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A rapid Flp-In system for expression of secreted H5N1 influenza hemagglutinin vaccine immunogen in mammalian cells.

Lu H, Khurana S, Verma N, Manischewitz J, King L, Beigel JH, Golding H - PLoS ONE (2011)

Bottom Line: Both proteins were properly folded as confirmed by binding to H5N1-neutralizing conformation-dependent human monoclonal antibodies.The HA0 (with unmodified cleavage site) was monomeric, while the HA1 contained oligomeric forms.Our data suggest that the 293 Flp-In system could serve as a platform for rapid expression of HA immunogens in mammalian cells from emerging influenza strains.

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: Continuing transmissions of highly pathogenic H5N1 viruses in poultry and humans underscores the need for a rapid response to potential pandemic in the form of vaccine. Recombinant technologies for production of immunogenic hemagglutinin (HA) could provide an advantage over the traditional inactivated vaccine manufacturing process. Generation of stably transfected mammalian cells secreting properly folded HA proteins is important for scalable controlled manufacturing.

Methodology/principal findings: We have developed a Flp-In based 293 stable cell lines through targeted site-specific recombination for expression of secreted hemagglutinin (HA) proteins and evaluated their immunogenicity. H5N1 globular domain HA1(1-330) and HA0(1-500) proteins were purified from the supernatants of 293 Flp-In stable cell lines. Both proteins were properly folded as confirmed by binding to H5N1-neutralizing conformation-dependent human monoclonal antibodies. The HA0 (with unmodified cleavage site) was monomeric, while the HA1 contained oligomeric forms. Upon rabbit immunization, both HA proteins elicited neutralizing antibodies against the homologous virus (A/Vietnam/1203/2004, clade 1) as well as cross-neutralizing antibodies against heterologous H5N1 clade 2 strains, including A/Indonesia/5/2005. These results exceeded the human antibody responses against the inactivated sub-virion H5N1 vaccine.

Conclusions/significance: Our data suggest that the 293 Flp-In system could serve as a platform for rapid expression of HA immunogens in mammalian cells from emerging influenza strains.

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Related in: MedlinePlus

Analysis of HA protein by gel filtration chromatography.Superdex S-200 gel filtration chromatography of Flp-In derived H5N1 HA proteins. Purified H5N1 HA1 (1-330) protein (A) or the HA0 (1-500) protein (B) were subjected to gel filtration. The panels present superimposed elution profiles of purified HA proteins (blue line) overlaid with calibration standards (grey line). The elution volumes of protein species are shown in parenthesis. While purified HA 1-330 protein was presented in monomer, trimer and oligomer form (A), the HA1-500 protein was observed primarily in a monomeric form (B). Gel filtration profile of Subunit H5N1 vaccine (Sanofi Pasteur) (C) show predominance of oligomers.
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pone-0017297-g003: Analysis of HA protein by gel filtration chromatography.Superdex S-200 gel filtration chromatography of Flp-In derived H5N1 HA proteins. Purified H5N1 HA1 (1-330) protein (A) or the HA0 (1-500) protein (B) were subjected to gel filtration. The panels present superimposed elution profiles of purified HA proteins (blue line) overlaid with calibration standards (grey line). The elution volumes of protein species are shown in parenthesis. While purified HA 1-330 protein was presented in monomer, trimer and oligomer form (A), the HA1-500 protein was observed primarily in a monomeric form (B). Gel filtration profile of Subunit H5N1 vaccine (Sanofi Pasteur) (C) show predominance of oligomers.

Mentions: To better decipher the quaternary forms in the Flp-In derived HA1 and HA0, we subjected the purified HA proteins to Superdex S-200 gel filtration chromatography. While the HA0 (1-500) protein was predominantly monomeric (Fig. 3B), the HA1 (1-330) protein contained monomers and higher MW oligomers (Fig. 3A). In comparison, the subunit H5N1 HA vaccine contained predominantly oligomers (Fig. 3C).


A rapid Flp-In system for expression of secreted H5N1 influenza hemagglutinin vaccine immunogen in mammalian cells.

Lu H, Khurana S, Verma N, Manischewitz J, King L, Beigel JH, Golding H - PLoS ONE (2011)

Analysis of HA protein by gel filtration chromatography.Superdex S-200 gel filtration chromatography of Flp-In derived H5N1 HA proteins. Purified H5N1 HA1 (1-330) protein (A) or the HA0 (1-500) protein (B) were subjected to gel filtration. The panels present superimposed elution profiles of purified HA proteins (blue line) overlaid with calibration standards (grey line). The elution volumes of protein species are shown in parenthesis. While purified HA 1-330 protein was presented in monomer, trimer and oligomer form (A), the HA1-500 protein was observed primarily in a monomeric form (B). Gel filtration profile of Subunit H5N1 vaccine (Sanofi Pasteur) (C) show predominance of oligomers.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046144&req=5

pone-0017297-g003: Analysis of HA protein by gel filtration chromatography.Superdex S-200 gel filtration chromatography of Flp-In derived H5N1 HA proteins. Purified H5N1 HA1 (1-330) protein (A) or the HA0 (1-500) protein (B) were subjected to gel filtration. The panels present superimposed elution profiles of purified HA proteins (blue line) overlaid with calibration standards (grey line). The elution volumes of protein species are shown in parenthesis. While purified HA 1-330 protein was presented in monomer, trimer and oligomer form (A), the HA1-500 protein was observed primarily in a monomeric form (B). Gel filtration profile of Subunit H5N1 vaccine (Sanofi Pasteur) (C) show predominance of oligomers.
Mentions: To better decipher the quaternary forms in the Flp-In derived HA1 and HA0, we subjected the purified HA proteins to Superdex S-200 gel filtration chromatography. While the HA0 (1-500) protein was predominantly monomeric (Fig. 3B), the HA1 (1-330) protein contained monomers and higher MW oligomers (Fig. 3A). In comparison, the subunit H5N1 HA vaccine contained predominantly oligomers (Fig. 3C).

Bottom Line: Both proteins were properly folded as confirmed by binding to H5N1-neutralizing conformation-dependent human monoclonal antibodies.The HA0 (with unmodified cleavage site) was monomeric, while the HA1 contained oligomeric forms.Our data suggest that the 293 Flp-In system could serve as a platform for rapid expression of HA immunogens in mammalian cells from emerging influenza strains.

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: Continuing transmissions of highly pathogenic H5N1 viruses in poultry and humans underscores the need for a rapid response to potential pandemic in the form of vaccine. Recombinant technologies for production of immunogenic hemagglutinin (HA) could provide an advantage over the traditional inactivated vaccine manufacturing process. Generation of stably transfected mammalian cells secreting properly folded HA proteins is important for scalable controlled manufacturing.

Methodology/principal findings: We have developed a Flp-In based 293 stable cell lines through targeted site-specific recombination for expression of secreted hemagglutinin (HA) proteins and evaluated their immunogenicity. H5N1 globular domain HA1(1-330) and HA0(1-500) proteins were purified from the supernatants of 293 Flp-In stable cell lines. Both proteins were properly folded as confirmed by binding to H5N1-neutralizing conformation-dependent human monoclonal antibodies. The HA0 (with unmodified cleavage site) was monomeric, while the HA1 contained oligomeric forms. Upon rabbit immunization, both HA proteins elicited neutralizing antibodies against the homologous virus (A/Vietnam/1203/2004, clade 1) as well as cross-neutralizing antibodies against heterologous H5N1 clade 2 strains, including A/Indonesia/5/2005. These results exceeded the human antibody responses against the inactivated sub-virion H5N1 vaccine.

Conclusions/significance: Our data suggest that the 293 Flp-In system could serve as a platform for rapid expression of HA immunogens in mammalian cells from emerging influenza strains.

Show MeSH
Related in: MedlinePlus