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A Synthetic Conjugate Polysaccharide Vaccine Against Haemophilus influenzae Type b

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Science  23 Jul 2004:
Vol. 305, Issue 5683, pp. 522-525
DOI: 10.1126/science.1095209

Abstract

Glycoconjugate vaccines provide effective prophylaxis against bacterial infections. To date, however, no commercial vaccine has been available in which the key carbohydrate antigens are produced synthetically. We describe the large-scale synthesis, pharmaceutical development, and clinical evaluation of a conjugate vaccine composed of a synthetic capsular polysaccharide antigen of Haemophilus influenzae type b (Hib). The vaccine was evaluated in clinical trials in Cuba and showed long-term protective antibody titers that compared favorably to licensed products prepared with the Hib polysaccharide extracted from bacteria. This demonstrates that access to synthetic complex carbohydrate–based vaccines is feasible and provides a basis for further development of similar approaches for other human pathogens.

Haemophilus influenzae type b (Hib) is an important human pathogen and was prevalent in developed countries until the introduction of successful conjugate vaccines during the 1990s (1). However, in developing countries more than 600,000 infant deaths occur annually as a result of Hib-induced pneumonia or meningitis (2). Extensive use of the polysaccharides as vaccines has offered a useful way to protect adults and older children (35), and further improvement in generating long-lasting immunity, especially in infants, has been achieved by covalently coupling the polysaccharide to carrier proteins (6). In fact, the high level of success attained by Hib glycoconjugate vaccine (7) has been quickly followed by similar approaches to meningococcal group C (8) and Streptococcus pneumoniae (9). Many candidate vaccines against other pathogens using the same principles are currently at different stages of research (10, 11).

The fragment of the Hib capsular polysaccharide used in some of the licensed vaccines can be as short as five ribosylribitol-phosphate repeating units (12). The ability of synthetic carbohydrate chemistry to mimic such fragments has been demonstrated in several laboratories with the use of stepwise multistep preparation (1315); the resulting synthetic antigens have served as components of candidate vaccines that have proven efficient in generating immunity in animals (16, 17). We set out to develop a synthetic methodology amenable to large-scale good manufacturing practice (GMP) production of antigens by reassembling Hib polysaccharide fragments. The previous process was redesigned to include a synthetic pathway with a reduced number of reaction and chromatography purification steps. We also identified a potentially superior method for oligomerization of the ribosylribitol-phosphate repeating unit, in which the saccharide fragment encompassing the key conformational epitope can be obtained in a single step.

To this end, we selected suitably protected ribitol derivative 1 and ribose acetate 2 because they best fit the criteria for large scale production as synthetic intermediates and could be more readily purified than others derived from D-glucose (Fig. 1). The use of crystalline peracetylated β-D-ribofuranose, 2, was found to readily glycosidate ribitol derivative 1 to provide 3a (18). The successful large-scale synthesis of ribosylribitol derivatives 4 and 5 from 3a as described in Fig. 1 thus represented one of the key features of our strategy.

Fig. 1.

Synthetic pathway leading to oligomeric polyribosylribitol phosphate 6. Reagents and conditions (Et, ethyl; Bn, benzyl; Bu, butyl; Ac, acetyl; and Piv, pivaloyl): (i) BF Et O and CH Cl; (ii) CH ONa and CH3OH; (iii) BnCl, Bu2SnO, NaH, and Bu4NI; (iv) tBuOK and dimethyl sulfoxide (DMSO) at 100°C; (v) PCl3, imidazole, CH3CN for 5 and N3(CH2)2OH for 4, followed by I2 oxidation; (vi) AcOH-H2O at 80°C; at (vii) PivCl and pyridine; (viii) I2, pyridine, H2O then gel filtration on LH-20; (ix) H2, Pd-C, and EtOH-H2O-EtOAc-AcOH at 1.5 atm; (x) cation exchange chromatography on Sephadex SP-C25.

Although construction of oligomers with controlled numbers of repeating units by solution and solid-phase techniques was possible in small quantities, their large-scale syntheses proved more difficult. To overcome this, we undertook a one-step polycondensation reaction with the use of H-phosphonate chemistry (19). Thus, the phosphate-containing end residue 4 and H-phosphonate derivative 5 were oligomerized in high yield and purity with the use of pivaloyl chloride as a polycondensation reagent (Fig. 1). Although this reaction is complex, it could be managed to generate the desired oligomers, avoiding several competing side reactions, such as O-acylation.

Synthetic oligomers of 6a with an average of eight repeating units were reproducibly obtained in high yield (80%) after purification by size exclusion chromatography. Deprotection and azide reduction of 6a to the amine 6b, which, after treatment with 3-maleimidopropionic acid N-hydroxysuccinimide ester, gave 7 (Fig. 2). The overall process accomplished under GMP proceeded with a high yield and was amenable to a 100-g scale per batch.

Fig. 2.

Conjugation of the maleimido-functionalized polyribosylribitol phosphate 7 obtained from 6b after coupling with 3-maleimidopropionic acid N-hydroxysuccinimide (DMSO, >95% conversion). 1,4-Conjugate addition of thiolated protein 8 onto 7 provided conjugate 9. The shift in the molecular weight for T T could be observed in size exclusion chromatography–high performance liquid chromatography (TSK-5000-1 column) for conjugate 9 (PRP/T T ratio of 1/2.6).

A vaccine prototype was subsequently produced by conjugating synthetic antigen 7 with thiolated human serum albumin (HSA) (20). The polyribosylribitol phosphate (sPRP)–HSA conjugate was used for coating enzyme-linked immunosorbent assay (ELISA) plates to screen for anti-Hib activity (21) of sera obtained from rabbits immunized with commercially available vaccines [Vaxem-Hib (Chiron, Emeryville, CA) and Hiberix (Glaxo Smith Kline Biologicals, Rixensart, Belgium)] and human antibodies against Hib obtained from immunized children (Vaxem-Hib). All sera showed equivalent recognition of sPRP-HSA and natural PRP–HSA conjugates, demonstrating that the synthetic oligosaccharide possessed the relevant antigenic epitopes for antibody binding recognition (22).

As a potentially more relevant protein carrier for synthetic oligosaccharides, a tetanus toxoid (TT) conjugate 9 was evaluated in animals. The glycoconjugate was obtained through the thiolation of TT lysine ϵ-amino groups as for HSA, and this sPRP-TT conjugate was immunogenic in rabbits with a wide range of sPRP/protein ratios, inducing a strong and specific antibody response (22).

At this point, we identified four key issues that allowed us to accomplish further development of sPRP-TT as a vaccine candidate: (i) a synthetic pathway to disaccharide derivative 5 with only one chromatographic purification step, making it amenable to large-scale GMP production, (ii) a single-step, high-yielding polycondensation reaction between 4 and 5 for the elongation of the oligosaccharide chain, (iii) a method for careful removal of protective groups, yielding highly pure sPRP 7, and lastly (iv) a conjugation process to TT carrier that incorporated sPRP in good yields.

The sPRP-TT conjugate vaccine with 10 μg of sPRP and a sPRP-to-TT ratio of 1/2.6 by weight was next used as part of a phase I clinical trial in 40 adult volunteers after rigorous toxicological assessment in animals (22). A second phase I trial in adults with the vaccine plus aluminum phosphate was also performed. Both formulations of sPRP-TT were shown to be safe in adults. The average immunoglobulin G (IgG) titers obtained from our synthetic antigens (23) displayed a behavior similar to that of the control vaccine (Vaxem-Hib); the results obtained from this initial evaluation (table S1) prompted the initiation of a full clinical evaluation. All trials were performed as recommended and according to good clinical practice (24). Generally, they were randomized and double-blind and used a control vaccine and at least two different batches of the test vaccine (25).

The next phase I clinical trial consisted of a single vaccine dose in 133 4- to 5-year-old children previously unvaccinated against Hib. This was followed by a phase II trial using 1041 children. A substantial postvaccination increase in the anti-Hib IgG titers was observed in both trials, and a significant increase in the bactericidal activities of the sera (26) was reached after the administration of a single vaccine dose in three groups of children. The similarities of these with previous studies (27) again indicated that the sPRP-TT vaccine was both as safe and as immunogenic as the commercial control vaccine (Fig. 3).

Fig. 3.

Bactericidal activity of immune serum against Hib (Eagan strain) obtained during a phase I trial conducted with children 4 to 5 years old in the province of Camaguey, Cuba, in the winter of 2002. The trial was conducted with 133 preschool children volunteers with the informed written consent of both parents. The trial was performed on a double-blind basis and all subjects were divided in four groups. Three groups (A to C) received a single dose of s-PRP-T T without any adjuvant (lots EH1024E, EH1026E, and EH1025E, respectively). The control vaccine (Vaxem-Hib) was administered to another group. The serum was obtained before and 4 weeks after the immunization. The plots are the reciprocal of the dilution killing 50% of the bacteria according to (26, 27).

The above results set the stage for more detailed clinical assessment of the vaccine in a targeted infant population. Thus, a phase I trial was initiated with 139 2-month-old infants who received three vaccine doses scheduled at 2, 4, and 6 months, as recommended for other conjugate anti-Hib vaccines. The test vaccine induced a strong and bactericidal antibody response against Hib in infants (Fig. 4) that fell to values ranging from 5 to 7 μg/mL at 18 months of age but remained at least five times that required for long-term protection (Fig. 4A). A booster dose with sPRP-TT applied to all groups increased the antibody against Hib titers by 10-fold. Thus, the capacity of sPRP-TT to prime an effective immune response against Hib was demonstrated.

Fig. 4.

Geometric means concentration (GMC) of serum IgG antibodies against Hib (A) and % of children having bactericidal anti-Hib in their serum (B) after the primary series of immunization (7 months), a year after the preimmunization (18 months), and after a booster dose (19 months). A phase I clinical trial was performed with 139 infants in the Camaguey province east of Havana. Infants were enrolled in five groups receiving three doses at 2, 4, and 6 months of age. One group received control vaccine (Vaxem-Hib). Two groups received s-PRP-T T plus aluminum phosphate, and two other groups received s-PRP-T T alone. Blood was drawn at 7, 18, and 19 months for the evaluation of the immune status. At 18 months, all infants received a single booster dose of sPRP-T T irrespective of group. Bars indicate the 95% confidence interval.

In a second phase II trial, a total of 1141 infants distributed in three groups received three doses of either sPRP-TT conjugate, sPRP-TT mixed with aluminum phosphate, or the control vaccine (Vaxem-Hib). Of the test infants, 99.7% reached antibody titers above 1 μg/mL, which is considered appropriate for long-lived protection against Hib (28, 29). The mean IgG anti-PRP titer was 27.4 μg/mL for all infants vaccinated with the sPRP-TT, which is consistent with previously reported clinical trials (between 7.67 and 35 μg/mL) for anti-Hib vaccines without adjuvant (30, 31).

The present study demonstrates that a synthetic capsular polysaccharide antigen can be produced on a large scale under GMP conditions and used to manufacture an effective vaccine for human use. The resulting conjugate vaccine incorporating a synthetic bacterial carbohydrate antigen was demonstrated to be as safe and immunogenic in humans as already-licensed vaccines incorporating the native polysaccharide (3234). Access to synthetic complex carbohydrate-based vaccines is therefore feasible and provides an alternative strategy in the fight against Hib infections. It also sets the stage for further development of similar approaches against other human pathogens.

Supporting Online Material

www.sciencemag.org/cgi/content/full/305/5683/522/DC1

Materials and Methods

Fig. S1

Table S1

References and Notes

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