Review
Precise protein conjugation technology for the construction of homogenous glycovaccines

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The introduction of vaccines for the treatment and prevention of bacterial or viral diseases in the early 19th century marked a crucial turning point in medical history. Since then, extensive immunization campaigns have eradicated smallpox and drastically reduced the number of diphtheria, tetanus, pertussis and measles cases worldwide. Although a broad selection of vaccines is available, there remains a need to develop additional vaccine candidates against a range of dangerous infectious diseases, preferably based on precise syntheses that lead to homogenous formulations. Different strategies for the construction of this type of vaccine candidates are being pursued. Glycoconjugate vaccines are successful in the fight against bacterial and viral infectious diseases. However, their exact mechanism of action remains largely unknown and the large-scale production of chemically defined constructs is challenging. In particular, the conjugation of the carbohydrate antigen to the protein carrier has proved to be crucial for the properties of these vaccines. This review highlights some of the latest findings and developments in the construction of glycoconjugate vaccines by means of site-specific chemical reactions.

Introduction

Vaccines are one of the most important tools in our fight against various infectious diseases. As resistance against many antibiotics increases and new viruses appear, the development of new vaccine candidates becomes ever more important. Glycoconjugate vaccines represent one of the most successful strategies to elicit a strong and long-lasting immune response, especially in infants. In general, the conjugation of a polysaccharide antigen to an immunogenic protein carrier generates a T-cell dependent immune response, which leads to the formation of enduring memory B-cells [1], [2]. By following this approach, glycoconjugate vaccines have been licensed for a number of infectious diseases, such as Haemophilus influenzae type b, Samonella Typhi, Neisseria meningitidis and Streptococcus pneumoniae [3], [4].

In recent years, efforts have been made to understand the exact mechanism by which glycoconjugates are processed by the immune system, and to develop more defined vaccine constructs. So far, glycoconjugate vaccines have been obtained largely through random conjugation reactions of polysaccharide antigens to immunogenic protein carriers [3], [5]. These reactions, like reductive aminations, active esters or carbodiimide-mediated couplings, commonly take place on functionalities of the carbohydrate or through the installation of a linker moiety between the sugar and a different amino acid, typically lysine, aspartic or glutamic residues, on the carrier protein (Fig. 1, I–III) [6]. Currently, there are six carrier proteins being used for licensed vaccines, namely tetanus toxoid (TT), diphtheria toxoid, CRM197, recombinant exotoxin A of P.aeruginosa, Protein D from H. influenzae and the outer membrane protein complex of meningococcus B. Despite the efficiency of the resulting constructs, the current approach comes with drawbacks like batch-to-batch variations and rather undefined products. To overcome these problems, new methods have been developed for the chemical synthesis of desired glycan antigens and for their site-selective conjugation on the protein carrier. The goal of a defined site-selective conjugation on a carrier can be addressed in different ways and we highlight some of them here, together with recent examples of chemically-defined glycoconjugate constructs.

Section snippets

Fully synthetic conjugate – Quimi-hib

The biggest success in synthetic glycoconjugate vaccine design so far is arguably the fully synthetic glycoconjugate vaccine Quimi-Hib against Haemophilus influenza type b in 2004 [7]. The authors identified four key points that facilitated the successful production of this defined glycoconjugate vaccine on a large scale: a synthetic route for a precursor disaccharide with just one chromatographic purification step, a high-yielding polycondensation reaction to obtain the necessary

Site-selective protein modification – non-canonical amino acids

There are a number of different routes available when it comes to the development of new site-selective modification strategies. The options include modifying proteinogenic amino acids, as in the selective glycoconjugates examples above, or introducing non-canonical amino acids. The use of non-canonical amino acids for modification reactions brings the advantage of new functional groups and usually minimizes unwanted side reactions [14]. A range of technologies introduce non-canonical amino

Selective lysine conjugates

Lysine represents the most abundant amino acid residue in carrier proteins like CRM197 or TT and presents a nucleophilic ε-amine sidechain. However, the high reactivity makes site-selective reactions on this sidechain particularly challenging and difficult to control. Nevertheless, methods have been developed to discriminate different lysine residues based on their reactivity and to use this differentiation for selective conjugations. Crotti et al. were able to map 37 of 39 lysine residues of

Effects of protein modification on function and immunogenicity of glycoconjugate vaccines [42]

The conjugation of carbohydrate antigens towards immunogenic protein carriers is a key point to enable the generation of polysaccharide specific IgG antibodies and the development of long-lasting T-cells [2], [43]. Although the exact mechanism of glycoconjugate vaccines in terms of immune recognition is little understood, different factors are known to influence the immunogenicity of these constructs. Length and density of the carbohydrate antigen, together with the conjugation method to the

Concluding remarks

These exemplary studies demonstrate the importance of site-selective conjugation for the development of efficient vaccine candidates. Ongoing research to introduce defined modifications in potential carrier proteins may provide useful tools on the road to a fully synthetic and defined conjugate vaccine. As bacterial resistance to current antibiotic treatments increases and new viruses emerge, vaccines will be crucial in our fight against them.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 675671 (A.K. and G.J.L.B.). Funding from the Royal Society (URFR180019 to G.J.L.B.), FCT Portugal (iFCT IF/00624/2015 to G.J.L.B.) and Agencia Estatal Investigacion of Spain (AEI; RTI2018-099592-B-C21 project to F.C.) is also acknowledged. We also thank Dr Vikki Cantrill for her help with the editing of this manuscript.

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