Limits...
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.

Show MeSH

Related in: MedlinePlus

Construction of expression vector for constitutively H5N1 HA secretion in Flp-In based mammalian expression system.(A) To enhance HA protein expression from the FRT-CMV vector (Invitrogen), an RNA splicing sequence from HTLV-I gene and a cassette containing NotI and PacI cloning sites was introduced after CMV promoter using Topo cloning system. (B) Schematic of HA1 (1-330) & HA0 (1-500) V5-His6 tagged fusion proteins expressed in Flp-In system. The H5N1/Vietnam HA sequence coding for either HA1 (1-330) or HA0 (1-500) was inserted into the vector as a NotI-PacI insert. (C–D) Expression and purification of H5N1 A/Vietnam/1203/2004 hemagglutinin proteins from 293 Flp-In stable cell lines. (C) Expression of HA proteins secreted into supernatant from 293 Flp-In cells [S], and in the cell lysates [C] was analyzed by western blot using anti-V5 MAb. (D) HA protein level from supernatant of 293 Flp-In cell culture in serum free medium, collected at different time points: 24, 48, 72 and 96 hours post culture splitting and was analyzed in SDS PAGE followed by western blot with anti-HA1 polyclonal sera.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3046144&req=5

pone-0017297-g001: Construction of expression vector for constitutively H5N1 HA secretion in Flp-In based mammalian expression system.(A) To enhance HA protein expression from the FRT-CMV vector (Invitrogen), an RNA splicing sequence from HTLV-I gene and a cassette containing NotI and PacI cloning sites was introduced after CMV promoter using Topo cloning system. (B) Schematic of HA1 (1-330) & HA0 (1-500) V5-His6 tagged fusion proteins expressed in Flp-In system. The H5N1/Vietnam HA sequence coding for either HA1 (1-330) or HA0 (1-500) was inserted into the vector as a NotI-PacI insert. (C–D) Expression and purification of H5N1 A/Vietnam/1203/2004 hemagglutinin proteins from 293 Flp-In stable cell lines. (C) Expression of HA proteins secreted into supernatant from 293 Flp-In cells [S], and in the cell lysates [C] was analyzed by western blot using anti-V5 MAb. (D) HA protein level from supernatant of 293 Flp-In cell culture in serum free medium, collected at different time points: 24, 48, 72 and 96 hours post culture splitting and was analyzed in SDS PAGE followed by western blot with anti-HA1 polyclonal sera.

Mentions: To enhance HA protein expression and protein secretion from the FRT-CMV vector, a RNA splicing sequence from HTLV-I gene and a cassette containing NotI and PacI cloning sites was introduced after the CMV promoter using Topo cloning system, followed by a secretory signal peptide from IgG kappa chain (Fig. 1A). The H5N1 A/Vietnam HA sequences coding for either HA1 (1-330) or HA0 (1-500) were inserted into the vector as NotI-PacI inserts. In the case of HA0, the polybasic cleavage site between HA1 and HA2 was not removed or modified, to ensure proper cleavage and folding of the secreted HA0 protein (Fig. 1B). Stable cell clones were selected by growing in DMEM with 150 µg/ml of hygromycin for 10–14 days followed by expansion of individual clones. The selected stable integrants were 100% homogeneous, an important attribute of the Flp-in system. All 40 of selected single–cell clones expressed HA protein at similar levels. For HA protein production, individual cell clones expressing either HA1 globular domain (1-330) or HA0 (1-500) were expanded in T175 flask in serum-free medium for 24 hours. Expression of HA proteins from 293 Flp-In cell lysates (C) and secreted proteins in supernatants (S) were resolved on SDS-PAGE under reducing conditions and detected by Western blot using anti-V5 MAb (Fig. 1C). As expected, under reducing conditions, a significant portion of the cleaved HA0 was separated into HA1 and HA2, only the later reacted with the anti-V5 tag (in the C-terminus of HA2) (Fig. 1C lanes 3, 4). Supernatants were collected at 24, 48, 72 and 96 hours post culture splitting and were analyzed in SDS PAGE followed by western blot with anti-HA1 polyclonal sera. In both HA1 and HA0 expressing cells, the highest level of HA secretion was observed at 24 and 48 hours following cell splitting (Fig. 1D).


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)

Construction of expression vector for constitutively H5N1 HA secretion in Flp-In based mammalian expression system.(A) To enhance HA protein expression from the FRT-CMV vector (Invitrogen), an RNA splicing sequence from HTLV-I gene and a cassette containing NotI and PacI cloning sites was introduced after CMV promoter using Topo cloning system. (B) Schematic of HA1 (1-330) & HA0 (1-500) V5-His6 tagged fusion proteins expressed in Flp-In system. The H5N1/Vietnam HA sequence coding for either HA1 (1-330) or HA0 (1-500) was inserted into the vector as a NotI-PacI insert. (C–D) Expression and purification of H5N1 A/Vietnam/1203/2004 hemagglutinin proteins from 293 Flp-In stable cell lines. (C) Expression of HA proteins secreted into supernatant from 293 Flp-In cells [S], and in the cell lysates [C] was analyzed by western blot using anti-V5 MAb. (D) HA protein level from supernatant of 293 Flp-In cell culture in serum free medium, collected at different time points: 24, 48, 72 and 96 hours post culture splitting and was analyzed in SDS PAGE followed by western blot with anti-HA1 polyclonal sera.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017297-g001: Construction of expression vector for constitutively H5N1 HA secretion in Flp-In based mammalian expression system.(A) To enhance HA protein expression from the FRT-CMV vector (Invitrogen), an RNA splicing sequence from HTLV-I gene and a cassette containing NotI and PacI cloning sites was introduced after CMV promoter using Topo cloning system. (B) Schematic of HA1 (1-330) & HA0 (1-500) V5-His6 tagged fusion proteins expressed in Flp-In system. The H5N1/Vietnam HA sequence coding for either HA1 (1-330) or HA0 (1-500) was inserted into the vector as a NotI-PacI insert. (C–D) Expression and purification of H5N1 A/Vietnam/1203/2004 hemagglutinin proteins from 293 Flp-In stable cell lines. (C) Expression of HA proteins secreted into supernatant from 293 Flp-In cells [S], and in the cell lysates [C] was analyzed by western blot using anti-V5 MAb. (D) HA protein level from supernatant of 293 Flp-In cell culture in serum free medium, collected at different time points: 24, 48, 72 and 96 hours post culture splitting and was analyzed in SDS PAGE followed by western blot with anti-HA1 polyclonal sera.
Mentions: To enhance HA protein expression and protein secretion from the FRT-CMV vector, a RNA splicing sequence from HTLV-I gene and a cassette containing NotI and PacI cloning sites was introduced after the CMV promoter using Topo cloning system, followed by a secretory signal peptide from IgG kappa chain (Fig. 1A). The H5N1 A/Vietnam HA sequences coding for either HA1 (1-330) or HA0 (1-500) were inserted into the vector as NotI-PacI inserts. In the case of HA0, the polybasic cleavage site between HA1 and HA2 was not removed or modified, to ensure proper cleavage and folding of the secreted HA0 protein (Fig. 1B). Stable cell clones were selected by growing in DMEM with 150 µg/ml of hygromycin for 10–14 days followed by expansion of individual clones. The selected stable integrants were 100% homogeneous, an important attribute of the Flp-in system. All 40 of selected single–cell clones expressed HA protein at similar levels. For HA protein production, individual cell clones expressing either HA1 globular domain (1-330) or HA0 (1-500) were expanded in T175 flask in serum-free medium for 24 hours. Expression of HA proteins from 293 Flp-In cell lysates (C) and secreted proteins in supernatants (S) were resolved on SDS-PAGE under reducing conditions and detected by Western blot using anti-V5 MAb (Fig. 1C). As expected, under reducing conditions, a significant portion of the cleaved HA0 was separated into HA1 and HA2, only the later reacted with the anti-V5 tag (in the C-terminus of HA2) (Fig. 1C lanes 3, 4). Supernatants were collected at 24, 48, 72 and 96 hours post culture splitting and were analyzed in SDS PAGE followed by western blot with anti-HA1 polyclonal sera. In both HA1 and HA0 expressing cells, the highest level of HA secretion was observed at 24 and 48 hours following cell splitting (Fig. 1D).

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