Ig [13]. Orthophenylenediamine was used as substrate; the absorbance was read at 492 nm. 3.7. Rocket immunoelectrophoresis Rocket immunoelectrophoresis [25] was performed in 1.2% HSA agarose gels (Litex, Glostrup, Denmark) on 12 x 8 cm Gelbond film (FMC, Rockland, MD) using Tris-Veronal (pH 8.8) buffer with an ionic strength of 0.04. The gels were divided into two parts, containing 0.2ml rabbit anti-PS4/cm 2 gel area or 1.3/~1 sheep anti-TT/cm 2. A current was applied for 3 h at 150V at 15 °C. Gels were stained with 5% Coomassie Brilliant Blue.

4. Results 4.1. Coupling of native type 4 pneumococcal polysaccharide to tetanus toxoid with or without the use of a C6 spacer In the first series of experiments, the effects of the introduction of spacer molecules on the immunogenicity of conjugates was investigated. Two sets of PS4TT conjugates were prepared using variable concentrations of EDC coupling reagent on cyanogen bromide-activated PS4, with or without the C6 spacer 6-aminohexanoic acid. Over the whole range of EDC concentrations tested, conjugates prepared with 6-aminohexanoic acid-coupled PS4 resulted in conjugates with a higher incorporation of TT (Table 1). The two sets of PS4TT conjugates were injected into groups of eight adult NIH mice. Mice were bled three weeks after primary immunization and two weeks after secondary immunization. The results, summarized in Table 1, demonstrated that most conjugates could induce anti-PS4 IgG antibodies. The highest antibody levels were obtained with PS4TT conjugates coupled with the use of a C6 spacer. Immunization with native PS4 or a mixture of PS4 and TT did not induce anti-PS4 IgG antibodies (Table 1 and data not shown). As far as anti-TT antibodies are concerned, the highest antibody levels were also obtained with conjugates containing 6-aminohexanoic acid (not shown). On the basis of these reults, subsequent conjugates

TABLE 1 PS4TT conjugates were prepared by using variable concentrations of N-ethyl-N'-(3-dimethylamino-propyl) carbodiimide (EDC) for coupling of T T to cyanogen bromide-activated PS4 to which the C6 spacer 6-aminohexanoic acid spacer was or was not coupled. The PS4:TT ratio in conjugates is expressed as a weight/weight ratio. N I H / R I V M mice were injected i,p. with 2.5/~g conjugate (on a polysaccharide basis) adsorbed to 0.1 mg AIPO 4. A second immunization was given 9 weeks later. Blood was withdrawn 3 weeks after primary and 2 weeks after secondary immunization. Serum anti-PS4 IgG antibodies were determined in pooled sera of groups of 8 mice by a PS4 specific ELISA and are expressed as percentages of a reference serum. EDC (mg/ml)

PS4 without C6 spacer PS4:TT ratio

0.75 1.5 3.0 6.0 unconjugated PS4

4.5:1 N.D. 8.2:1 9.0:1

PS4 with C6 spacer Anti-PS4 IgG

PS4:TT ratio

Prim.

Sec.