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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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Analytical ultracentrifugation of HA63–286-RBD.(A) The sedimentation velocity profiles (fringe displacement) were fitted to a continuous sedimentation coefficient distribution model c(s). The experiment was conducted at a loading protein concentration of 0.45 mg/mL in 10 mM Tris pH 8.0, 100 mM NaCl at 20°C and at a rotor speed of 60,000 rpm. The s-values of the proteins are listed in Table 1. (B) Absorbance scans at 280 nm at equilibrium are plotted versus the distance from the axis of rotation. The protein was centrifuged in the above buffer at 4°C for at least 24 h at each rotor speed of 20, 30 and 38 k rpm. The solid lines represent the global nonlinear least squares best-fit of all the data sets to a monomer-dimer self-association model with a very weak KD (288 mM). For clarity, only the loading protein concentration of 5 µM is shown. The r.m.s. deviation for this fit was 0.0037 absorbance units.
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pone-0011694-g004: Analytical ultracentrifugation of HA63–286-RBD.(A) The sedimentation velocity profiles (fringe displacement) were fitted to a continuous sedimentation coefficient distribution model c(s). The experiment was conducted at a loading protein concentration of 0.45 mg/mL in 10 mM Tris pH 8.0, 100 mM NaCl at 20°C and at a rotor speed of 60,000 rpm. The s-values of the proteins are listed in Table 1. (B) Absorbance scans at 280 nm at equilibrium are plotted versus the distance from the axis of rotation. The protein was centrifuged in the above buffer at 4°C for at least 24 h at each rotor speed of 20, 30 and 38 k rpm. The solid lines represent the global nonlinear least squares best-fit of all the data sets to a monomer-dimer self-association model with a very weak KD (288 mM). For clarity, only the loading protein concentration of 5 µM is shown. The r.m.s. deviation for this fit was 0.0037 absorbance units.

Mentions: We further characterized the folded state of protein HA63–286-RBD in solution by analytical ultracentrifugation, specifically using sedimentation velocity and equilibrium analysis assays. Both experiments showed that HA-RBD exists mainly as a monomer in solution (Figure 4), and there are no dimmers observed in the c(s) distribution profile (Figure 4a) at the concentration used. The analytical results are presented in Table 1. The frictional ratio value (f/f0 – value) of 1.30 reflects a slightly elongated globular protein, consistent with the predicted three-dimensional structure. The standard s-value, s020,w (water as solvent at 20°C and zero concentration), and frictional ratio calculated with the standard s-value (in parenthesis) are also listed in Table 1. The sedimentation equilibrium data do not fit quite as well to a discrete single monomer species model which predicts a mass value of 28,585 Da, slightly larger than the monomeric molecular weight. The dissociation equilibrium constant of the monomer-dimmer self-association model determined from the equilibrium data is KD = 288 mM, and this suggests a very weak dimerization interaction (root mean square deviation of the model was 0.0037 absorbance units at 280 nm; Figure 4b).


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)

Analytical ultracentrifugation of HA63–286-RBD.(A) The sedimentation velocity profiles (fringe displacement) were fitted to a continuous sedimentation coefficient distribution model c(s). The experiment was conducted at a loading protein concentration of 0.45 mg/mL in 10 mM Tris pH 8.0, 100 mM NaCl at 20°C and at a rotor speed of 60,000 rpm. The s-values of the proteins are listed in Table 1. (B) Absorbance scans at 280 nm at equilibrium are plotted versus the distance from the axis of rotation. The protein was centrifuged in the above buffer at 4°C for at least 24 h at each rotor speed of 20, 30 and 38 k rpm. The solid lines represent the global nonlinear least squares best-fit of all the data sets to a monomer-dimer self-association model with a very weak KD (288 mM). For clarity, only the loading protein concentration of 5 µM is shown. The r.m.s. deviation for this fit was 0.0037 absorbance units.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908544&req=5

pone-0011694-g004: Analytical ultracentrifugation of HA63–286-RBD.(A) The sedimentation velocity profiles (fringe displacement) were fitted to a continuous sedimentation coefficient distribution model c(s). The experiment was conducted at a loading protein concentration of 0.45 mg/mL in 10 mM Tris pH 8.0, 100 mM NaCl at 20°C and at a rotor speed of 60,000 rpm. The s-values of the proteins are listed in Table 1. (B) Absorbance scans at 280 nm at equilibrium are plotted versus the distance from the axis of rotation. The protein was centrifuged in the above buffer at 4°C for at least 24 h at each rotor speed of 20, 30 and 38 k rpm. The solid lines represent the global nonlinear least squares best-fit of all the data sets to a monomer-dimer self-association model with a very weak KD (288 mM). For clarity, only the loading protein concentration of 5 µM is shown. The r.m.s. deviation for this fit was 0.0037 absorbance units.
Mentions: We further characterized the folded state of protein HA63–286-RBD in solution by analytical ultracentrifugation, specifically using sedimentation velocity and equilibrium analysis assays. Both experiments showed that HA-RBD exists mainly as a monomer in solution (Figure 4), and there are no dimmers observed in the c(s) distribution profile (Figure 4a) at the concentration used. The analytical results are presented in Table 1. The frictional ratio value (f/f0 – value) of 1.30 reflects a slightly elongated globular protein, consistent with the predicted three-dimensional structure. The standard s-value, s020,w (water as solvent at 20°C and zero concentration), and frictional ratio calculated with the standard s-value (in parenthesis) are also listed in Table 1. The sedimentation equilibrium data do not fit quite as well to a discrete single monomer species model which predicts a mass value of 28,585 Da, slightly larger than the monomeric molecular weight. The dissociation equilibrium constant of the monomer-dimmer self-association model determined from the equilibrium data is KD = 288 mM, and this suggests a very weak dimerization interaction (root mean square deviation of the model was 0.0037 absorbance units at 280 nm; Figure 4b).

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