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An influenza A/H1N1/2009 hemagglutinin vaccine produced in Escherichia coli.

Aguilar-Yáñez JM, Portillo-Lara R, Mendoza-Ochoa GI, García-Echauri SA, López-Pacheco F, Bulnes-Abundis D, Salgado-Gallegos J, Lara-Mayorga IM, Webb-Vargas Y, León-Angel FO, Rivero-Aranda RE, Oropeza-Almazán Y, Ruiz-Palacios GM, Zertuche-Guerra MI, DuBois RM, White SW, Schultz-Cherry S, Russell CJ, Alvarez MM - PLoS ONE (2010)

Bottom Line: It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model.Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture.Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biotecnología-FEMSA, Tecnológico de Monterrey at Monterrey, Monterrey, México.

ABSTRACT

Background: The A/H1N1/2009 influenza pandemic made evident the need for faster and higher-yield methods for the production of influenza vaccines. Platforms based on virus culture in mammalian or insect cells are currently under investigation. Alternatively, expression of fragments of the hemagglutinin (HA) protein in prokaryotic systems can potentially be the most efficacious strategy for the manufacture of large quantities of influenza vaccine in a short period of time. Despite experimental evidence on the immunogenic potential of HA protein constructs expressed in bacteria, it is still generally accepted that glycosylation should be a requirement for vaccine efficacy.

Methodology/principal findings: We expressed the globular HA receptor binding domain, referred to here as HA(63-286)-RBD, of the influenza A/H1N1/2009 virus in Escherichia coli using a simple, robust and scalable process. The recombinant protein was refolded and purified from the insoluble fraction of the cellular lysate as a single species. Recombinant HA(63-286)-RBD appears to be properly folded, as shown by analytical ultracentrifugation and bio-recognition assays. It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model.

Conclusions/significance: Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture. Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy.

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Related in: MedlinePlus

Expression of HA63–286-RBD in E. coli.(A) Protein profile of cell lysates from culture experiments of E. coli C41, BL21 (DE3) pLysS or Rosetta-gami transformed with genes to produce (1) GFP+histidine tag (clone C41 1); (2) GFP+histidine tag (clone C41 2); (3) GFP+histidine tag (clone C41 3); (4) negative control, C41(5) HA63–286-RBD (clone C41 1); (6) HA63–286-RBD (clone C41 2); (7) HA63–286-RBD (clone Rosetta-gami clone 1); (8) HA63–286-RBD (clone Rosetta-gami clone 2). (9) Precision Plus Kaleidoscope molecular mass ruler showing 25 kD (pink) and 20 kD (blue) bands. The blue arrow indicates the 26 kD band corresponding to HA63–286-RBD. (B) SDS-PAGE showing (1) the soluble and (2) insoluble fraction of the C41 strain lysate after 8 hours induction with 1mM IPTG. (C) SDS-PAGE showing the protein profiles at different stages of recovery, purification and on-column refolding. (1) Crude lysate of the 8M urea solubilized inclusion bodies, (2) Unbound fraction, (3) 1st wash step, (4) 2nd wash step, (5,6) refolding steps, (7–12) Elution fraction using imidazole 300 mM, (13) chromatographic resin. (M) Precision Plus Kaleidoscope molecular mass ruler.
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pone-0011694-g003: Expression of HA63–286-RBD in E. coli.(A) Protein profile of cell lysates from culture experiments of E. coli C41, BL21 (DE3) pLysS or Rosetta-gami transformed with genes to produce (1) GFP+histidine tag (clone C41 1); (2) GFP+histidine tag (clone C41 2); (3) GFP+histidine tag (clone C41 3); (4) negative control, C41(5) HA63–286-RBD (clone C41 1); (6) HA63–286-RBD (clone C41 2); (7) HA63–286-RBD (clone Rosetta-gami clone 1); (8) HA63–286-RBD (clone Rosetta-gami clone 2). (9) Precision Plus Kaleidoscope molecular mass ruler showing 25 kD (pink) and 20 kD (blue) bands. The blue arrow indicates the 26 kD band corresponding to HA63–286-RBD. (B) SDS-PAGE showing (1) the soluble and (2) insoluble fraction of the C41 strain lysate after 8 hours induction with 1mM IPTG. (C) SDS-PAGE showing the protein profiles at different stages of recovery, purification and on-column refolding. (1) Crude lysate of the 8M urea solubilized inclusion bodies, (2) Unbound fraction, (3) 1st wash step, (4) 2nd wash step, (5,6) refolding steps, (7–12) Elution fraction using imidazole 300 mM, (13) chromatographic resin. (M) Precision Plus Kaleidoscope molecular mass ruler.

Mentions: The selection of this precise HA subdomain purposely excludes all residues of the metastable HA2 stalk domain including the hydrophobic fusion peptide and transmembrane domain. Computer simulations predict that the isolated HA1 receptor-binding domain has much less surface hydrophobicity than the entire HA protein ectodomain (compare Figure 2a and 2b) while still preserving its antigenic structure (Figure 2c). For these simulations, the full length HA of the Influenza A H1N1/1918 virus [23] was taken as a template for the estimation of the most probable structure of protein HA63–286-RBD. Given the close similitude in primary and tertiary structure between the HA H1N1/2009 and the HA H1N1/1918 protein [22], the accuracy of the predicted structure of the HA63–286-RBD is expected to be high. Hydrophobicity minimization is generally conducive to higher expression levels in E. coli which typically recognize prominent hydrophobic regions as being misfolded and subsequently degrade them [24]. Indeed, in our experiments, high production levels of the complete H1N1/2009 HA1 subunit were not achievable using either conventional E. coli strains or E. coli strains BL21 (DE3) pLysS variants C41 and C43 from Lucigen® Corporation (Middleton, WI) which are known to successfully express transmembrane proteins [25], [26]. Protein HA63–286-RBD was expressed in both E. coli Rosetta-gami and C41, but not in C43. Producer strains were deposited at ATCC® under Patent Deposit Designation PTA-10320. In terms of global yield and growth rate, clones derived from the E. coli C41 strain were more suitable for large-scale production (Figure 3a). Specific growth rates of 1.69 h−1 and average yields of 3.4 g/L were observed at 5L scale settings after 12 h cultivation.


An influenza A/H1N1/2009 hemagglutinin vaccine produced in Escherichia coli.

Aguilar-Yáñez JM, Portillo-Lara R, Mendoza-Ochoa GI, García-Echauri SA, López-Pacheco F, Bulnes-Abundis D, Salgado-Gallegos J, Lara-Mayorga IM, Webb-Vargas Y, León-Angel FO, Rivero-Aranda RE, Oropeza-Almazán Y, Ruiz-Palacios GM, Zertuche-Guerra MI, DuBois RM, White SW, Schultz-Cherry S, Russell CJ, Alvarez MM - PLoS ONE (2010)

Expression of HA63–286-RBD in E. coli.(A) Protein profile of cell lysates from culture experiments of E. coli C41, BL21 (DE3) pLysS or Rosetta-gami transformed with genes to produce (1) GFP+histidine tag (clone C41 1); (2) GFP+histidine tag (clone C41 2); (3) GFP+histidine tag (clone C41 3); (4) negative control, C41(5) HA63–286-RBD (clone C41 1); (6) HA63–286-RBD (clone C41 2); (7) HA63–286-RBD (clone Rosetta-gami clone 1); (8) HA63–286-RBD (clone Rosetta-gami clone 2). (9) Precision Plus Kaleidoscope molecular mass ruler showing 25 kD (pink) and 20 kD (blue) bands. The blue arrow indicates the 26 kD band corresponding to HA63–286-RBD. (B) SDS-PAGE showing (1) the soluble and (2) insoluble fraction of the C41 strain lysate after 8 hours induction with 1mM IPTG. (C) SDS-PAGE showing the protein profiles at different stages of recovery, purification and on-column refolding. (1) Crude lysate of the 8M urea solubilized inclusion bodies, (2) Unbound fraction, (3) 1st wash step, (4) 2nd wash step, (5,6) refolding steps, (7–12) Elution fraction using imidazole 300 mM, (13) chromatographic resin. (M) Precision Plus Kaleidoscope molecular mass ruler.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908544&req=5

pone-0011694-g003: Expression of HA63–286-RBD in E. coli.(A) Protein profile of cell lysates from culture experiments of E. coli C41, BL21 (DE3) pLysS or Rosetta-gami transformed with genes to produce (1) GFP+histidine tag (clone C41 1); (2) GFP+histidine tag (clone C41 2); (3) GFP+histidine tag (clone C41 3); (4) negative control, C41(5) HA63–286-RBD (clone C41 1); (6) HA63–286-RBD (clone C41 2); (7) HA63–286-RBD (clone Rosetta-gami clone 1); (8) HA63–286-RBD (clone Rosetta-gami clone 2). (9) Precision Plus Kaleidoscope molecular mass ruler showing 25 kD (pink) and 20 kD (blue) bands. The blue arrow indicates the 26 kD band corresponding to HA63–286-RBD. (B) SDS-PAGE showing (1) the soluble and (2) insoluble fraction of the C41 strain lysate after 8 hours induction with 1mM IPTG. (C) SDS-PAGE showing the protein profiles at different stages of recovery, purification and on-column refolding. (1) Crude lysate of the 8M urea solubilized inclusion bodies, (2) Unbound fraction, (3) 1st wash step, (4) 2nd wash step, (5,6) refolding steps, (7–12) Elution fraction using imidazole 300 mM, (13) chromatographic resin. (M) Precision Plus Kaleidoscope molecular mass ruler.
Mentions: The selection of this precise HA subdomain purposely excludes all residues of the metastable HA2 stalk domain including the hydrophobic fusion peptide and transmembrane domain. Computer simulations predict that the isolated HA1 receptor-binding domain has much less surface hydrophobicity than the entire HA protein ectodomain (compare Figure 2a and 2b) while still preserving its antigenic structure (Figure 2c). For these simulations, the full length HA of the Influenza A H1N1/1918 virus [23] was taken as a template for the estimation of the most probable structure of protein HA63–286-RBD. Given the close similitude in primary and tertiary structure between the HA H1N1/2009 and the HA H1N1/1918 protein [22], the accuracy of the predicted structure of the HA63–286-RBD is expected to be high. Hydrophobicity minimization is generally conducive to higher expression levels in E. coli which typically recognize prominent hydrophobic regions as being misfolded and subsequently degrade them [24]. Indeed, in our experiments, high production levels of the complete H1N1/2009 HA1 subunit were not achievable using either conventional E. coli strains or E. coli strains BL21 (DE3) pLysS variants C41 and C43 from Lucigen® Corporation (Middleton, WI) which are known to successfully express transmembrane proteins [25], [26]. Protein HA63–286-RBD was expressed in both E. coli Rosetta-gami and C41, but not in C43. Producer strains were deposited at ATCC® under Patent Deposit Designation PTA-10320. In terms of global yield and growth rate, clones derived from the E. coli C41 strain were more suitable for large-scale production (Figure 3a). Specific growth rates of 1.69 h−1 and average yields of 3.4 g/L were observed at 5L scale settings after 12 h cultivation.

Bottom Line: It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model.Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture.Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biotecnología-FEMSA, Tecnológico de Monterrey at Monterrey, Monterrey, México.

ABSTRACT

Background: The A/H1N1/2009 influenza pandemic made evident the need for faster and higher-yield methods for the production of influenza vaccines. Platforms based on virus culture in mammalian or insect cells are currently under investigation. Alternatively, expression of fragments of the hemagglutinin (HA) protein in prokaryotic systems can potentially be the most efficacious strategy for the manufacture of large quantities of influenza vaccine in a short period of time. Despite experimental evidence on the immunogenic potential of HA protein constructs expressed in bacteria, it is still generally accepted that glycosylation should be a requirement for vaccine efficacy.

Methodology/principal findings: We expressed the globular HA receptor binding domain, referred to here as HA(63-286)-RBD, of the influenza A/H1N1/2009 virus in Escherichia coli using a simple, robust and scalable process. The recombinant protein was refolded and purified from the insoluble fraction of the cellular lysate as a single species. Recombinant HA(63-286)-RBD appears to be properly folded, as shown by analytical ultracentrifugation and bio-recognition assays. It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model.

Conclusions/significance: Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture. Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy.

Show MeSH
Related in: MedlinePlus