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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

Characterization of purified HA proteins from 293 Flp-In cell.(A) Proper protein folding as demonstrated by steady-state binding equilibrium analysis of conformational dependent human H5N1 neutralizing MAb FLA5.10 (10 µg/ml) to purified Flp-In expressed H5N1 HA1 proteins immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of peptide. Purified mammalian cell derived H5N1 HA1 or the HA0 proteins obtained from Immune Technology Corp were also analyzed. Binding was recorded using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA). (B–C) Analysis of purified H5N1 protein from Flp-In cells in SDS-PAGE under reducing conditions (B) and non-reducing conditions (C). Purified HA protein from Flp-In cell has higher order protein structure as analyzed by coomassie staining of the reducing SDS PAGE (B) and by coomassie stained non-reducing SDS PAGE (C). Subunit H5N1 vaccine (Sanofi Pasteur) was run as comparator. Western blot analysis of non-reducing SDS PAG using an anti-H5N1 HA1 antibody confirmed the identity of bands observed in coomassie stained gel in Fig. 1C.
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pone-0017297-g002: Characterization of purified HA proteins from 293 Flp-In cell.(A) Proper protein folding as demonstrated by steady-state binding equilibrium analysis of conformational dependent human H5N1 neutralizing MAb FLA5.10 (10 µg/ml) to purified Flp-In expressed H5N1 HA1 proteins immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of peptide. Purified mammalian cell derived H5N1 HA1 or the HA0 proteins obtained from Immune Technology Corp were also analyzed. Binding was recorded using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA). (B–C) Analysis of purified H5N1 protein from Flp-In cells in SDS-PAGE under reducing conditions (B) and non-reducing conditions (C). Purified HA protein from Flp-In cell has higher order protein structure as analyzed by coomassie staining of the reducing SDS PAGE (B) and by coomassie stained non-reducing SDS PAGE (C). Subunit H5N1 vaccine (Sanofi Pasteur) was run as comparator. Western blot analysis of non-reducing SDS PAG using an anti-H5N1 HA1 antibody confirmed the identity of bands observed in coomassie stained gel in Fig. 1C.

Mentions: Proper protein folding is critical for preservation of HA antigenicity and immunogenicity. Since the majority of influenza neutralizing antibodies recognizes conformational epitopes, we used a panel of human H5N1 neutralizing antibodies generated from B cells of H5N1 A/Vietnam/1203/2004 recovered individuals, that recognized conformational dependent epitopes in HA1 domain of H5N1 A/Vietnam virus [12], [13]. Steady-state binding equilibrium analysis with conformation-dependent human H5N1 neutralizing MAb demonstrated that 293 Flp-In secreted H5N1 HA1 and HA0 proteins were properly folded (Fig. 2A). The licensed inactivated subunit H5N1 vaccine (Sanofi Pasteur) was used as positive controls (Fig. 2A). Similar binding was measured with additional two human MAbs (data not shown).


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)

Characterization of purified HA proteins from 293 Flp-In cell.(A) Proper protein folding as demonstrated by steady-state binding equilibrium analysis of conformational dependent human H5N1 neutralizing MAb FLA5.10 (10 µg/ml) to purified Flp-In expressed H5N1 HA1 proteins immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of peptide. Purified mammalian cell derived H5N1 HA1 or the HA0 proteins obtained from Immune Technology Corp were also analyzed. Binding was recorded using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA). (B–C) Analysis of purified H5N1 protein from Flp-In cells in SDS-PAGE under reducing conditions (B) and non-reducing conditions (C). Purified HA protein from Flp-In cell has higher order protein structure as analyzed by coomassie staining of the reducing SDS PAGE (B) and by coomassie stained non-reducing SDS PAGE (C). Subunit H5N1 vaccine (Sanofi Pasteur) was run as comparator. Western blot analysis of non-reducing SDS PAG using an anti-H5N1 HA1 antibody confirmed the identity of bands observed in coomassie stained gel in Fig. 1C.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017297-g002: Characterization of purified HA proteins from 293 Flp-In cell.(A) Proper protein folding as demonstrated by steady-state binding equilibrium analysis of conformational dependent human H5N1 neutralizing MAb FLA5.10 (10 µg/ml) to purified Flp-In expressed H5N1 HA1 proteins immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of peptide. Purified mammalian cell derived H5N1 HA1 or the HA0 proteins obtained from Immune Technology Corp were also analyzed. Binding was recorded using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA). (B–C) Analysis of purified H5N1 protein from Flp-In cells in SDS-PAGE under reducing conditions (B) and non-reducing conditions (C). Purified HA protein from Flp-In cell has higher order protein structure as analyzed by coomassie staining of the reducing SDS PAGE (B) and by coomassie stained non-reducing SDS PAGE (C). Subunit H5N1 vaccine (Sanofi Pasteur) was run as comparator. Western blot analysis of non-reducing SDS PAG using an anti-H5N1 HA1 antibody confirmed the identity of bands observed in coomassie stained gel in Fig. 1C.
Mentions: Proper protein folding is critical for preservation of HA antigenicity and immunogenicity. Since the majority of influenza neutralizing antibodies recognizes conformational epitopes, we used a panel of human H5N1 neutralizing antibodies generated from B cells of H5N1 A/Vietnam/1203/2004 recovered individuals, that recognized conformational dependent epitopes in HA1 domain of H5N1 A/Vietnam virus [12], [13]. Steady-state binding equilibrium analysis with conformation-dependent human H5N1 neutralizing MAb demonstrated that 293 Flp-In secreted H5N1 HA1 and HA0 proteins were properly folded (Fig. 2A). The licensed inactivated subunit H5N1 vaccine (Sanofi Pasteur) was used as positive controls (Fig. 2A). Similar binding was measured with additional two human MAbs (data not shown).

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