Bioconjugates made from recombinant N-glycosylated proteins from procaryotic cells
The present invention is directed to a bioconjugate vaccine, such as an O1-bioconjugate vaccine, comprising: a protein carrier comprising a protein carrier containing at least one consensus sequence, D/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid except proline; at least one antigenic polysaccharide from at least one pathogenic bacterium, linked to the protein carrier; and, optionally, an adjuvant. In another aspect, the present invention is directed to a method of producing an O1-bioconjugate in a bioreactor comprising a number steps.
1. A method of producing a composition comprising a bioconjugate in a bioreactor, wherein said bioconjugate comprises a protein linked to an O antigen, comprising the steps:
(i) expressing in bacteria: modified EPA protein containing at least one consensus sequence, D/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid except proline, or the AcrA protein; an oligosaccharyl transferase; and one or more O antigens;
(ii) growing the bacteria for a period of time to produce an amount of the bioconjugate comprising the AcrA protein or the modified EPA protein linked to the one more O antigens;
(iii) extracting periplasmic proteins; and
(iv) separating the bioconjugate from the extracted periplasmic proteins by anionic exchange.
2. The method of claim 1 , wherein the O antigen is from Shigella, E. coli , or Pseudomonas aeruginosa.
3. The method of claim 1 , wherein said modified EPA has an amino acid sequence comprising SEQ. ID NO.: 6.
4. The method of claim 1 , wherein said modified EPA has an amino acid sequence comprising SEQ. ID NO.: 7.
5. The method of claim 2 , wherein the O antigen is Shigella dysenteriae O1, Pseudomonas aeruginosa O1, or E. coli O1.
6. The method of claim 2 , wherein the O antigen is from extraintestinal pathogenic E. coli (ExPEC).
7. The method of claim 2 , wherein the O antigen is from S. flexneri 2a, S. flexneri 3a, S. flexneri 3b, S. flexneri 6 or S. sonnei.
8. The method of claim 2 , wherein the O antigen is from E. coli O4:K52 (ExPEC), E. coli O4:K6 (ExPEC), E. coli O6:K2 (ExPEC); E. coli O6:K54 (ExPEC), E. coli O22 (ExPEC), E. coli O75 (ExPEC), E. coli O83 (ExPEC), E. coli O 7, E. coli O 9, E. coli O16, E. coli O121 or E. coli O157 (EHEC).
9. The method of claim 1 , wherein the O antigen has the following structure:
10. The method of claim 1 , wherein said bacteria is E. coli.
11. The method of claim 1 , wherein said oligosaccharyl transferase is from Campylobacter spp.
12. The method of claim 11 , wherein said oligosaccharyl transferase is from Campylobacter jejuni.
13. The method of claim 1 , wherein said composition is at least 98% pure.