Immunogenicity in a Mouse Model of a Conjugate Vaccine Made with a Synthetic Single Repeating Unit of Type 14 Pneumococcal Polysaccharide Coupled to CRM197

Abstract

Oligosaccharides (OSs) related to the pneumococcal type 14 capsular polysaccharide (Pn14PS) were studied for their ability to inhibit the binding between anti-PS14 antisera and native PS14. A synthetic tetrasaccharide corresponding to the repeating unit of the Pn14PS, a hexasaccharide mimic, and an octasaccharide fragment obtained by Pn14PS depolymerization were good inhibitors. CRM197 conjugates of the tetrasaccharide and an octasaccharide mimic were prepared by using either adipic acid diester or diethyl squarate linkers. The conjugate with the tetrasaccharide chains induced anti-Pn14PS antibodies when injected subcutaneously into mice, as determined by an enzyme-linked immunosorbent assay, and antibody titers increased with oligosaccharide loading. The adipic acid-linked tetrasaccharide conjugates elicited higher antibody titers than those prepared with a squarate spacer. The lower anti-Pn14PS antibody response of the octasaccharide mimic conjugate indicates the importance of the backbone galactose residue for an appropriate antibody response. The OS-CRM197 conjugate prepared from a single repeat unit of the Pn14PS is a potential vaccine candidate.Streptococcus pneumoniae is a major cause of morbidity and mortality in children and adults in both industrialized and developing countries (10). Although the licensed 14-and 23-valent pneumococcal capsular polysaccharide (PnPS) vaccines are safe and effective in reducing the incidence of invasive disease in healthy adults (5, 24), they are weakly immunogenic in children less than 2 years old and in the elderly, the two groups at highest risk (8,22,25,31). Whereas PSs are T-cellindependent immunogens, conjugates in which the PS is covalently attached to a protein carrier elicit a T-cell-dependent antisaccharide response even in infants, as evidenced by a booster effect upon subsequent immunizations. A number of PnPS serotypes have been conjugated to various carrier proteins and have been shown to be immunogenic and protective in various animal models (13,20,30) and in humans (1, 32). Intact PS, small oligosaccharides (OSs), or OSs of undefined length have been used to prepare those conjugates (3, 13, 36). The capsular polysaccharide of S. pneumoniae type 14 (Fig. 1) consists of a branched tetrasaccharide repeating unit (21) which is identical to the asialo core antigen of the type III group B Streptococcus PS (38). The poor immunogenicity of the Pn14PS compared to other PnPSs (18) may be due to structural similarities between antigenic determinants of the Pn14PS and human OS structures (e.g., human milk OSs and blood group carbohydrate structures). To avoid cross-reactivity with human tissue and the induction of autoreactive antibodies (6), we have been investigating the synthesis of well-defined OS fragments (26,27) corresponding to the Pn14PS to enable specific protective epitopes to be defined. Inhibition studies were performed both with synthetic OS structures and fragments from PS degradation to determine optimal OSs for conjugation. The protein carrier used in...

Vaccines based on the cell surface carbohydrates of pathogenic bacteria

“…In many cases, the glycans attached in the conjugate are oligosaccharides prepared by degradation of the original polysaccharide (Anderson et al 1986, Costantino et al 1999. In addition, glycoconjugates can be produced from relatively low molecular weight oligosaccharides related to the repeating polymers (Mawas et al 2002, Benaissa-Trouw et al 2001, Jansen et al 2001, Jansen and Snippe 2004, or the short glycans of LPS O-chain (Gupta et al 1998), or low molecular weight capsular polysaccharides such as those expressed by Staphylococcus aureus (Fattom et al 2004). It has been shown possible to make effective glycoconjugate immunogens from low molecular weight oligosaccharides such as those present on the lipo-oligosaccharides of pathogens such as a Neisseria meningitidis (Mieszala et al 2003).…”

Saccharide/protein conjugate vaccines for Bordetella species: Preparation of saccharide, development of new conjugation procedures, and physico-chemical and immunological characterization of the conjugates

“…The choice of conjugation methods is restricted by the pH and temperature sensitivities of both vaccine components. The types of chemical linkages between the O-SP and the protein as well as the saccharide chain length and the chain density on the protein were shown to influence the serum antibody responses to both vaccine components [35,36,37,38,39,40]. Here we studied two conjugation methods of O-SP either by their Kdo or their glucosamine residues.…”

The Future of Synthetic Carbohydrate Vaccines: Immunological Studies on Streptococcus pneumoniae Type 14

“…It was reported that a synthetic branched tetrasaccharide, corresponding to a single structural repeating unit of Pn14PS conjugated to the cross-reactive material of diptheria toxin (CRM197), was found to induce anti-polysaccharide type 14 antibodies by Mawas, F. et al [74]. We continued to investigate further how small the minimal structure in Pn14PS can be and still produce specific antibodies against native polysaccharide type 14 [73].…”

“…Meanwhile we and other groups have been working on improving the immunogenicity of neoglycoconjugates against different S. pneumoniae serotypes in animal models: Di-, tri-, and tetrasaccharides related to polysaccharide type 17F conjugated to keyhole limpet hemocyanin (KLH) protein [69,70] and tri-and tetrasaccharides related to type 23 conjugated to KLH protein [71]; Di-, tri-, and tetrasaccharides related to type 6B conjugated to KLH protein [72]; Di-, tri-, and tetrasaccharide related to type 3 conjugated to the crossreactive material of diphteria toxin (CRM197) protein [60] and most recently overlapping oligosaccharide varying from tri-to dodecasaccharides related to polysaccharide type 14 conjugated to CRM197 protein [73,74].…”