Multivalent pneumococcal capsular polysaccharide conjugate vaccines employing genetically detoxified pneumolysin as a carrier protein

Vaccine, Vol. 16, No. 18, pp. 1732-1741, 1998 0 1998 Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain PII:SO264-410X(98)00225-4 0264-410X/98 $19+0.00 ELSEVIER

Multivalent pneumococcal capsular polysaccharide conjugate vaccines employing genetically detoxified pneumolysin as a carrier protein Francis Michon*$, Peter C. FUSCO”,Concei@o A.S.A. Minetti*, Maryline Laude-Sharp*, Catherine Uitz”, Chun-Hsien Huang”, Anello J. D’Ambra*, Samuel Moore*, David P. Remeta?, Iver Heron*, M.S. Blake* A genetically detoxified pneumolysin, pneumolysoid (PLD), was investigated as a carrier protein for pneumococcal capsular polysaccharide (CPS). Such a CPS-PLD conjugate might provide additional protection against pneumococcal infections and resultant tissue damage. A single point mutant of pneumolysin was selected, which lacked measurable haemolytic activity, but exhibited the overall structural and immunological properties of the wild type. PLD conjugates were prepared porn CPS serotypes 6B, 14, 198 and 23F by reductive amination. The structural features of free PLD, as well as the corresponding CPS-PLD, as assessed by circular dichroism spectroscopy, were virtually indistinguishable from the wild &pe counterpart. Each of the CPS monovalent and tetravalent conjugate formulations were examined for immunogenicity in mice at both 0.5 and 2.0 ug CPS per dose. Tetanus toxoid (TT) conjugates were similarly created and used for comparison. The resultant conjugate vaccines elicited high levels of CPS-specific IgG that was opsonophagocytic for all serotypes tested. Opsonophagocytic titres, expressed as reciprocal dilutions resulting in 50% killing using HL-60 cells, ranged from 100 to 30000, depending on the serotype and formulation. In general, the lower dose and tetravalent formulations yielded the best responses for all serotypes (i.e., either equivalent or better than the higher dose and monovalent formulations). The PLD conjugates were also generally equivalent to or better in CPS-specific responses than the TT conjugates. In particular both the PLD conjugate and the tetravalent formulations induced responses for type 23F CPS that were approximately an order of magnitude greater than that of the corresponding TT conjugate and monovalent formulations. In addition, all the PLD conjugates elicited high levels of pneumolysin-specific IgG which were shown to neutralize pneumolysininduced haemolytic activity in vitro. As a result of these findings, PLD appears to provide an advantageous alternative to conventional carrier proteins for pneumococcal multivalent CPS conjugate vaccines. 0 1998 Published by Elsevier Science Ltd. All rights reserved Keywords:

Pneumococcus:

pneumolysin: polysaccharide

conjugate

INTRODUCTION Streptococcus pneumoniae is the major cause of bacterial pneumonia, bacteraemia, meningitis, and otitis media’,‘. Even with appropriate antibiotic therapy, pneumococcal infections have been estimated to result in as many as 40000 deaths a year in the

*North American Vaccine, Inc., Beltsville, Maryland, USA. Wepartment of Biology and Biocalorimetry Center, The Johns Hopkins University, Baltimore, Maryland, USA. #Author to whom all correspondence should be addressed at: North American Vaccine, Inc., 13150 Old Columbia Rd, Columbia, MD 21046, USA. Tel.: 410-309-7135; Fax: 41 O-381 -3385; E-mail:[email protected]

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United StategV4. In addition, pneumococci have gained increased resistance to penicillin and other antibiotics making the development of an effective vaccine to prevent pneumococcal infections a public health priori@. Since the current 23-valent pneumococcal capsular polysaccharide vaccine is ineffective in children younger than 2 year?.‘, numerous groups are developing multivalent conjugate vaccines to prevent otitis media, which is the major indication in this age group. Pneumolysin (PL) is a sulfhydryl-activated cytolytic toxin that is produced by all types of Streptococcus pneumoniue8 and is considered a major virulence factor in pneumococcal infection’. Genetically engineered PL-negative mutant strains of S. pneumoniae have been

Pneumococcal

polysaccharide-pneumolysin

shown to be significantly less virulent for mice’““. Cytotoxicity of PL to pulmonary endothelial and epithelial cells has been demonstrated in vitro”. In addition, PL may be the principal cause of hearing loss and cochlear damage in a guinea pig model of pneumococcal meningitis’“. CPS conjugates of a detoxified PL, pneumolysoid (PLD), and a recombinant PL have been produced and used as experimental vaccines, which have conferred significant protection in mice against lethal challenge with the homologous pneumococci’J-‘“. In the current study, we report the preclinical evaluation of a multivalent pneumococcal CPS conjugate vaccine using a PLD as an alternative to conventional carrier proteins such as diphtheria and tetanus toxoids. Conjugates were prepared with a novel single point mutant PLD that was devoid of haemolytic activity but still maintained the overall structural and immunological properties of wild type PL. Individual PLD conjugates were synthesized from CPS serotypes 6B, 14, 19F and 23F, representing the penicillin-resistant clinical isolates most frequently found”, using reductive amination technology. Each of these CPS-PLD conjugates were then formulated into a multivalent vaccine for evaluation. MATERIALS AND METHODS Preparation

of PL and PLD

The cloning of PL and the generation of PLD, as well as the expression, refolding and purification of each, will be described in detail elsewhere (manuscript in preparation). A single point mutant PLD was selected on the basis that it retained all the properties of the wild type PL, with the additional advantages of greater conformational stability and undetectable levels of haemolytic activity. Preparation

of conjugates

Each of the CPS were first depolymerized and functional aldehydes were introduced into the fragmented CPS by oxidation with sodium metaperiodate. Following the oxidation process, the excess periodate was destroyed with ethylene glycol, and the oxidized polysaccharides were dialysed against DI water and lyophilised. Each of the oxidized CPS and the PLD in 0.2 M sodium phosphate buffer (PBS) were combined in a ratio of approx. 2.5:1 by weight. Sodium cyanoborohydride (2 parts) was then added and the conjugation mixtures were incubated at 37°C for 2 days. Residual aldehydes were eliminated with NaBH4. With the exception of conjugates made with type 23F CPS, each of the conjugates were purified by molecular sieve chromatography using a Superdex 200 PG column (Pharmacia, Uppsala, Sweden) using PBS containing 0.01% thimerosal. The type 23F conjugates were loaded onto a Q Sepharose Fast Flow column (Pharmacia), and eluted with 10 mM Tris-HCl, 0.5 M NaCl, pH 7.5 in sequence. Fractions from each of the columns corresponding to the conjugates were pooled and analysed for proteinlx and carbohydrate content”. Similar conjugates were constructed with tetanus as previously amination, reductive toxoid bY described”.“.

Inhibition

conjugate

vaccines:

F. Michon et al.

ELBA assay

Microtitre plates (NUNC Polysorp) were coated with PL (20 ng in 100 ~1 to each well), in PBS (50 mM sodium phosphate, 150 mM NaCl, pH 7.4) for 1 h at 37°C. After washing the plates with PBS +0.05% Tween 20 (PBST), the plates were subsequently coated with 150 ~1 of PBS +O.l% BSA, rewashed, and stored at 4°C until used. Hyperimmune rabbit anti-PL was diluted in PBST, added to the PL coated plates, and incubated at room temperature for 1 h. After washing, 100 ~1 of goat antirabbit Ig-HRP conjugate (KPL), diluted in PBST according to the manufacturer’s instructions, were added to each well. The plate was incubated at room temperature for 1 h and then washed again. One hundred microlitres of TMB microwell substrate (KPL) were added to each well. The reaction was stopped after 10 min by the addition of TMB one-component stop solution (KPL), and the A15,,nmwas immediately read. The dilution corresponding to half the maximum signal was chosen for the inhibition study. PLD mutants, as well as PL for a control, were diluted serially in threefold increments in PBST containing the rabbit antiserum (diluted such that the final mixture contained the dilution which gave half-maximal activity) and applied immediately to the coated microtitre plates in duplicate. The plates were incubated at room temperature for 1 h and processed as previously described. Inhibition was determined as the per cent of maximum signal achieved with diluted antiserum in the absence of any inhibitor. Immunization

of mice

Six- to &week-old female outbred CD-I mice (Charles River, Raleigh, NC, USA) were immunized with monovalent or tetravalent Streptococcus pneumoniar polysaccharides (PS) types 6B, 14, 19F, and 23F conjugated to TT or PLD (0.5 /lg PSIO.2 ml or 2 pg PSIO.2 ml) in 1 mg ml -’ alum. The vaccines were given subcutaneously, on days 0, 28. and 49, and blood samples were collected on days 0, 14, 28, 38 and 59. ELISA titres against polysaccharides and the carrier protein were determined using CPS-HSA conjugates and wild type PL. The opsonophagocytic activity of the sera was determined in a phagocytic assay usmg the HL-60 cell line as described previously”. Haemolysis

and haemolysis

neutralization

assay

The PL activity was assessed according to the procedure of Paton et al.“, with some modifications. In brief, on standard U-bottomed microtitre plates, PL and mutant PLDs were serially diluted in TBS (15 mM Tris, 0.15 M NaCl, pH 7.5) plus 1 mM DTT as a cofactor, in twofold increments, with a final volume of 100 ~1. Then 100 ~1 of 1% sheep erythrocytes in suspension with TBS were added, and the reaction was conducted at 37°C for 30 min. After centrifuging the cells, the extent of erythrocyte lysis was monitored in the supernatant at 405 nm using a microtitre plate reader. The end-point of the assay was taken as the reciprocal dilution at which 50% of erythrocytes were lysed, based on a 0.5% cell suspension that was lysed hypotonically. Inhibition of the haemolytic activity was tested as before’“, but with some variations. Before dilution, the

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Pneumococcal polysaccharide-pneumolysin

conjugate vaccines: F. Michon et al.

mouse antisera were treated twice with chloroform to eliminate the cholesterol. A twofold serial dilution of 50 ~1 of the mouse antisera was performed and 50 ,rd of a stock toxin solution at 4 HU (haemolytic units) were added. The haemolytic activity of the toxin was assessed immediately before the neutralization assay. After a 15min incubation at 37°C to allow serum antibody to bind to PL, 100 ~11of sheep RBCs (1% in TBS) (ICN, Costa Mesa, CA, USA) were added to each of the wells. The plates were incubated for 30 min at 37°C and analysed as previously described. The antihaemolytic titre was measured as the highest reciprocal dilution of serum which afforded complete inhibition of the haemolysis. Circular dichroism (CD) spectroscopy The secondary and tertiary structures of the free wild type and mutant pneumolysin, and their respective conjugates, were evaluated by circular dichroism (CD) spectroscopy in the far UV (180-250 nm) and near UV (250 to 350 nm) regions, respectively. Concentrated stock solutions of protein and conjugate were dialysed exhaustively against a buffer system comprising of 10 mM NaPOj (pH 8.0). Spectra of samples containing 1.0 mg ml-’ protein were recorded at 0.1 nm wavelength intervals on a JASCO Model 710 circular dichroism spectropolarimeter (JASCO, Easton, MD. USA) employing a scan speed of 5 nm mini ’ and average response time of 1 s. A minimum of four consecutive scans were accumulated and the average spectra stored. The temperature of the samples was

maintained at 25°C through the use of water-jacketed 0.01 cm and 1.0 cm path-length cells in the far and near UV, respectively.

RESULTS Basic structural and immunological features of PL, PLD and CPS-PLD conjugates as assessed by circular dichroism and competitive inhibition ELISA PL overexpressed in E. coZi and refolded from inclusion bodies exhibited a typical far UV CD spectrum characteristic of a high content of P-sheets with a minimum elliptic&y observed at approx. 215 nm 25. The far UV CD spectra of PL and the single point mutant PLD were essentially identical (manuscript in preparation). Likewise, chemical conjugation of either PL or PLD with CPS did not affect the overall secondary structure of the proteins (Figure IA). The near UV CD spectrum (Figure Ill), which derives from the relative asymmetry of tyrosyl and tryptophanyl residues in the protein, revealed a highly ordered structure with the mutant PLD, resembling the wild type free protein. The conjugate, however exhibited minor changes in the near UV CD profile (Figure IB), which may have resulted from the presence of the polysaccharide on the surface of interfering specific these complexes, with the Tyr-specific signal (i.e., negative ellipticity with a minimum centered at approx. 280 nm). Additional corroboration of the structural integrity and identity of the PLD mutants is that most of their antigenicity was

6

-5 -

180

I 200

I 220

I 240

I 260

I 280

I 300

I 320

I 340

Wavelength (nm) Figure 1 Circular dichroism studies of a free mutant pneumolysoid (PLD) carrier protein (PLD mutant A) (upper sponding type 14 CPS-PLD conjugate (lower panel), depicting the Far-UV (A) and Near-UV CD spectrum (B)

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panels) and a corre-

Pneumococcal

polysaccharide-pneumolysin

conjugate

vaccines:

F. Michon et al.

80

O-

1000

100

10

1

10000

inhibitor Concentration (ng/well) Figure 2 Competitive inhibition ELlSA studies protein, using soluble wild type PL, pneumolysoid

between a rabbit polyclonal antibody (PLD) mutant A, and PLD mutant B

retained

to wild type pneumolysin

(PL) and wild type PL

when compared with native PL, as shown in

Figure 2. Table 1

Composition

of CPS-pneumolysoid

conjugates

Pneumococcal serotype

Approximate MW of CPS

Protein (mg ml-‘)

CPS (mg ml-‘)

CPS/Protein

6B

41000

0.24

0.14

0.58

19F 14

41000 10000

0.46 0.13

0.08 0.14

0.62 0.30

23F

90 000

0.44

0.10

0.23

4

Dose response for pneumolysoid conjugates The compositions of the CPS-PLD conjugates, representing each of four serotypes (6B, 14, 19F, and 23F) are shown in Table I. Both the tetravalent and monovalent formulations were tested in mice at two different CPS doses, 0.5 and 2.0 /lg. Figure 3 shows the

1,000

in i W

100 i

19F

Polysaccharide 0 0.5 pg - Monovalent L-10.5 pg - Tetravalent

23F

Type

n 2.0 pg - Monovalent W 2.0 pg - Tetravalent

Figure 3 Polysaccharide dose response, in terms of CPS-specific IgG, for pneumococcal pneumolysoid conjugates in mice after two injections. Monovalent versus tetravalent adsorbed conjugate formulations were compared at 0.5 and 2.0 pg polysaccharide doses per type. Two subcutaneous injections were given, 4 weeks apart. ELISA IgG titres represent pooled sera collected 70 days after booster from groups of 10 mice

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19F 14 Polysaccharide

6B 0

0.5

pg PS n 2.0

23F Type

Tetra

pg PS

Figure 4 Pneumolysin-specific IgG elicited by monovalent and tetravalent adsorbed CPS-pneumolysoid conjugates in mice after two injections, compared at 0.5 and 2.0 pg polysaccharide doses per type. Two subcutaneous injections were given, 4 weeks apart. ELBA IgG titres represent pooled sera collected 10 days after booster from groups of 10 mice

dose response of each group, measured as IgG by ELISA in pooled sera from 10 mice 10 days after two subcutaneous injections that were 4 weeks apart. For the monovalent vaccines (F@re .?), the CPS-specific IgG titres ranged from 6700 to 130000 for types 6B, 14, and 19F, but the titres were much lower for type 23F at 370-800. However, when in combination as a tetravalent vaccine, the IgG titres were much closer together for all types, ranging from 15000 to 39000 at the lower dose. The higher dose generally

resulted in similar or lower titres, particularly for the tetravalent vaccine which ranged from 2100 to 26000. A third injection, 3 weeks later, showed no significant increase in overall IgG (i.e., no greater than a fourfold difference for any one type, with most less than twofold different, in either direction). Figure 4 shows the PL-specific IgG response for the same vaccines from Figure 3, again after two injections. These PL-specific IgG titres occurred within a fourfold range of 110000-400000 for types 6B, 19F, 23F, and

100,000

r

L 30,000 aJ z 10,000 1

:

m i

W

1mo 300 100

1

30 1

23F Polysaccharide 0 0.5 I3 2.0

pg PS - Pneumolysoid pg PS - Pneumolysoid

Type

n 0.5 I.rg PS - Tetanus n 2.0 pg PS - Tetanus

Toxoid Toxoid

Figure 5 CPS-specific IgG for tetravalent pneumococcal conjugates in mice after two injections with adsorbed conjugates containing pneumolysoid versus tetanus toxoid carriers, at 0.5 and 2.0 gg polysaccharide doses per type. Two subcutaneous injections were given, 4 weeks apart. ELISA IgG titres represent pooled sera collected 10 days after booster from groups of 10 mice

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Pneumococcal polysaccharide-pneumoiysin

6B

14

19

Polysaccharide q Pneumolysoid

conjugate vaccines: F. Michon et al.

23

Type

n Tetanus

Toxoid

Figure 6 CPS-specific opsonophagocytic activity elicited by tetravalent CPS-pneumolysoid and CPS-tetanus toxoid conjugate vaccines in mice after two injections. The CPS dose was 0.5 pg per type. Two subcutaneous injections were given, 4 weeks apart. ELBA IgG titres represent pooled sera collected 10 days after booster from groups of 10 mice

the tetravalent formulation, but were somewhat lower for the monovalent type 14 at 42000-71000. However, the higher dose resulted in titres that were approximately the same or within 2.5-fold higher. Also for PL, the third injection showed no significant increase in IgG (i.e., less than twofold in either direction) (data not shown). PLD vs TT as carrier The CPS-specific IgG response after two injections for PLD versus TT as the carrier in the tetravalent formulation, and at different CPS doses, is shown in Figure 5. The CPS-specific titres for the monovalent formulations, at both the 0.5 and 2 pg doses, were not significantly different for each serotype examined, but the magnitude of the response was much higher for 6B, 14, and 19F than for type 23F. No clear significant differences in titres were observed between the monovalent and the tetravalent formulations. However. in the case of the tetravalent vaccine, the lower dose generally resulted in a higher titre, and the IgG response to type 23F was also significantly higher in the combination as compared with the stand-alone vaccine. Figure 6 shows the opsonophagocytic (OP) activity of the CPS-specific antibodies elicited by either the tetravalent CPS-PLD or the tetravalent CPS-TT vaccines after two injections. The OP activity was measured in an assay using HL-60 cells”. The OP activities elicited to each type by either the PLD- or the TT-tetravalent conjugate were comparable, except for type 23F, where significantly higher activity was elicited by the PLD vaccine. Immunogenicity

time course studies

An immunogenicity time course study for the tetravalent CPS-PLD was performed as shown in Figure 7A. The animals received three injections on days 0,

28, and 49, and blood samples were obtained on days 0, 14, 28, 38, and 59. Each dose contained 0.5 /lg CPS of each type. The CPS-specific IgG response to each type increased over time to peak just after the second injection, and then plateau (titres ranging between 10000 and 60000). Booster effects after the second injection were observed for some types where the response had not peaked too quickly (e.g., 23F). Figure 7B shows the time course for the CPS-specific IgG response of the tetravalent TT conjugate. As with the PLD combination vaccine, the animals were similarly immunized using the same schedule and dose of vaccine. Again, the IgG response to each type polysaccharide increased after each injection with similar magnitude (final titres between 30000 and 200000) except for type 23F which exhibited a signilicantly lower titre (approx. 4000). Booster effects were also observed as before for some types after the second injection. For comparison with the CPS-PLD combination vaccine, the immunogenicity time course for the monovalent CPS-PLD conjugates are shown in Figure 7C. The animals received identical doses of 0.5 /lg of CPS and had the same immunization schedule as mentioned above. The IgG response to the CPS in the monovalent conjugates was very similar to the one observed for the combination, with the exception of type 23F CPS which gave rise to a shallower curve, yielding antibody titres approximately two orders of magnitude lower than those of the three other types. Anti-haemolytic

activity for pneumolysoid

conjugates

The ability of anti-pneumolysin antibodies, elicited by the pneumolysoid conjugates, to neutralize the haemolytic activity of PL is shown in Figure 8. The antihaemolytic titres were estimated on mouse antisera raised with both monovalent and tetravalent CPS-PLD

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66

I

14 _._*___ 19F -*-

23F _..*._

20

Time

30

40

60

(days)

B 100,000 L

10,000

@fj

cl

2

s

1,000

i

w

100

10 0

10

20

Time

30

40

50

60

50

60

(days)

C 100,000

% 1,000

0

10

20

Time

30

40

(days)

Figure 7 CPS-specific IgG response over time in mice towards (A) tetravalent CPS-pneumolysoid adsorbed conjugate vaccine; (B) tetravalent CPS-tetanus toxoid adsorbed conjugate vaccine; and (C) monovalent CPS-pneumolysoid adsorbed conjugate vaccines. Three subcutaneous injections were given on days 0, 28, and 49, with sera collected and pooled from groups of 10 mice on days 0, 14, 28, 38, and 49. CPS dose was 0.5 pg per type. Arrows represent booster times

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Pneumococcal polysaccharide-pneumolysin

conjugate vaccines: F, Michon et al.

a GB-PLD

1CPLD

19F-PLD

23F-PLD Tetra-PLD

Polysaccharide (0

0.5

pg PS n

2.0

Tetra-?T

Type p9

ps 1

Figure 8 Anti-haemolytic pneumolysin-specific activity elicited by monovalent and tetravalent (tetra) pneumolysoid (PLD) and tetravalent tetanus toxoid (7) adsorbed polysaccharide (PS) conjugates in mice after three injections. Three subcutaneous injections were given on days 0, 28, and 49, with sera collected and pooled from groups of 10 mice at 10 days after the last booster. CPS doses were 0.5 or 2.0 /(g per type

conjugates at both 0.5 and 2 pg PS doses. Antiserum against a tetravalent CPS-TT conjugate was used as a control. As can be seen, each of the formulations, except for the TT control, had elicited significantly high levels of haemolysin neutralizing antibody. This is an indication that the conjugated PLD retained most of its native antigenic structure, as initially predicted from the circular dichroism studies and the antibody inhibition assays (Figure I and Figure 2, respectively).

DISCUSSION In a prospective study of pneumococcal colonization and infection in children, it was found that pneumococcal serotypes 6, 14, 19F, and 23F are the most commonly carried as well as the most frequent cause of infection in infants, resulting mainly as otitis mediaz6. In addition, it was recently found that these same strains more frequently occur among penicillinresistant clinical isolates’ , Clmical studies carried out in young infants with a tetravalent pneumococcal conjugate vaccine that included the above types clearly showed a reduction in the carriage of vaccine-related strains”. These results suggest that transmission of specific pneumococcal types most often associated with disease and antibiotic resistance might at least be controlled by immunization. For the above reasons we decided to include these four types in our first multivalent conjugate vaccine. An effective vaccine against these types would provide an excellent basis for a paediatric pneumococcal vaccine. The present study was initiated to determine whether the recombinant PLD would function as a carrier protein for more than one pneumococcal type in a multivalent formulation of conjugates. We also wanted to determine whether antibodies elicited to the

conjugated PLD would recognize native PL, be functional in their ability to neutralize its haemolytic activity, and perhaps provide additional protection against pneumococcal infection. Finally, we sought to circumvent the potential problems associated with combining too many conventional carriers (e.g., TT) into one vaccine ‘7-3’. The finding that recombinant refolded PL achieved functional activity, and that the single point mutant PLD lacked haemolytic activity but retained all the immunological and structural features of the PL, makes this protein attractive as a carrier for CPS conjugate vaccines. Moreover, the basic structural features of the resultant conjugated protein were well preserved, as demonstrated in the circular dichroism studies. Spectroscopic methods represent a powerful tool in the evaluation of the integrity of proteins. In the particular case of conjugate vaccines which employ proteins as carriers, these methods, in conjunction with functional and immunological techniques may facilitate monitoring batch-to-batch variations, as well as the molecular basis for vaccine efficacy3’,32. We have previously determined that the mutations selected in the present study rendered the protein atoxic, but retained the ability to refold to a native-like structure indistinguishable from the parent molecule (manuscript in preparation). The nearly superimposable far UV CD spectra of the free mutant protein and the corrcsponding pneumococcal 14 conjugate, as seen by both amplitude and crossover points, are indications that the secondary structure of the protein within this macromolecular complex remains intact. These results contrast with previous studies conducted with other polysaccharide-protein conjugates in which minor variations in the secondary structure were noticeable following conjugation”.

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Although we have not specifically addressed the issue of identifying those lysyl residues that are committed to te chemical conjugation by reductive amination, our results suggest that tyrosyl residues in the vicinity of such sites may be perturbed by the presence of the polysaccharide, as manifested in the differences observed in the near UV CD spectra at 280 nm. It is worth noting, however, that the characteristic tryptophanyl peak centered at 290 nm remained unaffected by the conjugation, which is another indication that the Trp-containing regions were not perturbed by the reductive amination procedure. Consequently, the tertiary structure of PLD was partially disrupted upon conjugation with no apparent loss of secondary structure (a finding that is generally consistent with a previous study on a mutant CPS-diphtheria conjugate”‘). Our preclinical studies demonstrated that conjugates consisting of polysaccharides derived from four pneumococcal strains (6, 14, 19F, and 23F) and PLD were highly immunogenic in animals, and elicited CPS-specific antibodies which compared favourably with those raised with a TT tetravalent conjugate. In addition, the PLD tetravalent conjugate was able to generate high levels of PL-specific IgG antibodies that neutralized haemolytic activity of wild type PL. In light of a recently published report on the clear pathogenic role of PL in hearing loss and cochlear damage in a pneumococcal experimental meningitis model, it would appear that PL vaccine-induced antibodies might be a useful adjunct to the capsular antibodies to ameliorate or perhaps prevent the serious complications associated with otitis media13. Overall, the spectroscopic and serological results provide substantial evidence that CPS-PLD conjugates represent suitable vaccine candidates for the prevention of pneumococcal diseases. ACKNOWLEDGEMENTS We thank Diane L. Hebblewaite, James W. Perry, John B. Rathmann, and M. Susan Walker for their technical assistance in performing serological assays. REFERENCES Baltimore, R.S., Shapiro, E.D., Pneumococcal infections. In: Evans, AS., Brachman, P.S. (Eds.), Bacterial Infections of Humans: Epidemiology and Control. Plenum Press, New York, 1989, pp. 525-546. Schuchat, A., Robinson, K. and Wenger, J.D. et al. Bacterial meningitis in the United States in 1995. Active Surveillance Team. N. Engl. J. Med. 1997,337,970-976. Fedson, D.S., Shapiro, E.D. and LaForce, F.M. et a/. Pneumococcal vaccine after 15 years of use: another view. Arch. Intern. Med. 1994, 154, 2531-2535. Fiebach, N. and Beckett, W. Prevention of respiratory infections in adults. Arch. Intern. Med. 1994, 154, 2545-2557. Farr, B.M., Johnston, B.L. and Cobb, D.K. et al. Preventing pneumococcal bacteremia in patients at risk: results of a matched case-control study. Arch. Intern. Med. 1995, 155, 2336-2340. Douglas, R.M., Paton, J.C., Duncan, S.J. and Hansman, D.J. Antibody response to pneumococcal vaccination in children younger than five years of age. J. Infect. Dis. 1983, 148, 131-137. Leinonen, M., Sakkinen, A., Kalliokoski, Ft., Luotonen, J., Timonen, M. and Make@ P.R.H. Antibody response to 14-valent pneumococcal capsular polysaccharide vaccine in

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