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Modifications of cysteine residues in the transmembrane and cytoplasmic domains of a recombinant hemagglutinin protein prevent cross-linked multimer formation and potency loss.

Holtz KM, Robinson PS, Matthews EE, Hashimoto Y, McPherson CE, Khramtsov N, Reifler MJ, Meghrous J, Rhodes DG, Cox MM, Srivastava IK - BMC Biotechnol. (2014)

Bottom Line: During purification and storage of the rHA, disulfide mediated cross-linking of the trimers within the rosette occurs and results in reduced potency.Modification of these cysteine residues prevents disulfide bond cross-linking in the TM and CT, and the resulting rHA maintains potency for at least 12 months at 25 °C.Substitution of carboxy terminal cysteines is an alternative to using reducing agents, and permits room temperature storage of the vaccine.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Recombinant hemagglutinin (rHA) is the active component in Flublok®; a trivalent influenza vaccine produced using the baculovirus expression vector system (BEVS). HA is a membrane bound homotrimer in the influenza virus envelope, and the purified rHA protein assembles into higher order rosette structures in the final formulation of the vaccine. During purification and storage of the rHA, disulfide mediated cross-linking of the trimers within the rosette occurs and results in reduced potency. Potency is measured by the Single Radial Immuno-diffusion (SRID) assay to determine the amount of HA that has the correct antigenic form.

Results: The five cysteine residues in the transmembrane (TM) and cytoplasmic (CT) domains of the rHA protein from the H3 A/Perth/16/2009 human influenza strain have been substituted to alanine and/or serine residues to produce three different site directed variants (SDVs). These SDVs have been evaluated to determine the impact of the TM and CT cysteines on potency, cross-linking, and the biochemical and biophysical properties of the rHA. Modification of these cysteine residues prevents disulfide bond cross-linking in the TM and CT, and the resulting rHA maintains potency for at least 12 months at 25 °C. The strategy of substituting TM and CT cysteines to prevent potency loss has been successfully applied to another H3 rHA protein (from the A/Texas/50/2012 influenza strain) further demonstrating the utility of the approach.

Conclusion: rHA potency can be maintained by preventing non-specific disulfide bonding and cross-linked multimer formation. Substitution of carboxy terminal cysteines is an alternative to using reducing agents, and permits room temperature storage of the vaccine.

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Related in: MedlinePlus

A and B SDS-PAGE purity and trypsin resistance gels for the H3 rHA proteins. A. Each rHA protein was loaded in duplicate to give 1 μg total protein per lane. The samples were separated using 4-12% gradient Nu-PAGE gels and stained with Coomassie Blue. Molecular weights of the proteins standards are shown. B. Shown are the reducing SDS-PAGE gels from the trypsin resistance assay. Each H3 rHA protein was analyzed neat (Lane 1), after trypsin treatment (Lane 2), after heat treatment (Lane 3), and after heat and trypsin treatment (Lane 4). The trypsin enzyme is loaded as a control and molecular weights of the protein standards are shown. HA1 and HA2 are indicated by arrows.
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Fig2: A and B SDS-PAGE purity and trypsin resistance gels for the H3 rHA proteins. A. Each rHA protein was loaded in duplicate to give 1 μg total protein per lane. The samples were separated using 4-12% gradient Nu-PAGE gels and stained with Coomassie Blue. Molecular weights of the proteins standards are shown. B. Shown are the reducing SDS-PAGE gels from the trypsin resistance assay. Each H3 rHA protein was analyzed neat (Lane 1), after trypsin treatment (Lane 2), after heat treatment (Lane 3), and after heat and trypsin treatment (Lane 4). The trypsin enzyme is loaded as a control and molecular weights of the protein standards are shown. HA1 and HA2 are indicated by arrows.

Mentions: Purity of rHA was determined by reducing SDS-PAGE. The full length rHA proteins migrates as monomers (HA0) having an approximate molecular weight of 62 kDa (Figure 2A) under denaturing and reducing conditions. The purity of the rHA proteins exceeds 99%.Figure 2


Modifications of cysteine residues in the transmembrane and cytoplasmic domains of a recombinant hemagglutinin protein prevent cross-linked multimer formation and potency loss.

Holtz KM, Robinson PS, Matthews EE, Hashimoto Y, McPherson CE, Khramtsov N, Reifler MJ, Meghrous J, Rhodes DG, Cox MM, Srivastava IK - BMC Biotechnol. (2014)

A and B SDS-PAGE purity and trypsin resistance gels for the H3 rHA proteins. A. Each rHA protein was loaded in duplicate to give 1 μg total protein per lane. The samples were separated using 4-12% gradient Nu-PAGE gels and stained with Coomassie Blue. Molecular weights of the proteins standards are shown. B. Shown are the reducing SDS-PAGE gels from the trypsin resistance assay. Each H3 rHA protein was analyzed neat (Lane 1), after trypsin treatment (Lane 2), after heat treatment (Lane 3), and after heat and trypsin treatment (Lane 4). The trypsin enzyme is loaded as a control and molecular weights of the protein standards are shown. HA1 and HA2 are indicated by arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4320835&req=5

Fig2: A and B SDS-PAGE purity and trypsin resistance gels for the H3 rHA proteins. A. Each rHA protein was loaded in duplicate to give 1 μg total protein per lane. The samples were separated using 4-12% gradient Nu-PAGE gels and stained with Coomassie Blue. Molecular weights of the proteins standards are shown. B. Shown are the reducing SDS-PAGE gels from the trypsin resistance assay. Each H3 rHA protein was analyzed neat (Lane 1), after trypsin treatment (Lane 2), after heat treatment (Lane 3), and after heat and trypsin treatment (Lane 4). The trypsin enzyme is loaded as a control and molecular weights of the protein standards are shown. HA1 and HA2 are indicated by arrows.
Mentions: Purity of rHA was determined by reducing SDS-PAGE. The full length rHA proteins migrates as monomers (HA0) having an approximate molecular weight of 62 kDa (Figure 2A) under denaturing and reducing conditions. The purity of the rHA proteins exceeds 99%.Figure 2

Bottom Line: During purification and storage of the rHA, disulfide mediated cross-linking of the trimers within the rosette occurs and results in reduced potency.Modification of these cysteine residues prevents disulfide bond cross-linking in the TM and CT, and the resulting rHA maintains potency for at least 12 months at 25 °C.Substitution of carboxy terminal cysteines is an alternative to using reducing agents, and permits room temperature storage of the vaccine.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Recombinant hemagglutinin (rHA) is the active component in Flublok®; a trivalent influenza vaccine produced using the baculovirus expression vector system (BEVS). HA is a membrane bound homotrimer in the influenza virus envelope, and the purified rHA protein assembles into higher order rosette structures in the final formulation of the vaccine. During purification and storage of the rHA, disulfide mediated cross-linking of the trimers within the rosette occurs and results in reduced potency. Potency is measured by the Single Radial Immuno-diffusion (SRID) assay to determine the amount of HA that has the correct antigenic form.

Results: The five cysteine residues in the transmembrane (TM) and cytoplasmic (CT) domains of the rHA protein from the H3 A/Perth/16/2009 human influenza strain have been substituted to alanine and/or serine residues to produce three different site directed variants (SDVs). These SDVs have been evaluated to determine the impact of the TM and CT cysteines on potency, cross-linking, and the biochemical and biophysical properties of the rHA. Modification of these cysteine residues prevents disulfide bond cross-linking in the TM and CT, and the resulting rHA maintains potency for at least 12 months at 25 °C. The strategy of substituting TM and CT cysteines to prevent potency loss has been successfully applied to another H3 rHA protein (from the A/Texas/50/2012 influenza strain) further demonstrating the utility of the approach.

Conclusion: rHA potency can be maintained by preventing non-specific disulfide bonding and cross-linked multimer formation. Substitution of carboxy terminal cysteines is an alternative to using reducing agents, and permits room temperature storage of the vaccine.

Show MeSH
Related in: MedlinePlus