A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host

Abstract

Chemical synthesis of conjugate vaccines, consisting of a polysaccharide linked to a protein, can be technically challenging, and in vivo bacterial conjugations (bioconjugations) have emerged as manufacturing alternatives. Bioconjugation relies upon an oligosaccharyltransferase to attach polysaccharides to proteins, but currently employed enzymes are not suitable for the generation of conjugate vaccines when the polysaccharides contain glucose at the reducing end, which is the case for ~75% of Streptococcus pneumoniae capsules. Here, we use an O-linking oligosaccharyltransferase to generate a polyvalent pneumococcal bioconjugate vaccine with polysaccharides containing glucose at their reducing end. In addition, we show that different vaccine carrier proteins can be glycosylated using this system. Pneumococcal bioconjugates are immunogenic, protective and rapidly produced within E. coli using recombinant techniques. These proof-of-principle experiments establish a platform to overcome limitations of other conjugating enzymes enabling the development of bioconjugate vaccines for many important human and animal pathogens.

A promising bioconjugate vaccine against hypervirulent Klebsiella pneumoniae

“…However, both PglB and PglL are unable to conjugate polysaccharides containing glucose at the reducing end (the first sugar in the growing polysaccharide chain), such as most of the Streptococcus pneumoniae (28) and the K1 and K2 Klebsiella capsules (12). Recently, we identified a new class of conjugating enzyme, termed PglS, that is capable of transferring a diverse array of polysaccharides, including those that contain glucose as the reducing end sugar (23, 29). Importantly, more than 50% of all K. pneumoniae capsular serotypes are composed of polysaccharides with glucose at the reducing end, including both the K1 and K2 serotypes (12).…”

Synthetic Glycobiology: Parts, Systems, and Applications

“…PglS glycosylation has only been demonstrated to target its native pilin-like ComP. 90 While these complex GlycTag sequence and structure requirements make it more difficult to direct glycosylation by O -linked OSTs onto recombinant proteins, these enzymes still hold great promise for engineering due to their promiscuity in the sugars that they can attach to proteins. 124 For example, PglS is the only OST known to be able to transfer LLOs with glucose at the reducing end 90 and PglL has been shown to transfer a single N ′-diacetylbacillosamine from a nucleotide-activated sugar.…”

“… 352 , 353 As discussed previously, limitations in the diversity of sugar donor substrates that can be utilized by PglB have been circumvented by using O -linked OSTs to produce glycoconjugate vaccines in vivo . Specifically, PglL has been used to produce vaccine candidates against Shigella flexneri 2a 88 and Salmonella enterica serovar Paratyphi, 354 while PglS has been used to recombinantly produce vaccine candidates against Streptococcus pneumoniae ( 90 ) as well as hypervirulent Klebsiella pneumoniae . 355 …”

Recent advances in the production of recombinant glycoconjugate vaccines

“…27 Recently, it has been demonstrated that a newly identified O -linked OST from Acinetobacter species, PglS, can couple S. pneumoniae capsular polysaccharides with glucose as a reducing end sugar, which could not be coupled with CjPglB. 54 This paves the way for a polyvalent vaccine produced using bioconjugation as ~70% of serotypes have glucose at the reducing end 55 ; including 9/13 serotypes in the currently licensed pneumococcal Prevnar13 glycoconjugate vaccine.…”