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

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.

Autonomous functions for the Sec14p/spectrin-repeat region of Kalirin.

Kalirin is a GDP/GTP exchange factor (GEF) for Rho proteins that modulates the actin cytoskeleton in neurons. Alternative splicing generates Delta-isoforms, which encode the RhoGEF domain, but lack the N-terminal Sec14p domain and first 4 spectrin-like repeats of the full-length isoforms. Splicing has functional consequences, with Kal7 but not DeltaKal7 causing formation of dendritic spines. Cells lacking endogenous Kalirin were used to explore differences between these splice variants. Expression of DeltaKal7 in this system induces extensive lamellipodial sheets, while expression of Kal7 induces formation of adherent compact, round cells with abundant cortical actin. Based on in vitro and cell-based assays, Kal7 and DeltaKal7 are equally active GEFs, suggesting that other domains are involved in controlling cell morphology. Catalytically inactive Kal7 and a Kalirin fragment which includes only Sec14p and spectrin-like domains retain the ability to produce compact, round cells and fractionate as high molecular weight complexes. Separating the Sec14p domain from the spectrin-like repeats eliminates the ability of Kal7 to cause this response. The isolated Sec14p domain binds PI(3,5)P2 and PI3P, but does not alter cell morphology. We conclude that the Sec14p and N-terminal spectrin-like domains of Kalirin play critical roles in distinguishing the actions of full-length and Delta-Kalirin proteins.

Development of a novel recombinant influenza vaccine in insect cells.

Influenza is a highly contagious viral respiratory illness which is best prevented through vaccination. Currently, all US licensed influenza vaccines are produced in embryonated chicken eggs. The Baculovirus Expression Vector System (BEVS) technology offers several advantages over existing technology, including an exact match between the circulating virus and the antigen in the vaccine, speed, safety, versatility, and reliable scale-up. The expresSF+ insect cells are grown in the absence of serum and have been extensively qualified for safety according to ICH and US FDA guidance and for suitability for the production of recombinant proteins using BEVS. FluBlok, a recombinant hemagglutinin influenza vaccine, is composed of purified hemagglutinin protein produced using the BEVS technology. FluBlok has been shown to be safe, effective, and efficacious in human clinical studies.

Multiple novel isoforms of Trio are expressed in the developing rat brain.

Mammalian Trio is a multifunctional, multidomain Rho guanine nucleotide exchange factor (GEF) closely related to Kalirin. Trio is important for proper axon guidance in Drosophila, and mice lacking Trio exhibit both skeletal muscle and neuronal disorders. Full length mammalian Trio and Kalirin both consist of a Sec14P-like domain, several spectrin-like domains, two Rho GEF domains each containing a Dbl-homology (DH) and a pleckstrin-homology (PH) domain, two src homology 3 domains (SH3), Ig/fibronectin-like domains (Ig/FN), and a kinase domain. We have previously described multiple isoforms of Kalirin derived through alternative splicing and multiple transcription start sites, but multiple isoforms of Trio containing different functional domains have not been described. Using a new antibody directed against the spectrin-like region of rat Trio coupled with reverse transcription PCR and cDNA sequencing, we have identified 4 novel isoforms of Trio expressed in rat cortex and cerebellum. Two isoforms, Trio 9S and Trio 9L, are derived through alternative splicing of Trio exon 48 and are abundantly expressed in rat brain. Trio 8 is expressed in postnatal day 30 and adult cerebellum, but not in cortex or skeletal muscle. Trio/duet is expressed in adult cortex and cerebellum. In the rat brain, each of these Trio isoforms is expressed at a higher level than full length Trio.

Improved stability of recombinant hemagglutinin using a formulation containing sodium thioglycolate.

This study was designed to improve the stability of liquid formulations of recombinant influenza hemagglutinin (rHA) and to understand the mechanism of early loss of potency for rHA. The potency of rHA derived from several influenza strains was determined using single radial immunodiffusion (SRID), and the structure of the rHA was characterized using SDS-PAGE and dynamic light scattering. rHA formed disulfide cross-linked multimers, and potency decreased during extended storage. To reduce disulfide-mediated cross-linking and early potency loss, rHA was formulated with sodium thioglycolate (STG) and citrate. Addition of 80 mM STG and 55 mM sodium citrate inhibited disulfide-mediated cross-linking without affecting protein function for each rHA tested. The shelf life of the rHA formulation with STG-citrate, based on potency as determined by SRID, was extended as much as 20-fold, compared to a control formulation without STG-citrate. STG-citrate did not have a significant effect on the immunogenicity of H1 A/California/7/2009 rHA in mice.

Genomic organization and differential expression of Kalirin isoforms.

Multidomain guanine nucleotide (GDP/GTP) exchange factor (GEF) proteins coordinate diverse inputs that signal the actin cytoskeleton. Mammals have two such proteins (Kalirin, Trio), while Drosophila has one, which plays essential roles within and outside the nervous system. For Kalirin, numerous isoforms containing different combinations of functional domains are generated through alternative splicing and use of alternative transcriptional start sites. These different isoforms potentially allow a wide variety of proteins to interact with Kalirin, thereby affecting the activity of the functional domains. Humans, like rats, express a large set of Kalirin isoform mRNAs, and we identified a novel Kalirin isoform, containing only the second GEF domain. Kalirin isoforms are predominantly expressed in the brain, while Trio is expressed in a wider variety of tissues. Alternative splicing and transcription of Kalirin are differentially regulated during development in rats and humans, resulting in expression of isoforms of Kalirin containing different functional domains at different times and locations. The prevalence of Kalirin in the cortex throughout life suggests roles in axonal development and the mature brain.

Kalirin expression is regulated by multiple promoters.

Kalirin is a multidomain guanine nucleotide exchange factor for small GTPbinding proteins of the Rho family. It is expressed in multiple isoforms that contain different combinations of functional domains and display a complex pattern of expression during brain development. In addition to the isoforms generated through alternative splicing, we have identified multiple transcriptional start sites in rats and humans. These multiple transcriptional start sites result in full-length Kalirin transcripts possessing different 5' ends encoding proteins with differing amino termini. These alternative first exons display different patterns of expression in developing rats and humans and in cultured cells. Most of these alternate first exons lie >100 kb upstream of exon 2 in both rats and humans. Comparisons of the rat and human Kalirin promoter regions reveal numerous shared potential regulatory elements.

Mechanism of a decrease in potency for the recombinant influenza A virus hemagglutinin H3 antigen during storage.

The recombinant hemagglutinin (rHA)-based influenza vaccine Flublok® has recently been approved in the United States as an alternative to the traditional egg-derived flu vaccines. Flublok is a purified vaccine with a hemagglutinin content that is threefold higher than standard inactivated influenza vaccines. When rHA derived from an H3N2 influenza virus was expressed, purified, and stored for 1 month, a rapid loss of in vitro potency (∼50%) was observed as measured by the single radial immunodiffusion (SRID) assay. A comprehensive characterization of the rHA protein antigen was pursued to identify the potential causes and mechanisms of this potency loss. In addition, the biophysical and chemical stability of the rHA in different formulations and storage conditions was evaluated over time. Results demonstrate that the potency loss over time did not correlate with trends in changes to the higher order structure or hydrodynamic size of the rHA. The most likely mechanism for the early loss of potency was disulfide-mediated cross-linking of rHA, as the formation of non-native disulfide-linked multimers over time correlated well with the observed potency loss. Furthermore, a loss of free thiol content, particularly in specific cysteine residues in the antigen's C-terminus, was correlated with potency loss measured by SRID.