Compositions and methods for rapid in vitro synthesis of bioconjugate vaccines in vitro via production and N-glycosylation of protein carriers in detoxified prokaryotic cell lysates
Disclosed are methods, systems, components, and compositions for cell-free synthesis of glycosylated carrier proteins. The glycosylated carrier proteins may be utilized in vaccines, including anti-bacterial vaccines. The glycosylated carrier proteins may include a bacterial polysaccharide conjugated to a carrier, which may be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. The glycosylated carrier proteins may be synthesized in cell-free glycoprotein synthesis (CFGpS) systems using prokaryote cell lysates that are enriched in components for glycoprotein synthesis such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) including OSTs and LLOs associated with synthesis of bacterial O antigens.
1. A method for synthesizing an N-glycosylated carrier protein for a polysaccharide via coordinated transcription, translation, and N-glycosylation in vitro, in a single vessel, the method comprising:
transcribing and translating a carrier protein in a cell-free protein synthesis reaction, the carrier protein comprising an inserted consensus sequence, D/E-X 1 -N-X 2 -SIT or N-X-S/T, wherein X, X 1 and X 2 may be any natural or unnatural amino acid except proline;
(ii) glycosylating the carrier protein in the cell-free protein synthesis reaction with at least one polysaccharide, wherein the at least one polysaccharide is at least one bacterial O-antigen;
wherein the single vessel comprises a template encoding the carrier protein, an exogenous lipid-linked oligosaccharide (LLO) comprising the bacterial O-antigen, and one or more Escherichia coli ( E. coli ) cell lysates from engineered E. coli strains, wherein the engineered E. coli strains comprise:
(a) a nucleic acid encoding an orthogonal or heterologous oligosaccharyltransferase (OST) which is expressed in the engineered E. coli strains;
(b) one or both of:
(1) a mutation of the endogenous lpxM gene wherein the mutation results in the reduced expression or and/or activity of the encoded myristoyltransferase;
(2) a nucleic acid comprising an orthogonal or heterologous LpxE gene and encoding a lipidA 1-phosphatase which is expressed in the engineered E. coli strains;
(c) a mutation in the endogenous waaL gene, wherein the mutation results in the reduced expression and/or activity of the encoded O-antigen ligase;
(d) prokaryotic transcription and translation machinery.
2. The method of claim 1 , wherein the bacterial O-antigen is from E. coli.
3. The method of claim 1 , wherein the bacterial O-antigen is from Franciscella tularensis.
4. The method of claim 1 , further comprising formulating the N-glycosylated carrier protein as an antigenic composition comprising the N-glycosylated carrier protein.
5. The method of claim 1 , further comprising formulating the N-glycosylated carrier protein as a vaccine composition comprising the N-glycosylated carrier protein.
6. The method of claim 5 , wherein the vaccine composition further comprises an adjuvant.
7. The method of claim 1 , wherein the carrier protein is an engineered variant of E. coli maltose binding protein (MBP).
8. The method of claim 1 , wherein the carrier protein is selected from a detoxified variant of the toxin from Clostridium tetani , a detoxified variant of the toxin from Corynebacterium diptheriae, Haemophilus influenzae protein D (PD) or a variant thereof, and Neisseria meningitidis porin protein (PorA) or a variant thereof.
9. The method of claim 1 , wherein the glycosylating step utilizes an oligosaccharyltransferase (OST) which is a naturally occurring bacterial homolog of C. jejuni Pg1B, and wherein the consensus sequence comprises D/E-X 1 -N-X 2 -S/T.
10. The method of claim 1 , wherein the glycosylating step utilizes an OST that is an engineered variant of C. jejuni Pg1B, and wherein the consensus sequence comprises D/E-X 1 -N-X 2 -S/T.
11. The method of claim 1 , wherein the wherein the glycosylating step utilizes an OST that is a naturally occurring archaeal OST, and wherein the consensus sequence comprises N-X-S/T.
12. The method of claim 1 , wherein the wherein the glycosylating step utilizes an OST which is a naturally occurring single-subunit eukaryotic OST, and wherein the consensus sequence comprises N-X-S/T.
13. The method of claim 1 , wherein the heterologous LpxE gene is from F. tularensis.
14. The method of claim 1 , wherein the one or more cell lysates have an endotoxin unit (EU) concentration of less than about 180,000 EU/ml.
15. A method of vaccinating a subject in need thereof against a bacterial O-antigen, the method comprising administering to the subject the vaccine composition of claim 5 .