Semisynthetic glycoconjugate based on dual role protein/PsaA as a pneumococcal vaccine

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Abstract

Pneumococcal infections remain a major public health concern worldwide. The currently available vaccines in the market are based on pneumococcal capsular polysaccharides but they still need to be improved to secure an optimal coverage notably in population at risk. To circumvent this, association of virulence pneumococcal proteins to the polysaccharide valencies has been proposed with the hope to observe an additive - if not synergistic - protective effect. Along this line, the use of the highly conserved and ubiquitous pneumococcal surface adhesin A (PsaA) as a protein carrier for a synthetic pneumococcal oligosaccharide is demonstrated herein for the first time. A tetrasaccharide mimicking functional antigenic determinants from the S. pneumoniae serotype 14 capsular polysaccharide (Pn14TS) was chemically synthesised. The mature PsaA (mPsaA) was expressed in E. coli and purified using affinity chromatography. The Pn14PS was conjugated to mPsaA using maleimide-thiol coupling chemistry to obtain mPsaA-Pn14PS conjugate (protein/sugar molar ratio: 1/5.4). The mPsaA retained the structural conformation after the conjugation and lyophilisation. The prepared glycoconjugate adjuvanted with α-galactosylceramide, a potent activator of invariant Natural Killer T cells, was tested in mice for its immunological response upon subcutaneous injection in comparison with mPsaA alone and a model BSA conjugate (BSA-Pn14PS, used here as a control). Mice immunised with the mPsaA-Pn14TS produced a robust IgG response against mPsaA and against the capsular polysaccharide from pneumococcal serotype 14. These data provide the basis for novel pneumococcal vaccine development.

Introduction

Streptococcus pneumoniae is a leading cause of mortality and morbidity in children and the elderly population. It is responsible for infections like pneumonia, otitis media, meningitis and bacteraemia which accounts for an estimated number of 800,000 deaths in children annually (Wilson et al., 2015). The polysaccharide subunits expressed by pneumococci are anchored to the cell wall surface and preventing the clearance of pneumococci from the lung and protecting them from complement-mediated opsonophagocytosis (Hyams et al., 2010). Polysaccharide chains act as early target antigens as their production is essential for pneumococcal colonisation and virulence. These polysaccharides are highly immunogenic and induce the production of antibodies that react with homologous serotype (Daniels et al., 2016).

Capsular polysaccharide (CP) vaccines currently in use for adults have only weak or no efficacy towards pneumonia and bronchitis caused by S. pneumoniae (Daniels et al., 2016). The protection level of a vaccinated individual varies depending on the location as the prevalence of serotype varies significantly among the continents. Due to this phenomenon, the capsular serotypes used as vaccine antigens are monitored internationally (Wilson et al., 2015). The inability of the polysaccharide vaccines to elicit a protective immune response in toddlers and young children has given way for the development of improved vaccines. Polysaccharides are T-cell independent antigens, and they do not elicit any protective immune response in infants and children, but conjugating them to a protein makes them T-cell dependent and immunogenic in the same population (Jakobsen and Jonsdottir, 2003). Researchers have tried conjugating capsular polysaccharide to carrier proteins like tetanus toxoid, diphtheria toxin, and cross-reactive material of diphtheria toxoid (CRM197) to overcome this issue. The prepared glycoconjugates can generate T-cell dependent antibody response and higher antibody titers in children and high-risk individuals (Jakobsen et al., 2006; Lai and Schreiber, 2009). Glycoconjugate vaccines such as the heptavalent (PCV7) vaccine and the more recent tridecavalent (PCV13) pneumococcal vaccine are promising in preventing pneumonia caused by the corresponding serotypes. However, it is still not clear whether it is by preventing nasopharyngeal colonisation or by improving adaptive immune response in the lungs. The overall protection mechanism against the S. pneumoniae infection is not clearly understood, which hinders the design of more efficacious novel vaccines (Entwisle et al., 2017).

There is a need for pneumococcal vaccines with broader coverage against the pneumococcal serotypes. As capsular polysaccharides are serotype specific, researchers have focused on identifying protein immunogens which can offer complete serotype coverage and generate enhanced protection. Along with this line, pneumococcal surface adhesin A (PsaA) is a membrane-associated metal binding lipoprotein (Li et al., 2014), which is highly conserved and essential for the survival and virulence of the bacteria (Rajam et al., 2008). PsaA is involved in the adhesion and colonisation of nasopharyngeal epithelium by the pneumococci. Its presence in all serotypes of pneumococci along with its ability to generate robust responses in infants or elderly population further contribute to make it an ideal candidate for pneumococcal vaccine formulations (Morrison et al., 2000; Rapola et al., 2000). Indeed, PsaA used as immunogen was able to prevent entry of the pathogen noticeably by reducing the nasal carriage of the pathogen and even to confer protection when combined with adjuvants in animal models (Gor et al., 2011; Lu et al., 2015; Olafsdottir et al., 2012; Wang et al., 2010). Alternatively, PsaA has also been used as a carrier protein in glycoconjugate vaccines which has already demonstrated protection in animal models (Chen, 2016; Lin et al., 2010). Additionally, the concomitant administration of PsaA with PCV7 reduced the colonisation in the murine models (Whaley et al., 2010). In these reports, glycoconjugates were acquired from bacterial source (Chen, 2016; Lin et al., 2010), but the utility of PsaA as a carrier for synthetic oligosaccharides mimicking full-length CP remains to be documented. To this aim, the capsular polysaccharide of the S. Pneumoniae type 14 consists of the repeating units of {6)-[β-d-Galp-(1 → 4)-]β-d-GlcpNAc-(1 → 3)-β-d-Galp-(1 → 4)-β-d-Glcp-(1→}n (Laferriere et al., 1998). The tetrasaccharide β-d-Galp-(1 → 4)-β-d-Glcp-(1 → 6)-[β-d-Galp-(1 → 4)]-β-d-GlcpNAc (referred to as Pn14TS) has been identified as the smallest oligosaccharide of the serotype 14 that is capable of inducing opsonophagocytic response when coupled to the carrier proteins like CRM197 (Safari et al., 2008). It possesses higher protective properties as compared to related hexa- or octasaccharides when conjugated (Kurbatova et al., 2017). In this study, we thus propose to produce this synthetic mimic of S. pneumoniae type 14 CP and to conjugate it to pneumococcal PsaA, using thiol maleimide coupling chemistry to obtain a semisynthetic glycoconjugate vaccine. The immunogenicity of the obtained glycoconjugate was studied in BALB/c mice. As an adjuvant, we chose α-galactosylceramide (α-GalCer), a potent activator of invariant Natural Killer T (iNKT) cells (Bendelac et al., 2007; Paget and Trottein, 2013).

Section snippets

Mice and ethics statement

Specific pathogen-free C57BL/6 mice (6 week-old, female) were purchased from Janvier (Le Genest-St-Isle, France). Mice were maintained in a biosafety level 2 facility in the Animal Resource Centre at the Lille Pasteur Institute for at least two weeks prior to usage, to allow appropriate acclimation. Mice were fed a standard rodent chow (SAFE A04) (SAFE, Augy, France) and water ad libitium. The diet contains ~11.8% fiber including ~10% water-insoluble fiber (3.6% cellulose) and 1.8%

Synthesis of antigen tetrasaccharide

Chemo-enzymatic and chemical synthesis of the pneumococcal serotype 14 tetrasaccharides have been previously reported in the literature. The former relies on the beta-(1 → 4)-connection of the galactose residue to the N-acetyl-β-d-glucosamine residue by using bovine milk β-1,4-galactosyltransferase (Joosten et al., 2003; Niggemann et al., 1998). Chemical synthesis is based on an obvious [2 + 2] approach consisting in the glycosylation of lactose donor with a lactosamine acceptor which, in turn,

Discussion

The advent of glycoconjugate vaccines has been a breakthrough for the prophylactic treatment of infectious diseases caused by encapsulated bacteria including S. pneumoniae (Costantino et al., 2011). The pneumococcal glycoconjugate vaccine is safe and effective but faces serotype replacement phenomenon: Initially composed of 7 polysaccharides corresponding to 7 different serotypes, it has been implemented to protect against 13 pneumococcal and soon 15 serotypes. These have been selected by

Conclusion

In summary, the first semi-synthetic glycoconjugate vaccine solely composed of pneumococcal antigens, i.e., of a synthetic tetrasaccharide mimicking the capsule of serotype 14 coupled to PsaA has been reported. The latter has been selected as it should contribute to control pneumococcal colonisation and ensure herd immunity. The conjugate has been prepared in high yield and purity and proved to be stable during long-term storage. The conjugate was able to induce the production of IgGs against

Acknowledgements

Maruthi Prasanna acknowledges his doctoral fellowship from the European Commission, Education, Audiovisual and Culture Executive Agency (EACEA), under the Erasmus Mundus programme, “NanoFar: European Doctorate in Nanomedicine and Pharmaceutical Innovation” (Project: 2015-01-C4). We would like to thank Dr. B. Frisch (University of Strasbourg) for the production of α-GalCer.

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