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Properly folded bacterially expressed H1N1 hemagglutinin globular head and ectodomain vaccines protect ferrets against H1N1 pandemic influenza virus.

Khurana S, Verma S, Verma N, Crevar CJ, Carter DM, Manischewitz J, King LR, Ross TM, Golding H - PLoS ONE (2010)

Bottom Line: Both proteins induced neutralizing antibodies, and reduced viral loads in nasal washes.However, the HA1 (1-330) protein that had higher content of multimeric forms provided better protection from fever and weight loss at a lower vaccine dose compared with HA (1-480).Protein yield for the HA1 (1-330) ranged around 40 mg/Liter, while the HA (1-480) yield was 0.4-0.8 mg/Liter.

View Article: PubMed Central - PubMed

Affiliation: Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America.

ABSTRACT

Background: In the face of impending influenza pandemic, a rapid vaccine production and mass vaccination is the most effective approach to prevent the large scale mortality and morbidity that was associated with the 1918 "Spanish Flu". The traditional process of influenza vaccine production in eggs is time consuming and may not meet the demands of rapid global vaccination required to curtail influenza pandemic.

Methodology/principal findings: Recombinant technology can be used to express the hemagglutinin (HA) of the emerging new influenza strain in a variety of systems including mammalian, insect, and bacterial cells. In this study, two forms of HA proteins derived from the currently circulating novel H1N1 A/California/07/2009 virus, HA1 (1-330) and HA (1-480), were expressed and purified from E. coli under controlled redox refolding conditions that favoured proper protein folding. However, only the recombinant HA1 (1-330) protein formed oligomers, including functional trimers that bound receptor and caused agglutination of human red blood cells. These proteins were used to vaccinate ferrets prior to challenge with the A/California/07/2009 virus. Both proteins induced neutralizing antibodies, and reduced viral loads in nasal washes. However, the HA1 (1-330) protein that had higher content of multimeric forms provided better protection from fever and weight loss at a lower vaccine dose compared with HA (1-480). Protein yield for the HA1 (1-330) ranged around 40 mg/Liter, while the HA (1-480) yield was 0.4-0.8 mg/Liter.

Conclusions/significance: This is the first study that describes production in bacterial system of properly folded functional globular HA1 domain trimers, lacking the HA2 transmembrane protein, that elicit potent neutralizing antibody responses following vaccination and protect ferrets from in vivo challenge. The combination of bacterial expression system with established quality control methods could provide a mechanism for rapid large scale production of influenza vaccines in the face of influenza pandemic threat.

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Related in: MedlinePlus

Biochemical and functional characterization of bacterially expressed and purified H1N1 HA proteins.(A) Purified E. coli derived HA proteins were analyzed by SDS-PAGE. DNA encoding HA1 (1–330) and HA (1–480) from HA gene segment of A/California/07/2009 (H1N1) generated from egg-grown virus were used for cloning in a T7 promoter based expression vector (pSK) where the desired polypeptide can be expressed as fusion protein with His6 tag at the C-terminus. The proteins were expressed, denatured and refolded under controlled redox conditions and purified using His-Trap fast flow chromatography to >90% purity (see Materials and Methods). The purified proteins run at their corresponding molecular weight in reducing SDS-PAGE. (B-C) CD melt spectroscopy shows that both H1N1 HA1 (1–330) (B) and H1N1 HA (1–480) (C) are properly folded. Both H1N1 HA proteins, at a concentration of 0.5 mg/ml in 20 mM PBS, pH 7.2, were subjected to heating at 0.5°C/min increments. The protein unfolding kinetics was measured at 222 nm using a J-715 Circular Dichroism system (JASCO corp., Easton, MD). (D-E) Superdex S-200 gel filtration chromatography of purified H1N1 HA proteins from E.coli. The panels present superimposed elution profiles of purified HA proteins (red line) overlaid with calibration standards (grey line). (D) The H1N1 HA (1–330) protein purified from bacterial cells existed as approximately 20% high-molecular-mass oligomer (>600 kDa), 45% trimer (∼110 kDa) and 35% monomer (34kDa) (red line). (E) H1N1 HA (1–480) is present only as a monomer (50kDa). (F-G) Binding kinetics of purified H1N1 HA proteins in a SPR based receptor binding assay. Steady-state equilibrium analysis of different H1N1-HA proteins to fetuin and its asialylated counterpart (Asialo-fetuin) was analyzed at 25°C using a ProteOn surface plasmon resonance biosensor (BioRad Labs). Samples of purified H1N1-HA proteins (10 µg/ml) were injected simultaneously over a mock surface to which no protein was bound, followed by the Asialofetuin in (F) or Fetuin in (G) immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of protein. Binding kinetics and data analysis were performed using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA). (H) Agglutination of human RBCs by properly folded bacterial H1N1 HA (1–330) protein. Serial dilutions of purified HA proteins or virus were mixed with washed RBC and incubated to analyze the receptor binding and cross-linking of human RBC. Virus H1N1xPR8 A/California/07/2009 (X-179A) was used as a control. Strong hemagglutination was observed for bacterial H1N1 HA (1–330) but not with either bacterial H1N1 HA (1–480) or mammalian H1N1 HA0.
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pone-0011548-g001: Biochemical and functional characterization of bacterially expressed and purified H1N1 HA proteins.(A) Purified E. coli derived HA proteins were analyzed by SDS-PAGE. DNA encoding HA1 (1–330) and HA (1–480) from HA gene segment of A/California/07/2009 (H1N1) generated from egg-grown virus were used for cloning in a T7 promoter based expression vector (pSK) where the desired polypeptide can be expressed as fusion protein with His6 tag at the C-terminus. The proteins were expressed, denatured and refolded under controlled redox conditions and purified using His-Trap fast flow chromatography to >90% purity (see Materials and Methods). The purified proteins run at their corresponding molecular weight in reducing SDS-PAGE. (B-C) CD melt spectroscopy shows that both H1N1 HA1 (1–330) (B) and H1N1 HA (1–480) (C) are properly folded. Both H1N1 HA proteins, at a concentration of 0.5 mg/ml in 20 mM PBS, pH 7.2, were subjected to heating at 0.5°C/min increments. The protein unfolding kinetics was measured at 222 nm using a J-715 Circular Dichroism system (JASCO corp., Easton, MD). (D-E) Superdex S-200 gel filtration chromatography of purified H1N1 HA proteins from E.coli. The panels present superimposed elution profiles of purified HA proteins (red line) overlaid with calibration standards (grey line). (D) The H1N1 HA (1–330) protein purified from bacterial cells existed as approximately 20% high-molecular-mass oligomer (>600 kDa), 45% trimer (∼110 kDa) and 35% monomer (34kDa) (red line). (E) H1N1 HA (1–480) is present only as a monomer (50kDa). (F-G) Binding kinetics of purified H1N1 HA proteins in a SPR based receptor binding assay. Steady-state equilibrium analysis of different H1N1-HA proteins to fetuin and its asialylated counterpart (Asialo-fetuin) was analyzed at 25°C using a ProteOn surface plasmon resonance biosensor (BioRad Labs). Samples of purified H1N1-HA proteins (10 µg/ml) were injected simultaneously over a mock surface to which no protein was bound, followed by the Asialofetuin in (F) or Fetuin in (G) immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of protein. Binding kinetics and data analysis were performed using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA). (H) Agglutination of human RBCs by properly folded bacterial H1N1 HA (1–330) protein. Serial dilutions of purified HA proteins or virus were mixed with washed RBC and incubated to analyze the receptor binding and cross-linking of human RBC. Virus H1N1xPR8 A/California/07/2009 (X-179A) was used as a control. Strong hemagglutination was observed for bacterial H1N1 HA (1–330) but not with either bacterial H1N1 HA (1–480) or mammalian H1N1 HA0.

Mentions: DNA fragments encoding amino acid sequence 1–330 and 1–480 of HA from A/California/07/2009 were cloned as NotI-PacI inserts in the T7 promoter based expression vector with His6 tag at the C-terminus [13]. Both fragments of H1N1 HA expressed in E. coli Rosetta Gami cells (Novagen) localized to insoluble fraction (inclusion bodies). IBs were refolded in vitro under controlled redox conditions and purified by HisTrap Fast flow chromatography. This process was previously shown to generate highly purified properly folded HA1 fragments from H5N1 [13]. The purified HA1 (1–330) and HA (1–480) proteins ran as a single band on SDS-PAGE with the anticipated MW of approximately 30 and 50 kDa, respectively (Fig. 1A).


Properly folded bacterially expressed H1N1 hemagglutinin globular head and ectodomain vaccines protect ferrets against H1N1 pandemic influenza virus.

Khurana S, Verma S, Verma N, Crevar CJ, Carter DM, Manischewitz J, King LR, Ross TM, Golding H - PLoS ONE (2010)

Biochemical and functional characterization of bacterially expressed and purified H1N1 HA proteins.(A) Purified E. coli derived HA proteins were analyzed by SDS-PAGE. DNA encoding HA1 (1–330) and HA (1–480) from HA gene segment of A/California/07/2009 (H1N1) generated from egg-grown virus were used for cloning in a T7 promoter based expression vector (pSK) where the desired polypeptide can be expressed as fusion protein with His6 tag at the C-terminus. The proteins were expressed, denatured and refolded under controlled redox conditions and purified using His-Trap fast flow chromatography to >90% purity (see Materials and Methods). The purified proteins run at their corresponding molecular weight in reducing SDS-PAGE. (B-C) CD melt spectroscopy shows that both H1N1 HA1 (1–330) (B) and H1N1 HA (1–480) (C) are properly folded. Both H1N1 HA proteins, at a concentration of 0.5 mg/ml in 20 mM PBS, pH 7.2, were subjected to heating at 0.5°C/min increments. The protein unfolding kinetics was measured at 222 nm using a J-715 Circular Dichroism system (JASCO corp., Easton, MD). (D-E) Superdex S-200 gel filtration chromatography of purified H1N1 HA proteins from E.coli. The panels present superimposed elution profiles of purified HA proteins (red line) overlaid with calibration standards (grey line). (D) The H1N1 HA (1–330) protein purified from bacterial cells existed as approximately 20% high-molecular-mass oligomer (>600 kDa), 45% trimer (∼110 kDa) and 35% monomer (34kDa) (red line). (E) H1N1 HA (1–480) is present only as a monomer (50kDa). (F-G) Binding kinetics of purified H1N1 HA proteins in a SPR based receptor binding assay. Steady-state equilibrium analysis of different H1N1-HA proteins to fetuin and its asialylated counterpart (Asialo-fetuin) was analyzed at 25°C using a ProteOn surface plasmon resonance biosensor (BioRad Labs). Samples of purified H1N1-HA proteins (10 µg/ml) were injected simultaneously over a mock surface to which no protein was bound, followed by the Asialofetuin in (F) or Fetuin in (G) immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of protein. Binding kinetics and data analysis were performed using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA). (H) Agglutination of human RBCs by properly folded bacterial H1N1 HA (1–330) protein. Serial dilutions of purified HA proteins or virus were mixed with washed RBC and incubated to analyze the receptor binding and cross-linking of human RBC. Virus H1N1xPR8 A/California/07/2009 (X-179A) was used as a control. Strong hemagglutination was observed for bacterial H1N1 HA (1–330) but not with either bacterial H1N1 HA (1–480) or mammalian H1N1 HA0.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2902520&req=5

pone-0011548-g001: Biochemical and functional characterization of bacterially expressed and purified H1N1 HA proteins.(A) Purified E. coli derived HA proteins were analyzed by SDS-PAGE. DNA encoding HA1 (1–330) and HA (1–480) from HA gene segment of A/California/07/2009 (H1N1) generated from egg-grown virus were used for cloning in a T7 promoter based expression vector (pSK) where the desired polypeptide can be expressed as fusion protein with His6 tag at the C-terminus. The proteins were expressed, denatured and refolded under controlled redox conditions and purified using His-Trap fast flow chromatography to >90% purity (see Materials and Methods). The purified proteins run at their corresponding molecular weight in reducing SDS-PAGE. (B-C) CD melt spectroscopy shows that both H1N1 HA1 (1–330) (B) and H1N1 HA (1–480) (C) are properly folded. Both H1N1 HA proteins, at a concentration of 0.5 mg/ml in 20 mM PBS, pH 7.2, were subjected to heating at 0.5°C/min increments. The protein unfolding kinetics was measured at 222 nm using a J-715 Circular Dichroism system (JASCO corp., Easton, MD). (D-E) Superdex S-200 gel filtration chromatography of purified H1N1 HA proteins from E.coli. The panels present superimposed elution profiles of purified HA proteins (red line) overlaid with calibration standards (grey line). (D) The H1N1 HA (1–330) protein purified from bacterial cells existed as approximately 20% high-molecular-mass oligomer (>600 kDa), 45% trimer (∼110 kDa) and 35% monomer (34kDa) (red line). (E) H1N1 HA (1–480) is present only as a monomer (50kDa). (F-G) Binding kinetics of purified H1N1 HA proteins in a SPR based receptor binding assay. Steady-state equilibrium analysis of different H1N1-HA proteins to fetuin and its asialylated counterpart (Asialo-fetuin) was analyzed at 25°C using a ProteOn surface plasmon resonance biosensor (BioRad Labs). Samples of purified H1N1-HA proteins (10 µg/ml) were injected simultaneously over a mock surface to which no protein was bound, followed by the Asialofetuin in (F) or Fetuin in (G) immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of protein. Binding kinetics and data analysis were performed using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA). (H) Agglutination of human RBCs by properly folded bacterial H1N1 HA (1–330) protein. Serial dilutions of purified HA proteins or virus were mixed with washed RBC and incubated to analyze the receptor binding and cross-linking of human RBC. Virus H1N1xPR8 A/California/07/2009 (X-179A) was used as a control. Strong hemagglutination was observed for bacterial H1N1 HA (1–330) but not with either bacterial H1N1 HA (1–480) or mammalian H1N1 HA0.
Mentions: DNA fragments encoding amino acid sequence 1–330 and 1–480 of HA from A/California/07/2009 were cloned as NotI-PacI inserts in the T7 promoter based expression vector with His6 tag at the C-terminus [13]. Both fragments of H1N1 HA expressed in E. coli Rosetta Gami cells (Novagen) localized to insoluble fraction (inclusion bodies). IBs were refolded in vitro under controlled redox conditions and purified by HisTrap Fast flow chromatography. This process was previously shown to generate highly purified properly folded HA1 fragments from H5N1 [13]. The purified HA1 (1–330) and HA (1–480) proteins ran as a single band on SDS-PAGE with the anticipated MW of approximately 30 and 50 kDa, respectively (Fig. 1A).

Bottom Line: Both proteins induced neutralizing antibodies, and reduced viral loads in nasal washes.However, the HA1 (1-330) protein that had higher content of multimeric forms provided better protection from fever and weight loss at a lower vaccine dose compared with HA (1-480).Protein yield for the HA1 (1-330) ranged around 40 mg/Liter, while the HA (1-480) yield was 0.4-0.8 mg/Liter.

View Article: PubMed Central - PubMed

Affiliation: Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America.

ABSTRACT

Background: In the face of impending influenza pandemic, a rapid vaccine production and mass vaccination is the most effective approach to prevent the large scale mortality and morbidity that was associated with the 1918 "Spanish Flu". The traditional process of influenza vaccine production in eggs is time consuming and may not meet the demands of rapid global vaccination required to curtail influenza pandemic.

Methodology/principal findings: Recombinant technology can be used to express the hemagglutinin (HA) of the emerging new influenza strain in a variety of systems including mammalian, insect, and bacterial cells. In this study, two forms of HA proteins derived from the currently circulating novel H1N1 A/California/07/2009 virus, HA1 (1-330) and HA (1-480), were expressed and purified from E. coli under controlled redox refolding conditions that favoured proper protein folding. However, only the recombinant HA1 (1-330) protein formed oligomers, including functional trimers that bound receptor and caused agglutination of human red blood cells. These proteins were used to vaccinate ferrets prior to challenge with the A/California/07/2009 virus. Both proteins induced neutralizing antibodies, and reduced viral loads in nasal washes. However, the HA1 (1-330) protein that had higher content of multimeric forms provided better protection from fever and weight loss at a lower vaccine dose compared with HA (1-480). Protein yield for the HA1 (1-330) ranged around 40 mg/Liter, while the HA (1-480) yield was 0.4-0.8 mg/Liter.

Conclusions/significance: This is the first study that describes production in bacterial system of properly folded functional globular HA1 domain trimers, lacking the HA2 transmembrane protein, that elicit potent neutralizing antibody responses following vaccination and protect ferrets from in vivo challenge. The combination of bacterial expression system with established quality control methods could provide a mechanism for rapid large scale production of influenza vaccines in the face of influenza pandemic threat.

Show MeSH
Related in: MedlinePlus