Abstract
Enteric fever, mainly caused by Salmonella enterica serovar Paratyphi A, remains a common and serious infectious disease worldwide. As yet, there are no licensed vaccines against S. Paratyphi A. Biosynthesis of conjugate vaccines has become a promising approach against bacterial infection. However, the popular biosynthetic strategy using N-linked glycosylation systems does not recognize the specialized O-polysaccharide structure of S. Paratyphi A. Here, we describe an O-linked glycosylation approach, the only currently available glycosylation system suitable for an S. Paratyphi A conjugate vaccine. We successfully generated a recombinant S. Paratyphi A strain with a longer O-polysaccharide chain and transformed the O-linked glycosylation system into the strain. Thus, we avoided the need for construction of an O-polysaccharide expression vector. In vivo assays indicated that this conjugate vaccine could evoke IgG1 antibody to O-antigen of S. Paratyphi A strain CMCC 50973 and elicit bactericidal activity against S. Paratyphi A strain CMCC 50973 and five other epidemic strains. Furthermore, we replaced the peptides after the glycosylation site (Ser) with an antigenic peptide (P2). The results showed that the anti-lipopolysaccharide antibody titer, bactericidal activity of serum, and protective effect during animal challenge could be improved, indicating a potential strategy for further vaccine design. Our system provides an easier and more economical method for the production of S. Paratyphi A conjugate vaccines. Modification of the glycosylation site sequon provides a potential approach for the development of next-generation “precise conjugate vaccines.”
Highlights
Enteric fever, caused by several Salmonella enterica subspecies enterica serovars that are spread through contaminated water, food, flies, and cockroaches, remains an important cause of morbidity and mortality in developing countries, especially in Asia.[1]
Following proteinase K digestion, the glycopeptide was examined by liquid (Supplementary Fig. 1). These results indicated that a bioconjugate vaccine for S
Serum was collected pre-immunization and 10 days after each immunization, and an enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the induction of IgG antibody against SPA50973 LPS. Both CTB4573H-OPS and rEPA4573H-OPS could induce a stronger immune response in BALB/c mice than OPS alone, the average titer of the CTB4573H-OPS group was significantly higher (P < 0.001 after the third immunization) than that of the rEPA4573H-OPS group (Supplementary Fig. 6). These results indicated that at this dose, cholera toxin B subunit (CTB) had greater potential as a carrier than recombinant Pseudomonas aeruginosa exotoxin A (rEPA), which may be related to the pentameric form of glycosylated CTB (Supplementary Fig. 7)
Summary
Enteric fever, caused by several Salmonella enterica subspecies enterica serovars that are spread through contaminated water, food, flies, and cockroaches, remains an important cause of morbidity and mortality in developing countries, especially in Asia.[1]. Three vectors (expressing the polysaccharide gene cluster, glycosyltransferase, and the carrier protein) need to be expressed in E. coli, and the glycoprotein is produced following a one-step purification method after induction.[12] Compared with the earlier chemical methods, the bio-method is a low-cost, high-efficiency expressing CldLT2, and the number of repeat units was >20 (Supplementary Fig. 2B). This indicated that CldLT2 could control the length of OPS chains in S.
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