BACKGROUND
licensed, seasonal influenza vaccines available in the united states include trivalent and quadrivalent inactivated vaccines and a live attenuated influenza vaccine produced in embryonated chicken eggs  <cit> , a cell culture based trivalent vaccine produced in madin darby canine kidney  cells  <cit> , and most recently a recombinant trivalent vaccine  produced using the baculovirus-insect cell system  <cit> .
flublok vaccine has several distinct advantages over other flu vaccines including high purity of the ha protein, and absence of antibiotics, preservatives, gelatin, and egg proteins.
ha, the most abundant and immunogenic surface antigen of the influenza virus, is responsible for mediating viral attachment by binding to sialic acid residues on the host cell surface, and for fusing the viral envelope to the host cell membrane  <cit> .
the ha protein is a homotrimer extending 135 Å from the viral membrane, and consists of a stem-like region formed by three helices, one from each monomer, and a globular head domain containing antigenic epitopes.
these two domains form the ectodomain which has previously been solubilized by bromelain cleavage to determine its crystal structure  <cit> .
the tm domain sequence has a propensity to form alpha-helical oligomers in model systems  <cit>  and this tendency may extend to the alpha-helices of the stem region  <cit> ; however, the structure of this domain, as well as the conformation of c-terminal amino acids of the ct, has not been determined.
flublok includes three rha proteins  that are highly purified  using our universal purification process, and are updated according to the annual influenza strain selection process  <cit> .
by comparison, the whole virus vaccines produced using the traditional egg based system are chemically inactivated with either formaldehyde or beta-propiolactone  and partially purified by either column chromatography or sucrose gradient ultracentrifugation and filtration  <cit> .
split and subunit vaccines produced using both the egg and cell culture systems include a detergent extraction step, as well as an additional sucrose gradient or alternative purification step, to further reduce the lipid and contaminating protein content  <cit> .
despite considerable variation in the manufacturing processes and purity, the quantification or potency of the ha proteins produced either in eggs or in bevs is determined using the srid assay  <cit> .
the srid based potency assay, in use since  <dig>  is required to standardize ha content in inactivated licensed flu vaccines by the fda.
the srid assay uses strain specific anti-ha antibodies to quantify ha trimer in the presence of the surfactant zwittergent 3– <dig>
purified wild-type rha proteins, particularly h <dig> rhas, show an apparent initial loss of potency in the srid assay before leveling off within typically four weeks after production.
in the case of h <dig> rha from the a/perth/16/ <dig> influenza strain included in the 2010– <dig>  and 2011– <dig> flublok vaccines, this apparent initial loss of potency is as high as 40% and is correlated with an increase in disulfide bond cross-linking, and the oxidation of at least one conserved cysteine residue at position  <dig> of the primary sequence in the ct domain  <cit> .
ha proteins derived from human influenza viruses can contain 2– <dig> cysteine residues in the tm and ct domains depending on the subtype  in addition to twelve conserved cysteine residues located in the ectodomain which form six sets of disulfide bonds based on x-ray crystal structure analysis and are required for proper folding  <cit> .
these cysteine residues have been implicated in membrane fusion, viral assembly and replication  <cit> .
more recent studies have examined the importance of the tm and ct cysteine residues on thermal stability, heterotypic protection in mice, and other virological features  <cit> .
in the context of influenza viruses, these cysteines in ha proteins are modified with palmitic acid which may be important for biological function.
while the baculovirus-insect cell system supports palmitoylation  <cit> , such reversible, post-translational modifications may be incomplete, as the available data indicate that at least one of the two c-terminal cysteine is partially free and undergoes oxidation  <cit> .figure  <dig>
a and b amino acid sequence of the tm and ct domains.
a. shown is a sequence alignment of the transmembrane  and cytoplasmic tail  domains in the hemagglutinin protein.
protein sequences from different human influenza strains belonging to the h <dig>  b, and h <dig> subtypes have been included and the cysteine residues highlighted.
b. the amino acid sequences of the wild-type and site directed variants of h <dig> a/perth and h <dig> a/texas rha are shown.
the position of the cysteine residues and the modifications are indicated.
the sequences for the tm and ct domains are underlined and double underlined, respectively.
in this study, we evaluated the role of the tm and ct cysteine residues on the thermal stability as determined by dsf, functional stability as determined by potency, antigenicity, and tertiary and quaternary structure of the h <dig> rha protein from the a/perth/16/ <dig> influenza strain.
all or a portion of the tm and ct cysteine residues were substituted with either serine or alanine to produce sdv rha proteins.
our results demonstrate that this cysteine substitution strategy can prevent an apparent potency loss associated with disulfide mediated cross-linking, but does not impact the expression, purification, folding, or thermal stability of the molecule.
hemagglutination inhibition, an assay used to determine the antigenic identity of the vaccine viruses, has been used to show that the sdv rhas are antigenically similar to wild type rha, and that the cysteine substitution strategy does not impact the antigen specific antibody responses in mice upon immunization.
the sdv rhas are presented to the immune system in the same quaternary structural form as the wild-type rha based on the comparability of the rosette structure as determined by negative stain electron microscopy data.
finally, the cysteine substitution strategy has been applied to another h <dig> rha derived from the a/texas/50/ <dig>  influenza strain with comparable results demonstrating that the approach can be applied to address in vitro potency loss in h <dig> rhas caused by the formation of non-native disulfide bonds.
RESULTS
comparable expression and purification of the wild-type and site directed variants
to examine the role of the cysteine residues located in the tm and ct of the h <dig> a/perth rha protein, alanine or serine substitutions were introduced into the wild-type gene by site directed mutagenesis.
three different sdvs were produced having two, three, and five cysteine substitutions.
they are referred to by the number of cysteine substitutions or as 2cys, 3cys and 5cys, respectively .
alanine was selected based on its small size, similar hydrophobicity, and lack of chemistry including hydrogen bonding.
a cysteine to serine substitution was made at position  <dig> in the 2cys sdv based on amino acid sequence alignments consistently showing a serine at the equivalent position of ha proteins from h <dig> and b subtypes .
as a control in this study, a pair of conserved cysteine residues in the ectodomain known to form a disulfide bond, c <dig> and c <dig>  was disrupted by substitution with serine residues.
finally, to verify the relative potency findings obtained with the h <dig> a/perth 5cys sdv rha in this study, the same strategy was used to replace all five tm/ct cysteine residues with alanine residues in the h <dig> a/texas rha protein .
the universal purification process for the wild-type rha was applied to the sdv rha proteins without modification.
the expression level for the control sdv replacing the two conserved and paired cysteines in the ectodomain was too low to purify .
the potency of the soluble rha in the supernatant fraction was determined in the srid assay and was used to establish the starting yield.
the final yield after purification was determined using the total protein results obtained by the bca assay corrected for the rha purity determined by sds-page.
the results provided in table  <dig> show that the starting and purified yields for the 2cys, 3cys, and 5cys sdv rha proteins relative to the wild-type rha were equivalent or better compared to the wild-type rha protein.table  <dig>
starting and final purified yields for rha proteins
rha
starting yield
purified yield
% of wild-type
% of wild-type
*sdv contains modifications c64s, c76s in the ectodomain of the protein.
purity of rha was determined by reducing sds-page.
the full length rha proteins migrates as monomers  having an approximate molecular weight of 62 kda  under denaturing and reducing conditions.
the purity of the rha proteins exceeds 99%.figure  <dig>
a and b sds-page purity and trypsin resistance gels for the h <dig> 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 h <dig> rha protein was analyzed neat , after trypsin treatment , after heat treatment , and after heat and trypsin treatment .
the trypsin enzyme is loaded as a control and molecular weights of the protein standards are shown.
ha <dig> and ha <dig> are indicated by arrows.
rha folding and activity were unaffected by substitutions of the tm/ct cysteines
the trypsin resistance assay provides a qualitative assessment of the folded state of the rha protein.
all the trypsin recognition sites are masked in the correctly folded full-length, ha trimer with the exception of one site located toward the end of the stem region.
as a result, properly folded rha protein is cleaved by trypsin into ha <dig> and ha <dig>  while denatured or mis-folded rha degrades in the presence of trypsin.
each rha was trypsin treated with and without heat denaturation and analyzed by reducing sds-page alongside the untreated rha with and without heat denaturation .
all rhas were resistant to trypsin digestion and the trypsin resistance profiles obtained for the sdv rhas were comparable to that of the wild-type rha.
in the presence of trypsin, the monomeric rha band  was cleaved into ha <dig> and ha <dig> that migrated as ~55 kda and ~25 kda proteins, respectively .
boiling the rha sample prior to trypsin treatment denatured the protein and resulted in digestion of the rha by trypsin .
to determine whether the sdv rhas contain functionally active forms of the protein, they were evaluated in a hemagglutination assay using the wild-type h <dig> rha protein as control.
agglutination of red blood cells requires the ha trimers to be organized in higher order  structures.
the globular head of the rha trimer forms a receptor binding site for terminal sialic acids on red blood cells.
the arrangement of trimers in a rosette structure results in a lattice like structure of the bound cells which do not settle out.
unbound cells settle out and produce a button like or halo pattern of cells as observed in negative controls .
the ha activity of the h <dig> sdv rhas were within 2– <dig> -fold of the ha activity of the wild-type h <dig> rha .figure  <dig>
a and b hemagglutination activity and negative stain electron microscopy of the h <dig> rha proteins.
a. the hemagglutination assay was performed in 96-well u-bottom plates.
the rha proteins were assayed in triplicate at an initial concentration of 1 μg/ml loaded in the left-most lane.
two-fold serially dilutions were performed across the plate in pbs, and equal volume  <dig> % guinea pig red blood cells added.
endpoints are denoted by dotted lines.
b. the rha proteins were stored at 25°c for  <dig>  month at the time of em analysis.
the bar represents 100 nm.
hemagglutination activity results for wild-type and sdv rhas
rha protein
ha activity
units/μg
particle size is unaffected by cysteine substitutions
the particle size of the h <dig> rha proteins was determined by dynamic light scattering , and the results for the volume mean diameter are provided in table  <dig>  the mean volume diameter for the wild-type h <dig> rha, and the 5cys and 3cys sdv h <dig> rhas are comparable .
in contrast, the mean volume diameter of the 2cys sdv h <dig> rha was consistently larger in size  and may be due to the alternative cysteine to serine substitution in this sdv.
despite this difference, the mean volume diameter of the particles observed for the wild-type and sdv h <dig> rhas by dls  was consistent with the size of rosettes visible by em  <cit> .table  <dig>
volume mean diameter by dls for wild-type and cys sdv h <dig> rhas
sample name
volume mean diameter
day 0
day 7
day 28
average
the morphology of the rosettes was determined by electron microscopy .
for this analysis, aliquots of rha protein samples stored for  <dig>  month at 25°c were submitted to paragon bioservices for electron microscopy.
representative em images are shown in figure 3b.
rosette size, estimated to be approximately 30–50 nm, and spike densities were similar in the em images for the wild-type h <dig> rha and the sdvs.
sec was performed to determine the homogeneity of the preparations.
the sec elution profiles were similar for the rhas .
the retention time of the primary peak  and the extrapolated mws  were similar for all the rhas.
in addition, the extrapolated numbers of trimers per rosette for the wild-type rha and sdv rhas remained constant  <cit>  suggesting that the cysteine substitution did not affect the assembly of the trimers into rosettes.figure  <dig>
a-4c sec-hplc elution profiles, sec-uplc elution profiles, and thermal denaturation curves using dsf for the rha proteins.
a. the sec-hplc elution profiles for the wild-type and sdv rha proteins overlay.
the retention times and extrapolated mws are similar for the rha proteins.
the # of trimers per rosette was estimated using the extrapolated mw and a trimer mw of ~210 kda.
b. the sec-uplc elution profiles before  and after  zwittergent 3– <dig> pre-treatment are shown in for the wild-type  and 5cys sdv rha .
c. the melting temperature  was determined by dsf, and the second derivative thermal denaturation curves are shown for each rha.
both the wild type h <dig> rha and  <dig> cys sdv rha were analyzed by sec uplc before and after treatment with 1% zwittergent 3– <dig>  a surfactant which converts rha rosettes into the trimeric form.
in the absence of zwittergent treatment, the wild-type and 5cys sdv rhas gave a single broad peak by sec with a retention time of approximately 3 minutes .
pre-treatment of a 5cys sdv rha with 1% zwittergent 3– <dig> prior to sec-uplc analysis shifted the broad peak to a narrower peak having a retention time of  <dig>  minutes.
in contrast, the sec-uplc profile for the wild-type rha pre-treated with 1% zwittergent 3– <dig> resulted in multiple peaks with retention times between  <dig> and  <dig>  minutes.
these results support the presence of disulfide cross-linked multimers that are not disrupted by the zwittergent treatment in the wild-type rha.
in contrast, the 5cys sdv rha does not have disulfide mediated cross-linked structures and completely shifts to a trimeric conformation upon treatment with 1% zwittergent 3– <dig>
thermal stability of rha is unaffected by cysteine substitutions in the tm and ct domains
the wild-type h <dig> rha and cys sdv h <dig> rhas were analyzed from 25°c to 99°c by dsf in the presence of a molecular rotor dye  to determine thermal stability.
fluorescence intensity was monitored as a function of temperature and a single, large cooperative unfolding was observed for each protein .
these data showed that tms of sdv rha were all similar to the wild-type h <dig> rha , and these differences in the tms are not statistically significant.
since the thermal stability of the sdvs as determined by dsf was quite similar to the wild-type rha, this data further supports that the effect of cysteine substitutions on the overall structure of the rha is minimal.table  <dig>
melting temperatures for wild-type and cys sdv h <dig> rhas using dsf
protein
tm mean
standard deviation
cysteine modifications affect potency loss, cross-linking, and hydrophobicity
the wild-type h <dig> rha and cys sdv h <dig> rhas were stored at 25°c and potency by srid and cross-linking by sds-page were monitored.
the potency was determined by srid on the day of final purification , day  <dig>  day  <dig> or  <dig>  and day  <dig>  and after approximately  <dig> ,  <dig>   <dig>  and 12 months.
the results were plotted relative to the day  <dig> results for wild-type rha, as well as, for each h <dig> sdv rha .
the relative potencies of the 5cys and 3cys sdv h <dig> rhas showed little change over time and were significantly improved compared to the wild-type and 2cys sdv h <dig> rha proteins.
based on the results, the loss in potency decreases with the number of cysteine substitutions in the order wt > 2cys > 3cys > 5cys.figure  <dig>
potency determined by srid for the wild-type and sdv rha proteins stored at 25°c.
the rha proteins were placed at 25°c storage and the potency measured at different time points by srid.
shown are the potency results plotted relative to the day  <dig> value for each h <dig> a/perth rha in the study.
figure 6a shows the reducing and non-reducing sds-page data obtained on days  <dig> and  <dig> of the study.
under non-reducing conditions, rha proteins typically migrate as monomers and cross-linked oligomers which form a ladder of higher molecular weight bands.
the primary full-length ha <dig> migrates at approximately 62 kda, and dimer and trimer migrate at approximately  <dig> and 180 kda, respectively.
rha proteins migrating above the 210 kda marker are due to the formation of higher order cross-linked structures.
the cross-linking is disulfide mediated based on the sds-page profiles obtained under reducing conditions which show predominantly monomeric rha .figure  <dig>
a and b non-reducing and reducing sds-page gels and densitometry for rha proteins on days  <dig> and  <dig>  a. each rha  was analyzed by sds-page under non-reducing  and reducing  conditions on days  <dig>   <dig>   <dig> or  <dig>  and  <dig>  shown are gels obtained on day  <dig>  and on day  <dig> .
densitometry was performed on the non-reducing lanes on the left-hand-side of each gel using the carestream software program, and the corresponding intensity profiles are shown on the right.
b. the intensities of individual bands in the non-reducing sds-page gel profiles were measured using the carestream software program.
the ratio of intensities for cross-linked bands migrating above the  <dig> kda marker to the un-cross-linked monomeric band migrating at ~70 kda was calculated for each rha and time point and plotted as function of storage time below.
the day  <dig> results show essentially no disulfide mediated cross-linking by non-reducing sds-page for the 5cys and 3cys variants, while the wild-type and the 2cys sdv show evidence of cross-linked dimer, trimer and higher order oligomers.
after 1 month at 25°c, the 5cys sdv shows no sign of cross-linking, and the 3cys sdv shows a relatively small amount  of cross-linked dimer and trimer compared to an extensive increase in cross-linking for the 2cys sdv  and the wild-type rha  during the same time period.
a loss of intensity of ha <dig>  is observed for both the 2cys and the wild-type rha as a result of the cross-linking, while the intensity of ha <dig> protein remains comparable on days  <dig> and  <dig> for the 3cys and 5cys sdvs.
thus, the decrease in cross-linking observed with the number of cysteine substitutions in the tm/ct correlates with the corresponding increase in the relative potency for these sdvs.
the intensities of the individual bands in the sds-page gels were measured at each time point in the study, and the ratio of intensities of the cross-linked forms to the un-cross-linked rha  was determined for each rha.
these ratios were plotted as a function of time .
it is evident that cross-linking steadily increased for the wild-type h <dig> rha and the 2cys sdv for up to 14 days and then stabilized.
a slight increase in cross-linking was observed for the 3cys sdv on day  <dig>  while no cross-linking was observed for the 5cys sdv during the study period.
the rp-hplc profiles for the wild-type and 2cys sdv h <dig> rhas are comparable, but different from the rp-hplc profiles for the 3cys and 5cys h <dig> sdv rhas .
the 3cys and 5cys sdv h <dig> rhas elute as a single peak between  <dig> - <dig>  minutes, while the wild-type and 2cys sdv h <dig> rhas elute in three separate peaks  between 6– <dig> minutes.
the elution time for the first peak is very similar for all the three sdvs , while the first peak for the wild-type elutes slightly later .
additionally, retention times for peaks  <dig> and  <dig> are slightly shorter for the 2cys compared to the wild-type rha.
by day  <dig>  the three peaks decrease in intensity and broaden for both the wild-type and 2cys sdv rha suggesting a change in the conformation of the rha.
in contrast, the profiles for the 3cys and 5cys sdvs showing a single peak are similar at the beginning and end of the study with one exception.
a leading tail on the elution peak for the 3cys rha is observed on day  <dig>  and coincides with the appearance of cross-linked rha by sds-page on day  <dig> for the 3cys sdv.
these results suggest that 3cys sdv is effective in reducing the disulfide mediated cross-linking, but all five cysteine substitutions of the 5cys sdv are required to completely eliminate disulfide mediated cross-linking and prevent potency loss.figure  <dig>
rp-hplc profiles for the h <dig> rha proteins.
each h <dig> rha was analyzed in duplicate.
representative chromatograms from the day  <dig> and  <dig> time points are provided.
rhas were incubated with 25 mm dtt for at least 30 minutes prior to analysis.
~25 μg of each rha was injected onto a poros r <dig> column and an acetonitrile gradient applied.
the marked improvement in relative potency and the lack of disulfide mediated cross-linking for the 5cys sdv in this study was confirmed for another 5cys sdv rha and corresponding wild-type rha derived from the h <dig> a/texas influenza strain.
these rha proteins were purified and stored at two temperatures, 5°c and 25°c.
potency by srid, cross-linking by sds-page, and hydrophobicity by rp-hplc were monitored monthly up to 12 months .figure  <dig>
a-c experimental data for h <dig> a/texas 5cys sdv and wild-type rha stored at 5°c and 25°c.
potency by srid, cross-linking by sds-page, and hydrophobicity by rp-hplc were monitored monthly for 12 months.
a. shown are the potency results obtained at each time point for the rha proteins stored at 5°c  and 25°c .
for each rha, the potency results are plotted relative to the day  <dig> value.
b. shown are the non-reducing and reducing sds-page results obtained on day  <dig> and after 12 months for the sets of wild-type and 5cys sdv rha proteins stored at 5°c and 25°c.
c. shown are the rp-hplc profiles obtained on day  <dig> and 12 months for the sets of wild-type and 5cys sdv rha proteins stored at 5°c and 25°c.
antigenicity is unaffected by cysteine substitutions in the tm and ct domains
to determine the antigenicity of the rha proteins, the hemagglutination inhibition  assay was performed using two different polyclonal antisera.
in addition, the cber reference antigen for h <dig> a/perth/16/ <dig> ha  was also included in the assay for comparison with rha proteins.
specific binding of the antibodies to different antigenic sites in the ha proteins interferes with the ability of the ha protein to agglutinate red blood cells and forms the basis of the hai assay.
the hai endpoint or titer, the reciprocal of the last dilution of antiserum that completely inhibits hemagglutination, was determined for each rha protein using both antisera and compared to the cber reference ha antigen in table  <dig>  using both antisera, the hai endpoints of the cys sdv h <dig> rha were equivalent or within 2-fold of the wild-type h <dig> rha and the cber reference antigen for h <dig> a/perth ha.
the results confirm that cysteine substitutions introduced into the tm and ct domain of h <dig> rha did not alter the presentation of the antigenic sites.table  <dig>
hai titers for wild-type and cys sdv h <dig> rhas
h <dig> ha protein
hai titer
rabbit poly clonal anti-h <dig> rha ab
sheep polyclonal anti-h <dig> ha ab
antigen-specific antibody production is unaffected by cysteine substitutions in the tm and ct domains
a comparative study was performed in mice using the 5cys sdv rha and corresponding wild-type rha from the h <dig> a/texas strain.
the mouse study was performed by josman llc and included  <dig> cohorts, three for each rha antigen and one for a placebo, with  <dig> acclimated balb/c mice in each cohort.
on day  <dig> and day  <dig> of the study, mice were administered a 25 μl intramuscular injection of either the wild-type rha antigen at one of three doses  or the corresponding 5cys sdv antigen at one of three doses .
the placebo group received an equal volume dose of the formulation buffer.
pre-immune serum samples collected on day  <dig> were tested alongside serum samples collected on day  <dig> in the hai assay  using both the wild-type rha and sdv rha as the test antigens.
the number of responders having at least a 4-fold change in geometric titer on day  <dig> compared to day  <dig> were similar for all test groups receiving the two different rha antigens regardless of the antigen used in the assay .
further, the average geometric mean titers for all groups were comparable regardless of the antigen and dose delivered to the mice .
based on the results, c-terminal cysteine substitutions and the degree of disulfide cross-linking do not impact the levels of antigen-specific antibody responses upon immunization.figure  <dig>
a and b pre-clinical mouse immunogenicity study results.
sera samples from mice immunized with the h <dig> a/texas 5cys sdv rha antigen  and with the h <dig> a/texas wild-type rha antigen  were tested in the hai assay using both antigens, the h <dig> a/texas 5cys rha antigen  and the h <dig> a/texas wild-type rha antigen .
in each plot, the dotted line represents 4× the mean value for the day  <dig> titer.
discussion
the influenza ha protein is an integral membrane protein and the primary target for inducing neutralizing antibodies to influenza virus.
when purified to homogeneity, the protein forms a rosette structure which exposes the immunogenic, trimeric ectodomain, while the transmembrane domains and short cytoplasmic tails are most likely buried inside the rosette structure  <cit> .
it has long been demonstrated that formation of trimeric ha is not dependent on the tm or ct domains  <cit> , though it has been suggested that the tm domain may contribute to the stability of the trimer  <cit> .
similarly, the early stages of ha mediated membrane fusion are unaffected by deletion of the tm and ct domains and replacement by a non-proteinaceous gpi anchor  <cit> .
therefore, the wild-type rha and the tm and ct cysteine sdvs in this study are expected to form similar trimers and rosettes.
an evaluation of the human influenza strains which fall into  <dig> subtypes, h1n <dig> , h3n <dig>  and, b, shows that the h <dig> rhas have five cysteine residues in the tm and ct domains .
the two cysteine residues in the ct domain are highly conserved and are found in all human influenza strains.
the cysteine residue positioned at the interface between the tm and ct is found in h <dig> and h <dig> has, but not in b has.
the biological role of these cysteine residues has been the subject of several studies which have identified them as sites of palmitoylation/acylation involved in membrane fusion and viral replication  <cit> .
while these activities are indispensable for the virus, they are not required for expression and purification of the recombinant hemagglutinin protein during vaccine manufacture.
in fact, replacing all five of the tm/ct cysteines in the 5cys sdv or different subsets of them in the 3cys and 2cys sdvs  result in rha proteins having starting yields, final purities, and purified yields that are comparable to or better than the wild-type rha protein .
in contrast, substitution of the two conserved cysteine residues in the globular head domain of the rha protein, c <dig> and c <dig>  with serine residues results in low expression further confirming that the cysteine residues in the globular head domain play a critical role in the folding of hemagglutinin protein, and cannot be substituted .
the folding and functional activity of the wild-type rha and the tm/ct sdvs were compared by evaluating the trypsin resistance profiles, and hemagglutination activities.
trypsin resistance is a property of properly folded trimeric ha proteins, while hemagglutination requires the organization of these trimers into higher order rosette structures that can interact with red blood cells through their sialic acid and form a lattice like network.
trypsin treatment cleaves properly folded, full-length, trimeric ha into two fragments, an amino-terminal ha <dig> fragment and a carboxy-terminal ha <dig> fragment, while denatured or monomeric rha is digested by the endoprotease.
trypsin treatment of the wild-type and sdv rhas results in the same characteristic ha <dig> and ha <dig> cleavage with apparent molecular weights of ~ <dig> kda for ha <dig> and ~28 kda for ha <dig> .
hemagglutination occurs when the red blood cells interact via their sialic acid moiety with ha rosettes, and form a lattice like network that remains in suspension.
in the absence of hemagglutination, the cells do not form the lattice like network, and a halo or circle of settled cells is observed.
these results demonstrate that the hemagglutination activity of sdvs of rha is comparable to the wild-type rha .
further, these results suggest that the cysteine substitutions in the tm/ct domains do not impact the functional activity of h <dig> sdv rhas.
having established proper folding and function, the particle size and thermal stability of the wild-type and sdv rha proteins were determined.
the average particle size by dls  for the wild-type rha and the tm/ct sdvs is in the range expected for rosettes structures.
the volume mean diameter of the 3cys and 5cys sdvs is 35 nm and 37 nm, respectively, compared to 39 nm for the wild-type h <dig> rha protein.
the diameter of the particles for the 2cys sdv is consistently 10 nm larger than the wild-type, and may reflect that the replacement of cysteine to serine at position  <dig> is not optimal and leads to particles of larger size.
by em , the rosettes of the wild-type and sdv rhas are strikingly similar, and indistinguishable in terms of size and appearance.
in addition to particle size and morphology, the thermal stability of the rosettes in the sdv rhas as measured by dsf is similar to that of the wild-type rha .
the largest difference in tm, observed between the 5cys sdv and the wild-type, is only  <dig> °c.
decreased thermal stability has been reported previously for three h <dig> ha variants in which one or both of the two tm cysteine residues unique to this subtype were modified  <cit> .
however, the thermal stability in this study was determined in a fundamentally different way on crude extracts using the hemagglutination activity assay.
further, our thermal stability data are in close agreement with previously report tm values for rosettes suggesting that both the wt and cys sdv rhas are similarly highly stable  <cit> .
by sec, all the rha proteins have a single, primary peak with retention times differing by  <dig>  minute or less.
in addition, the wild-type and sdv rhas elute before the largest standard, thyroglobulin, having an approximate molecular weight of 660 kda .
these data also suggest that the rha trimers assemble into rosettes structures, and the ability to form the rosettes is unaffected by the cysteine substitutions in the tm/ct domains.
differences in the homogeneity of the rosette population in the wild-type and 5cys sdv rhas are detected by uplc sec analysis of the zwittergent 3– <dig> treated rha .
the zwittergent 3– <dig> surfactant efficiently converts the rosettes of the 5cys sdv, but not of the wild-type rha, to trimers.
due to its smaller size compared to the rosette, the rha trimer elutes off the sec column later and the peak is shifted.
in contrast, zwittergent 3– <dig> treatment of the wild-type rha protein results in multiple peaks corresponding to rosette, trimer, and various species of intermediate sizes.
disulfide mediating cross-linking occurs readily in the wild-type rha based on sds-page analysis , and the various cross-linked states of the protein result in multiple elution peaks as the zwittergent 3– <dig> surfactant cannot disrupt the disulfide bonds.
though the overall folding, thermal stability, size, and morphology are the same for the wild-type and tm/ct sdvs, differences in the formation of disulfide mediated cross-linked structures have been observed by sds-page .
disulfide mediate cross-linking occurs readily at the c-terminus of the ha molecule through free cysteines located in the tm and ct domains.
in the wild-type rha and 2cys sdv rha, this cross-linking occurs during the purification process and is extensive.
cross-linked dimers, trimers, as well as higher order cross-linked oligomers are observed immediately following purification on day  <dig>  albeit at a slightly reduced level in the 2cys sdv.
in the presence of reducing agent, the oligomers are converted to monomeric ha <dig> confirming that the cross-linking is disulfide mediated and due to the oxidation of free cysteine residues.
cross-linking of the 5cys sdv rha is not observed even after 28 days at room temperature, while cross-linked dimers and trimers of the 3cys sdv rha are first observed on the day  <dig> time point .
the non-reducing and reducing sds-page results for the 5cys sdv and corresponding wild-type rha from the h <dig> a/texas strain further support the findings obtained with the h <dig> a/perth sdvs .
the formation of non-native disulfide bonds is prevented in h <dig> rha proteins by replacing all five tm and ct cysteine residues.
based on the sds-page results, the rha protein is highly susceptible to oxidation through cysteines in both the tm and ct domain.
the subset of cysteines modified in the 3cys sdv  appears to be more susceptible to cross-linking than the subset of cysteines in the 2cys sdv .
the latter two substitutions have little effect on the sds-page profile of the 2cys sdv compared to the wild-type rha, while the former three substitutions markedly reduce the formation of cross-linked rha in 3cys sdv.
these results are not unexpected as the cysteines in the ct domain are more exposed at the c-terminus of the molecule.
free cysteine at this position may permit the cross-linking of monomers within a given trimer  and between monomers of different trimers .
the rp-hplc profiles for h <dig> a/perth rhas  and the h <dig> a/texas rhas  are consistent with the non-reducing sds-page analyses.
the multiple elution peaks observed for the wild-type rhas and 2cys sdv rhas may be due to different cross-linked populations.
alternatively, they may due to the covalent attachment of fatty acids  through thio-ester linkages involving the last three c-terminal cysteine residues present in both of these rha molecules  <cit> , but absent in the 3cys and 5cys sdv rha proteins.
both types of populations, cross-linked rha and acylated rha, are more likely to be retained on the column due the increased hydrophobic character and elute later in the gradient as observed here in the rp-hplc profiles for the wild-type and 2cys sdv rha.
concurrent with evaluation of the cross-linking, the potency of the wild-type and sdv rha proteins from the h <dig> a/perth and h <dig> a/texas strains were measured by the srid assay .
by this method, the relative potency is directly proportional to the number of tm/ct substitutions and inversely correlated with cross-linking.
in the 5cys sdv rha in which all five tm/ct cysteines have been replaced, the potency relative to day  <dig> remains at 100% even after 12 months at room temperature for both the h <dig> a/texas and h <dig> a/perth 5cys sdv rhas, and no disulfide mediated cross-linking is observed.
by comparison, the relative potency of the wild-type h <dig> rhas drop approximately 75% after 4 weeks of storage at 25°c, and demonstrates extensive cross-linking.
compared to the wild-type rhas, relative potency of the 2cys sdv  drops to 50% during the same time .
furthermore, the relative potency of the 3cys sdv , drops only 20% after 4 weeks storage at 25°c.
while cross-linking is markedly reduced for the 3cys sdv rha, the 2cys sdv shows only a marginal improvement over the wild-type rha protein.
these results clearly demonstrate that the substitution of cysteine residues in the ct domain has a significant impact on potency, but substituting cysteine residues in both the tm and ct domains has a synergistic effect on potency of the rha.
while including a reducing agent and redox buffer, stg and citrate, in the formulation buffer has been shown to prevent an apparent potency loss associated with the formation of non-native disulfide bonds and higher order cross-linked oligomers of the ha , the results of this study clearly show that replacing the five cysteines in the tm and ct domains with an alternative amino acid has the same effect and is a highly effective alternative solution to address potency loss.
the ability of these cysteines to cross-link appears to be a direct consequence of the production and purification process that releases the tm domain from the constrained environment of the membrane bilayer and the ct domain from the reducing environment of the cell.
since the three dimensional structure of the c-terminus  has not been solved by x-ray crystallography, a theoretical structural analysis of ha using a membrane protein topology prediction algorithm was performed assuming that the transmembrane region of the ha molecule adopts and continues the alpha- helical conformation of the proceeding ha <dig> domain  <cit> .
using the program tmhmm  <cit>  and the ha sequence from h <dig> a/perth, seven symmetrical alpha helical trimer configurations are possible .
based on this model, the tm and ct cysteines are in the correct orientation and geometry to form both intra-molecular cross-links within a trimer and inter-molecular cross-links between different trimers.
amino acids with a spacing of three or four may be found on the same face of an alpha helix and cysteine residues in those positions can form disulfide bonds between two adjacent helices, thus covalently linking helices.
cysteine residues on the outside of the helices may participate in the covalent crosslinking of higher order oligomers.
the association of ha trimers through their hydrophobic transmembrane domains in rosette structures also facilitates intermolecular cross-linking by positioning the c-terminal cysteines from different trimers in close proximity to each other.figure  <dig>
modeling of the tm and ct domains in the ha trimer.
the top shows the amino acid sequence of the carboxy terminus  for h <dig> a/perth ha with the tm and ct cysteines highlighted in yellow , and the  <dig> possible orientations of that sequence in a helix with the interfacial positions highlighted in pink .
the schematics depict the trimer configuration  with  <dig> positions labeled a through g on the bottom left and one possible configuration  on the bottom right.
while both intra- and inter-molecular cross-linking of the ha trimer are possible, the latter may reduce diffusion, and/or antibody recognition in the srid assay.
prior to analysis in the srid assay, the rha proteins are pre-treated with zwittergent 3– <dig> surfactant into order to dissociate non-covalently bound rha rosettes structures into soluble trimers.
sec-uplc analysis of rha before and after zwittergent 3– <dig> treatment clearly shows that only a portion of the wild-type rosettes are converted to trimers; in contrast, the rosettes of the 5cys sdv are quantitatively converted to trimers.
due to their small size , rha trimers can readily diffuse in to the agarose gel containing antibody and the size of the resulting precipitin ring is directly proportional to the antigen concentration.
in contrast, trimers covalently bound together in rosettes through disulfide bonds are resistant to zwittergent 3– <dig> pre-treatment.
depending on the extent of oxidation and/or the orientation of the cross-linked trimers, mobility of the particles through the gel may be hindered resulting in artificially small ring sizes.
alternatively, access to the epitopes may become increasingly hindered as the rosettes become covalently cross-linked through disulfide bonds resulting in a reduction in antibody binding and signal.
due to the inability of the srid assay to accurately measure the rosette in a cross-linked state, an artificial or apparent potency loss is observed as the number of these oxidized species increases with storage of the rha protein.
ha rosettes have been considered an indicator of immunogenicity because of their resemblance to the infectious influenza virions having ha trimeric spikes protruding from the viral envelope  <cit> .
additionally, the antigenic sites on the ha molecule responsible for eliciting neutralizing antibodies are located in the globular head domain approximately 135 Å away from the cysteine substitutions located at the opposite end of the elongate molecule in the tm and ct domains  <cit> .
despite this wide physical separation, structural perturbations from side chain mutations have been shown to be propagated over long distances through the peptide backbone.
for this reason, the antigenic and immunological affects of c-terminal substitutions on the rha protein were determined.
the hemagglutination inhibition assay was performed using the sdv and wild-type h <dig> a/perth rha proteins in order to determine and compare recognition and binding to antibodies in sera produced using the wild-type rha antigen.
the hai titers for the sdvs were comparable to the wild-type rha  indicating that the antigenic presentation of the epitopes on the sdvs has not been changed by the cysteine substitutions.
also, mice were immunized with either the h <dig> a/texas 5cys sdv or the corresponding wild-type rha antigen.
based on similar hai titers obtained in each group, the cysteine substitutions have no effect on the levels of rha-specific antibody responses induced upon immunization.
thus, the cross-linked trimers in the wild-type rha rosettes and the un-cross-linked trimers in the 5cys sdv rha rosettes induce comparable antibody responses in mice upon immunization.
the results suggest that the potency loss observed for the wild-type rha protein over time is largely due to the inability of the required potency assay to measure higher order cross-linked forms of the protein that retain their immunogenicity.
future work will include clinical trials to determine the impact on immunogenicity in human subjects.
in the manufacture of the rha vaccine, the unique  environment permits the formation of non-native disulfide bonds through free cysteines located in the tm and ct domains of the wild-type rha protein.
c-terminal cross-linking may occur to some extent in traditional split and subunit influenza vaccines as the purification process uses a detergent extraction step which may liberate the c terminus of the ha from the viral membrane.
while both inter- and intra-molecular cross-linking can occur, only the latter results in cross-linked rosette structures that remain immunogenic, but are not accurately measured in the srid assay.
as a result, an apparent potency loss is observed with storage of the wid-type rha vaccine.
cysteine mutagenesis is one way to overcome protein oxidation without the introduction of reducing agents in the formulation.
however, the utility of this strategy is largely limited to the recombinant influenza vaccine since site specific mutagenesis of the virus prior to amplification in chicken cells or mammalian cells would represent a significant challenge.
CONCLUSIONS
rha proteins form higher-order, defined oligomers  that become covalently cross-linked through cysteine residues in the tm and ct domains.
replacement of these cysteine residues prevents disulfide bond formation at the c-terminus, but does not impact the expression, purification, or folding of the molecule based on our work as well as others  <cit> .
moreover, the particle size and morphology, thermal stability, and antigenicity of the molecule are unaffected.
application of the cysteine mutagenesis strategy to rha proteins derived from the other seasonal influenza subtypes , and pandemic strains  is currently underway, and a clinical trial is being planned.
this technology may be extremely important for pandemic preparedness and would greatly enhance the value of the stock pile if the vaccine is not subject to potency loss at room temperature.

 licensed, seasonal influenza vaccines available in the united states include trivalent and quadrivalent inactivated vaccines and a live attenuated influenza vaccine produced in embryonated chicken eggs, a cell culture based trivalent vaccine produced in madin darby canine kidney cells, and most recently a recombinant trivalent vaccine produced using the baculovirus-insect cell system. flublok vaccine has several distinct advantages over other flu vaccines including high purity of the ha protein, and absence of antibiotics, preservatives, gelatin, and egg proteins. ha, the most abundant and immunogenic surface antigen of the influenza virus, is responsible for mediating viral attachment by binding to sialic acid residues on the host cell surface, and for fusing the viral envelope to the host cell membrane. the ha protein is a homotrimer extending 135 Å from the viral membrane, and consists of a stem-like region formed by three helices, one from each monomer, and a globular head domain containing antigenic epitopes. these two domains form the ectodomain which has previously been solubilized by bromelain cleavage to determine its crystal structure. the tm domain sequence has a propensity to form alpha-helical oligomers in model systems and this tendency may extend to the alpha-helices of the stem region; however, the structure of this domain, as well as the conformation of c-terminal amino acids of the ct, has not been determined. flublok includes three rha proteins that are highly purified using our universal purification process, and are updated according to the annual influenza strain selection process. by comparison, the whole virus vaccines produced using the traditional egg based system are chemically inactivated with either formaldehyde or beta-propiolactone and partially purified by either column chromatography or sucrose gradient ultracentrifugation and filtration. split and subunit vaccines produced using both the egg and cell culture systems include a detergent extraction step, as well as an additional sucrose gradient or alternative purification step, to further reduce the lipid and contaminating protein content. despite considerable variation in the manufacturing processes and purity, the quantification or potency of the ha proteins produced either in eggs or in bevs is determined using the srid assay. the srid based potency assay, in use since is required to standardize ha content in inactivated licensed flu vaccines by the fda. the srid assay uses strain specific anti-ha antibodies to quantify ha trimer in the presence of the surfactant zwittergent 3– purified wild-type rha proteins, particularly h rhas, show an apparent initial loss of potency in the srid assay before leveling off within typically four weeks after production. in the case of h rha from the a/perth/16/ influenza strain included in the 2010– and 2011– flublok vaccines, this apparent initial loss of potency is as high as 40% and is correlated with an increase in disulfide bond cross-linking, and the oxidation of at least one conserved cysteine residue at position of the primary sequence in the ct domain. ha proteins derived from human influenza viruses can contain 2– cysteine residues in the tm and ct domains depending on the subtype in addition to twelve conserved cysteine residues located in the ectodomain which form six sets of disulfide bonds based on x-ray crystal structure analysis and are required for proper folding. these cysteine residues have been implicated in membrane fusion, viral assembly and replication. more recent studies have examined the importance of the tm and ct cysteine residues on thermal stability, heterotypic protection in mice, and other virological features. in the context of influenza viruses, these cysteines in ha proteins are modified with palmitic acid which may be important for biological function. while the baculovirus-insect cell system supports palmitoylation, such reversible, post-translational modifications may be incomplete, as the available data indicate that at least one of the two c-terminal cysteine is partially free and undergoes oxidation.figure a and b amino acid sequence of the tm and ct domains. a. shown is a sequence alignment of the transmembrane and cytoplasmic tail domains in the hemagglutinin protein. protein sequences from different human influenza strains belonging to the h b, and h subtypes have been included and the cysteine residues highlighted. b. the amino acid sequences of the wild-type and site directed variants of h a/perth and h a/texas rha are shown. the position of the cysteine residues and the modifications are indicated. the sequences for the tm and ct domains are underlined and double underlined, respectively. in this study, we evaluated the role of the tm and ct cysteine residues on the thermal stability as determined by dsf, functional stability as determined by potency, antigenicity, and tertiary and quaternary structure of the h rha protein from the a/perth/16/ influenza strain. all or a portion of the tm and ct cysteine residues were substituted with either serine or alanine to produce sdv rha proteins. our results demonstrate that this cysteine substitution strategy can prevent an apparent potency loss associated with disulfide mediated cross-linking, but does not impact the expression, purification, folding, or thermal stability of the molecule. hemagglutination inhibition, an assay used to determine the antigenic identity of the vaccine viruses, has been used to show that the sdv rhas are antigenically similar to wild type rha, and that the cysteine substitution strategy does not impact the antigen specific antibody responses in mice upon immunization. the sdv rhas are presented to the immune system in the same quaternary structural form as the wild-type rha based on the comparability of the rosette structure as determined by negative stain electron microscopy data. finally, the cysteine substitution strategy has been applied to another h rha derived from the a/texas/50/ influenza strain with comparable results demonstrating that the approach can be applied to address in vitro potency loss in h rhas caused by the formation of non-native disulfide bonds.  comparable expression and purification of the wild-type and site directed variants to examine the role of the cysteine residues located in the tm and ct of the h a/perth rha protein, alanine or serine substitutions were introduced into the wild-type gene by site directed mutagenesis. three different sdvs were produced having two, three, and five cysteine substitutions. they are referred to by the number of cysteine substitutions or as 2cys, 3cys and 5cys, respectively. alanine was selected based on its small size, similar hydrophobicity, and lack of chemistry including hydrogen bonding. a cysteine to serine substitution was made at position in the 2cys sdv based on amino acid sequence alignments consistently showing a serine at the equivalent position of ha proteins from h and b subtypes. as a control in this study, a pair of conserved cysteine residues in the ectodomain known to form a disulfide bond, c and c was disrupted by substitution with serine residues. finally, to verify the relative potency findings obtained with the h a/perth 5cys sdv rha in this study, the same strategy was used to replace all five tm/ct cysteine residues with alanine residues in the h a/texas rha protein. the universal purification process for the wild-type rha was applied to the sdv rha proteins without modification. the expression level for the control sdv replacing the two conserved and paired cysteines in the ectodomain was too low to purify. the potency of the soluble rha in the supernatant fraction was determined in the srid assay and was used to establish the starting yield. the final yield after purification was determined using the total protein results obtained by the bca assay corrected for the rha purity determined by sds-page. the results provided in table  show that the starting and purified yields for the 2cys, 3cys, and 5cys sdv rha proteins relative to the wild-type rha were equivalent or better compared to the wild-type rha protein.table starting and final purified yields for rha proteins rha starting yield purified yield % of wild-type % of wild-type *sdv contains modifications c64s, c76s in the ectodomain of the protein. purity of rha was determined by reducing sds-page. the full length rha proteins migrates as monomers having an approximate molecular weight of 62 kda under denaturing and reducing conditions. the purity of the rha proteins exceeds 99%.figure a and b sds-page purity and trypsin resistance gels for the h 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 h rha protein was analyzed neat, after trypsin treatment, after heat treatment, and after heat and trypsin treatment. the trypsin enzyme is loaded as a control and molecular weights of the protein standards are shown. ha and ha are indicated by arrows. rha folding and activity were unaffected by substitutions of the tm/ct cysteines the trypsin resistance assay provides a qualitative assessment of the folded state of the rha protein. all the trypsin recognition sites are masked in the correctly folded full-length, ha trimer with the exception of one site located toward the end of the stem region. as a result, properly folded rha protein is cleaved by trypsin into ha and ha while denatured or mis-folded rha degrades in the presence of trypsin. each rha was trypsin treated with and without heat denaturation and analyzed by reducing sds-page alongside the untreated rha with and without heat denaturation. all rhas were resistant to trypsin digestion and the trypsin resistance profiles obtained for the sdv rhas were comparable to that of the wild-type rha. in the presence of trypsin, the monomeric rha band was cleaved into ha and ha that migrated as ~55 kda and ~25 kda proteins, respectively. boiling the rha sample prior to trypsin treatment denatured the protein and resulted in digestion of the rha by trypsin. to determine whether the sdv rhas contain functionally active forms of the protein, they were evaluated in a hemagglutination assay using the wild-type h rha protein as control. agglutination of red blood cells requires the ha trimers to be organized in higher order structures. the globular head of the rha trimer forms a receptor binding site for terminal sialic acids on red blood cells. the arrangement of trimers in a rosette structure results in a lattice like structure of the bound cells which do not settle out. unbound cells settle out and produce a button like or halo pattern of cells as observed in negative controls. the ha activity of the h sdv rhas were within 2– -fold of the ha activity of the wild-type h rha.figure a and b hemagglutination activity and negative stain electron microscopy of the h rha proteins. a. the hemagglutination assay was performed in 96-well u-bottom plates. the rha proteins were assayed in triplicate at an initial concentration of 1 μg/ml loaded in the left-most lane. two-fold serially dilutions were performed across the plate in pbs, and equal volume % guinea pig red blood cells added. endpoints are denoted by dotted lines. b. the rha proteins were stored at 25°c for  month at the time of em analysis. the bar represents 100 nm. hemagglutination activity results for wild-type and sdv rhas rha protein ha activity units/μg particle size is unaffected by cysteine substitutions the particle size of the h rha proteins was determined by dynamic light scattering, and the results for the volume mean diameter are provided in table  the mean volume diameter for the wild-type h rha, and the 5cys and 3cys sdv h rhas are comparable. in contrast, the mean volume diameter of the 2cys sdv h rha was consistently larger in size and may be due to the alternative cysteine to serine substitution in this sdv. despite this difference, the mean volume diameter of the particles observed for the wild-type and sdv h rhas by dls was consistent with the size of rosettes visible by em.table volume mean diameter by dls for wild-type and cys sdv h rhas sample name volume mean diameter day 0 day 7 day 28 average the morphology of the rosettes was determined by electron microscopy. for this analysis, aliquots of rha protein samples stored for  month at 25°c were submitted to paragon bioservices for electron microscopy. representative em images are shown in figure 3b. rosette size, estimated to be approximately 30–50 nm, and spike densities were similar in the em images for the wild-type h rha and the sdvs. sec was performed to determine the homogeneity of the preparations. the sec elution profiles were similar for the rhas. the retention time of the primary peak and the extrapolated mws were similar for all the rhas. in addition, the extrapolated numbers of trimers per rosette for the wild-type rha and sdv rhas remained constant suggesting that the cysteine substitution did not affect the assembly of the trimers into rosettes.figure a-4c sec-hplc elution profiles, sec-uplc elution profiles, and thermal denaturation curves using dsf for the rha proteins. a. the sec-hplc elution profiles for the wild-type and sdv rha proteins overlay. the retention times and extrapolated mws are similar for the rha proteins. the # of trimers per rosette was estimated using the extrapolated mw and a trimer mw of ~210 kda. b. the sec-uplc elution profiles before and after zwittergent 3– pre-treatment are shown in for the wild-type and 5cys sdv rha. c. the melting temperature was determined by dsf, and the second derivative thermal denaturation curves are shown for each rha. both the wild type h rha and cys sdv rha were analyzed by sec uplc before and after treatment with 1% zwittergent 3– a surfactant which converts rha rosettes into the trimeric form. in the absence of zwittergent treatment, the wild-type and 5cys sdv rhas gave a single broad peak by sec with a retention time of approximately 3 minutes. pre-treatment of a 5cys sdv rha with 1% zwittergent 3– prior to sec-uplc analysis shifted the broad peak to a narrower peak having a retention time of  minutes. in contrast, the sec-uplc profile for the wild-type rha pre-treated with 1% zwittergent 3– resulted in multiple peaks with retention times between and  minutes. these results support the presence of disulfide cross-linked multimers that are not disrupted by the zwittergent treatment in the wild-type rha. in contrast, the 5cys sdv rha does not have disulfide mediated cross-linked structures and completely shifts to a trimeric conformation upon treatment with 1% zwittergent 3– thermal stability of rha is unaffected by cysteine substitutions in the tm and ct domains the wild-type h rha and cys sdv h rhas were analyzed from 25°c to 99°c by dsf in the presence of a molecular rotor dye to determine thermal stability. fluorescence intensity was monitored as a function of temperature and a single, large cooperative unfolding was observed for each protein. these data showed that tms of sdv rha were all similar to the wild-type h rha, and these differences in the tms are not statistically significant. since the thermal stability of the sdvs as determined by dsf was quite similar to the wild-type rha, this data further supports that the effect of cysteine substitutions on the overall structure of the rha is minimal.table melting temperatures for wild-type and cys sdv h rhas using dsf protein tm mean standard deviation cysteine modifications affect potency loss, cross-linking, and hydrophobicity the wild-type h rha and cys sdv h rhas were stored at 25°c and potency by srid and cross-linking by sds-page were monitored. the potency was determined by srid on the day of final purification, day day or and day and after approximately, and 12 months. the results were plotted relative to the day results for wild-type rha, as well as, for each h sdv rha. the relative potencies of the 5cys and 3cys sdv h rhas showed little change over time and were significantly improved compared to the wild-type and 2cys sdv h rha proteins. based on the results, the loss in potency decreases with the number of cysteine substitutions in the order wt > 2cys > 3cys > 5cys.figure potency determined by srid for the wild-type and sdv rha proteins stored at 25°c. the rha proteins were placed at 25°c storage and the potency measured at different time points by srid. shown are the potency results plotted relative to the day value for each h a/perth rha in the study. figure 6a shows the reducing and non-reducing sds-page data obtained on days and of the study. under non-reducing conditions, rha proteins typically migrate as monomers and cross-linked oligomers which form a ladder of higher molecular weight bands. the primary full-length ha migrates at approximately 62 kda, and dimer and trimer migrate at approximately and 180 kda, respectively. rha proteins migrating above the 210 kda marker are due to the formation of higher order cross-linked structures. the cross-linking is disulfide mediated based on the sds-page profiles obtained under reducing conditions which show predominantly monomeric rha.figure a and b non-reducing and reducing sds-page gels and densitometry for rha proteins on days and a. each rha was analyzed by sds-page under non-reducing and reducing conditions on days or and shown are gels obtained on day and on day. densitometry was performed on the non-reducing lanes on the left-hand-side of each gel using the carestream software program, and the corresponding intensity profiles are shown on the right. b. the intensities of individual bands in the non-reducing sds-page gel profiles were measured using the carestream software program. the ratio of intensities for cross-linked bands migrating above the kda marker to the un-cross-linked monomeric band migrating at ~70 kda was calculated for each rha and time point and plotted as function of storage time below. the day results show essentially no disulfide mediated cross-linking by non-reducing sds-page for the 5cys and 3cys variants, while the wild-type and the 2cys sdv show evidence of cross-linked dimer, trimer and higher order oligomers. after 1 month at 25°c, the 5cys sdv shows no sign of cross-linking, and the 3cys sdv shows a relatively small amount of cross-linked dimer and trimer compared to an extensive increase in cross-linking for the 2cys sdv and the wild-type rha during the same time period. a loss of intensity of ha is observed for both the 2cys and the wild-type rha as a result of the cross-linking, while the intensity of ha protein remains comparable on days and for the 3cys and 5cys sdvs. thus, the decrease in cross-linking observed with the number of cysteine substitutions in the tm/ct correlates with the corresponding increase in the relative potency for these sdvs. the intensities of the individual bands in the sds-page gels were measured at each time point in the study, and the ratio of intensities of the cross-linked forms to the un-cross-linked rha was determined for each rha. these ratios were plotted as a function of time. it is evident that cross-linking steadily increased for the wild-type h rha and the 2cys sdv for up to 14 days and then stabilized. a slight increase in cross-linking was observed for the 3cys sdv on day while no cross-linking was observed for the 5cys sdv during the study period. the rp-hplc profiles for the wild-type and 2cys sdv h rhas are comparable, but different from the rp-hplc profiles for the 3cys and 5cys h sdv rhas. the 3cys and 5cys sdv h rhas elute as a single peak between -  minutes, while the wild-type and 2cys sdv h rhas elute in three separate peaks between 6– minutes. the elution time for the first peak is very similar for all the three sdvs, while the first peak for the wild-type elutes slightly later. additionally, retention times for peaks and are slightly shorter for the 2cys compared to the wild-type rha. by day the three peaks decrease in intensity and broaden for both the wild-type and 2cys sdv rha suggesting a change in the conformation of the rha. in contrast, the profiles for the 3cys and 5cys sdvs showing a single peak are similar at the beginning and end of the study with one exception. a leading tail on the elution peak for the 3cys rha is observed on day and coincides with the appearance of cross-linked rha by sds-page on day for the 3cys sdv. these results suggest that 3cys sdv is effective in reducing the disulfide mediated cross-linking, but all five cysteine substitutions of the 5cys sdv are required to completely eliminate disulfide mediated cross-linking and prevent potency loss.figure rp-hplc profiles for the h rha proteins. each h rha was analyzed in duplicate. representative chromatograms from the day and time points are provided. rhas were incubated with 25 mm dtt for at least 30 minutes prior to analysis. ~25 μg of each rha was injected onto a poros r column and an acetonitrile gradient applied. the marked improvement in relative potency and the lack of disulfide mediated cross-linking for the 5cys sdv in this study was confirmed for another 5cys sdv rha and corresponding wild-type rha derived from the h a/texas influenza strain. these rha proteins were purified and stored at two temperatures, 5°c and 25°c. potency by srid, cross-linking by sds-page, and hydrophobicity by rp-hplc were monitored monthly up to 12 months.figure a-c experimental data for h a/texas 5cys sdv and wild-type rha stored at 5°c and 25°c. potency by srid, cross-linking by sds-page, and hydrophobicity by rp-hplc were monitored monthly for 12 months. a. shown are the potency results obtained at each time point for the rha proteins stored at 5°c and 25°c. for each rha, the potency results are plotted relative to the day value. b. shown are the non-reducing and reducing sds-page results obtained on day and after 12 months for the sets of wild-type and 5cys sdv rha proteins stored at 5°c and 25°c. c. shown are the rp-hplc profiles obtained on day and 12 months for the sets of wild-type and 5cys sdv rha proteins stored at 5°c and 25°c. antigenicity is unaffected by cysteine substitutions in the tm and ct domains to determine the antigenicity of the rha proteins, the hemagglutination inhibition assay was performed using two different polyclonal antisera. in addition, the cber reference antigen for h a/perth/16/ ha was also included in the assay for comparison with rha proteins. specific binding of the antibodies to different antigenic sites in the ha proteins interferes with the ability of the ha protein to agglutinate red blood cells and forms the basis of the hai assay. the hai endpoint or titer, the reciprocal of the last dilution of antiserum that completely inhibits hemagglutination, was determined for each rha protein using both antisera and compared to the cber reference ha antigen in table  using both antisera, the hai endpoints of the cys sdv h rha were equivalent or within 2-fold of the wild-type h rha and the cber reference antigen for h a/perth ha. the results confirm that cysteine substitutions introduced into the tm and ct domain of h rha did not alter the presentation of the antigenic sites.table hai titers for wild-type and cys sdv h rhas h ha protein hai titer rabbit poly clonal anti-h rha ab sheep polyclonal anti-h ha ab antigen-specific antibody production is unaffected by cysteine substitutions in the tm and ct domains a comparative study was performed in mice using the 5cys sdv rha and corresponding wild-type rha from the h a/texas strain. the mouse study was performed by josman llc and included cohorts, three for each rha antigen and one for a placebo, with acclimated balb/c mice in each cohort. on day and day of the study, mice were administered a 25 μl intramuscular injection of either the wild-type rha antigen at one of three doses or the corresponding 5cys sdv antigen at one of three doses. the placebo group received an equal volume dose of the formulation buffer. pre-immune serum samples collected on day were tested alongside serum samples collected on day in the hai assay using both the wild-type rha and sdv rha as the test antigens. the number of responders having at least a 4-fold change in geometric titer on day compared to day were similar for all test groups receiving the two different rha antigens regardless of the antigen used in the assay. further, the average geometric mean titers for all groups were comparable regardless of the antigen and dose delivered to the mice. based on the results, c-terminal cysteine substitutions and the degree of disulfide cross-linking do not impact the levels of antigen-specific antibody responses upon immunization.figure a and b pre-clinical mouse immunogenicity study results. sera samples from mice immunized with the h a/texas 5cys sdv rha antigen and with the h a/texas wild-type rha antigen were tested in the hai assay using both antigens, the h a/texas 5cys rha antigen and the h a/texas wild-type rha antigen. in each plot, the dotted line represents 4× the mean value for the day titer. discussion the influenza ha protein is an integral membrane protein and the primary target for inducing neutralizing antibodies to influenza virus. when purified to homogeneity, the protein forms a rosette structure which exposes the immunogenic, trimeric ectodomain, while the transmembrane domains and short cytoplasmic tails are most likely buried inside the rosette structure. it has long been demonstrated that formation of trimeric ha is not dependent on the tm or ct domains, though it has been suggested that the tm domain may contribute to the stability of the trimer. similarly, the early stages of ha mediated membrane fusion are unaffected by deletion of the tm and ct domains and replacement by a non-proteinaceous gpi anchor. therefore, the wild-type rha and the tm and ct cysteine sdvs in this study are expected to form similar trimers and rosettes. an evaluation of the human influenza strains which fall into subtypes, h1n, h3n and, b, shows that the h rhas have five cysteine residues in the tm and ct domains. the two cysteine residues in the ct domain are highly conserved and are found in all human influenza strains. the cysteine residue positioned at the interface between the tm and ct is found in h and h has, but not in b has. the biological role of these cysteine residues has been the subject of several studies which have identified them as sites of palmitoylation/acylation involved in membrane fusion and viral replication. while these activities are indispensable for the virus, they are not required for expression and purification of the recombinant hemagglutinin protein during vaccine manufacture. in fact, replacing all five of the tm/ct cysteines in the 5cys sdv or different subsets of them in the 3cys and 2cys sdvs result in rha proteins having starting yields, final purities, and purified yields that are comparable to or better than the wild-type rha protein. in contrast, substitution of the two conserved cysteine residues in the globular head domain of the rha protein, c and c with serine residues results in low expression further confirming that the cysteine residues in the globular head domain play a critical role in the folding of hemagglutinin protein, and cannot be substituted. the folding and functional activity of the wild-type rha and the tm/ct sdvs were compared by evaluating the trypsin resistance profiles, and hemagglutination activities. trypsin resistance is a property of properly folded trimeric ha proteins, while hemagglutination requires the organization of these trimers into higher order rosette structures that can interact with red blood cells through their sialic acid and form a lattice like network. trypsin treatment cleaves properly folded, full-length, trimeric ha into two fragments, an amino-terminal ha fragment and a carboxy-terminal ha fragment, while denatured or monomeric rha is digested by the endoprotease. trypsin treatment of the wild-type and sdv rhas results in the same characteristic ha and ha cleavage with apparent molecular weights of ~ kda for ha and ~28 kda for ha. hemagglutination occurs when the red blood cells interact via their sialic acid moiety with ha rosettes, and form a lattice like network that remains in suspension. in the absence of hemagglutination, the cells do not form the lattice like network, and a halo or circle of settled cells is observed. these results demonstrate that the hemagglutination activity of sdvs of rha is comparable to the wild-type rha. further, these results suggest that the cysteine substitutions in the tm/ct domains do not impact the functional activity of h sdv rhas. having established proper folding and function, the particle size and thermal stability of the wild-type and sdv rha proteins were determined. the average particle size by dls for the wild-type rha and the tm/ct sdvs is in the range expected for rosettes structures. the volume mean diameter of the 3cys and 5cys sdvs is 35 nm and 37 nm, respectively, compared to 39 nm for the wild-type h rha protein. the diameter of the particles for the 2cys sdv is consistently 10 nm larger than the wild-type, and may reflect that the replacement of cysteine to serine at position is not optimal and leads to particles of larger size. by em, the rosettes of the wild-type and sdv rhas are strikingly similar, and indistinguishable in terms of size and appearance. in addition to particle size and morphology, the thermal stability of the rosettes in the sdv rhas as measured by dsf is similar to that of the wild-type rha. the largest difference in tm, observed between the 5cys sdv and the wild-type, is only °c. decreased thermal stability has been reported previously for three h ha variants in which one or both of the two tm cysteine residues unique to this subtype were modified. however, the thermal stability in this study was determined in a fundamentally different way on crude extracts using the hemagglutination activity assay. further, our thermal stability data are in close agreement with previously report tm values for rosettes suggesting that both the wt and cys sdv rhas are similarly highly stable. by sec, all the rha proteins have a single, primary peak with retention times differing by  minute or less. in addition, the wild-type and sdv rhas elute before the largest standard, thyroglobulin, having an approximate molecular weight of 660 kda. these data also suggest that the rha trimers assemble into rosettes structures, and the ability to form the rosettes is unaffected by the cysteine substitutions in the tm/ct domains. differences in the homogeneity of the rosette population in the wild-type and 5cys sdv rhas are detected by uplc sec analysis of the zwittergent 3– treated rha. the zwittergent 3– surfactant efficiently converts the rosettes of the 5cys sdv, but not of the wild-type rha, to trimers. due to its smaller size compared to the rosette, the rha trimer elutes off the sec column later and the peak is shifted. in contrast, zwittergent 3– treatment of the wild-type rha protein results in multiple peaks corresponding to rosette, trimer, and various species of intermediate sizes. disulfide mediating cross-linking occurs readily in the wild-type rha based on sds-page analysis, and the various cross-linked states of the protein result in multiple elution peaks as the zwittergent 3– surfactant cannot disrupt the disulfide bonds. though the overall folding, thermal stability, size, and morphology are the same for the wild-type and tm/ct sdvs, differences in the formation of disulfide mediated cross-linked structures have been observed by sds-page. disulfide mediate cross-linking occurs readily at the c-terminus of the ha molecule through free cysteines located in the tm and ct domains. in the wild-type rha and 2cys sdv rha, this cross-linking occurs during the purification process and is extensive. cross-linked dimers, trimers, as well as higher order cross-linked oligomers are observed immediately following purification on day albeit at a slightly reduced level in the 2cys sdv. in the presence of reducing agent, the oligomers are converted to monomeric ha confirming that the cross-linking is disulfide mediated and due to the oxidation of free cysteine residues. cross-linking of the 5cys sdv rha is not observed even after 28 days at room temperature, while cross-linked dimers and trimers of the 3cys sdv rha are first observed on the day time point. the non-reducing and reducing sds-page results for the 5cys sdv and corresponding wild-type rha from the h a/texas strain further support the findings obtained with the h a/perth sdvs. the formation of non-native disulfide bonds is prevented in h rha proteins by replacing all five tm and ct cysteine residues. based on the sds-page results, the rha protein is highly susceptible to oxidation through cysteines in both the tm and ct domain. the subset of cysteines modified in the 3cys sdv appears to be more susceptible to cross-linking than the subset of cysteines in the 2cys sdv. the latter two substitutions have little effect on the sds-page profile of the 2cys sdv compared to the wild-type rha, while the former three substitutions markedly reduce the formation of cross-linked rha in 3cys sdv. these results are not unexpected as the cysteines in the ct domain are more exposed at the c-terminus of the molecule. free cysteine at this position may permit the cross-linking of monomers within a given trimer and between monomers of different trimers. the rp-hplc profiles for h a/perth rhas and the h a/texas rhas are consistent with the non-reducing sds-page analyses. the multiple elution peaks observed for the wild-type rhas and 2cys sdv rhas may be due to different cross-linked populations. alternatively, they may due to the covalent attachment of fatty acids through thio-ester linkages involving the last three c-terminal cysteine residues present in both of these rha molecules, but absent in the 3cys and 5cys sdv rha proteins. both types of populations, cross-linked rha and acylated rha, are more likely to be retained on the column due the increased hydrophobic character and elute later in the gradient as observed here in the rp-hplc profiles for the wild-type and 2cys sdv rha. concurrent with evaluation of the cross-linking, the potency of the wild-type and sdv rha proteins from the h a/perth and h a/texas strains were measured by the srid assay. by this method, the relative potency is directly proportional to the number of tm/ct substitutions and inversely correlated with cross-linking. in the 5cys sdv rha in which all five tm/ct cysteines have been replaced, the potency relative to day remains at 100% even after 12 months at room temperature for both the h a/texas and h a/perth 5cys sdv rhas, and no disulfide mediated cross-linking is observed. by comparison, the relative potency of the wild-type h rhas drop approximately 75% after 4 weeks of storage at 25°c, and demonstrates extensive cross-linking. compared to the wild-type rhas, relative potency of the 2cys sdv drops to 50% during the same time. furthermore, the relative potency of the 3cys sdv, drops only 20% after 4 weeks storage at 25°c. while cross-linking is markedly reduced for the 3cys sdv rha, the 2cys sdv shows only a marginal improvement over the wild-type rha protein. these results clearly demonstrate that the substitution of cysteine residues in the ct domain has a significant impact on potency, but substituting cysteine residues in both the tm and ct domains has a synergistic effect on potency of the rha. while including a reducing agent and redox buffer, stg and citrate, in the formulation buffer has been shown to prevent an apparent potency loss associated with the formation of non-native disulfide bonds and higher order cross-linked oligomers of the ha, the results of this study clearly show that replacing the five cysteines in the tm and ct domains with an alternative amino acid has the same effect and is a highly effective alternative solution to address potency loss. the ability of these cysteines to cross-link appears to be a direct consequence of the production and purification process that releases the tm domain from the constrained environment of the membrane bilayer and the ct domain from the reducing environment of the cell. since the three dimensional structure of the c-terminus has not been solved by x-ray crystallography, a theoretical structural analysis of ha using a membrane protein topology prediction algorithm was performed assuming that the transmembrane region of the ha molecule adopts and continues the alpha- helical conformation of the proceeding ha domain. using the program tmhmm and the ha sequence from h a/perth, seven symmetrical alpha helical trimer configurations are possible. based on this model, the tm and ct cysteines are in the correct orientation and geometry to form both intra-molecular cross-links within a trimer and inter-molecular cross-links between different trimers. amino acids with a spacing of three or four may be found on the same face of an alpha helix and cysteine residues in those positions can form disulfide bonds between two adjacent helices, thus covalently linking helices. cysteine residues on the outside of the helices may participate in the covalent crosslinking of higher order oligomers. the association of ha trimers through their hydrophobic transmembrane domains in rosette structures also facilitates intermolecular cross-linking by positioning the c-terminal cysteines from different trimers in close proximity to each other.figure modeling of the tm and ct domains in the ha trimer. the top shows the amino acid sequence of the carboxy terminus for h a/perth ha with the tm and ct cysteines highlighted in yellow, and the possible orientations of that sequence in a helix with the interfacial positions highlighted in pink. the schematics depict the trimer configuration with positions labeled a through g on the bottom left and one possible configuration on the bottom right. while both intra- and inter-molecular cross-linking of the ha trimer are possible, the latter may reduce diffusion, and/or antibody recognition in the srid assay. prior to analysis in the srid assay, the rha proteins are pre-treated with zwittergent 3– surfactant into order to dissociate non-covalently bound rha rosettes structures into soluble trimers. sec-uplc analysis of rha before and after zwittergent 3– treatment clearly shows that only a portion of the wild-type rosettes are converted to trimers; in contrast, the rosettes of the 5cys sdv are quantitatively converted to trimers. due to their small size, rha trimers can readily diffuse in to the agarose gel containing antibody and the size of the resulting precipitin ring is directly proportional to the antigen concentration. in contrast, trimers covalently bound together in rosettes through disulfide bonds are resistant to zwittergent 3– pre-treatment. depending on the extent of oxidation and/or the orientation of the cross-linked trimers, mobility of the particles through the gel may be hindered resulting in artificially small ring sizes. alternatively, access to the epitopes may become increasingly hindered as the rosettes become covalently cross-linked through disulfide bonds resulting in a reduction in antibody binding and signal. due to the inability of the srid assay to accurately measure the rosette in a cross-linked state, an artificial or apparent potency loss is observed as the number of these oxidized species increases with storage of the rha protein. ha rosettes have been considered an indicator of immunogenicity because of their resemblance to the infectious influenza virions having ha trimeric spikes protruding from the viral envelope. additionally, the antigenic sites on the ha molecule responsible for eliciting neutralizing antibodies are located in the globular head domain approximately 135 Å away from the cysteine substitutions located at the opposite end of the elongate molecule in the tm and ct domains. despite this wide physical separation, structural perturbations from side chain mutations have been shown to be propagated over long distances through the peptide backbone. for this reason, the antigenic and immunological affects of c-terminal substitutions on the rha protein were determined. the hemagglutination inhibition assay was performed using the sdv and wild-type h a/perth rha proteins in order to determine and compare recognition and binding to antibodies in sera produced using the wild-type rha antigen. the hai titers for the sdvs were comparable to the wild-type rha indicating that the antigenic presentation of the epitopes on the sdvs has not been changed by the cysteine substitutions. also, mice were immunized with either the h a/texas 5cys sdv or the corresponding wild-type rha antigen. based on similar hai titers obtained in each group, the cysteine substitutions have no effect on the levels of rha-specific antibody responses induced upon immunization. thus, the cross-linked trimers in the wild-type rha rosettes and the un-cross-linked trimers in the 5cys sdv rha rosettes induce comparable antibody responses in mice upon immunization. the results suggest that the potency loss observed for the wild-type rha protein over time is largely due to the inability of the required potency assay to measure higher order cross-linked forms of the protein that retain their immunogenicity. future work will include clinical trials to determine the impact on immunogenicity in human subjects. in the manufacture of the rha vaccine, the unique environment permits the formation of non-native disulfide bonds through free cysteines located in the tm and ct domains of the wild-type rha protein. c-terminal cross-linking may occur to some extent in traditional split and subunit influenza vaccines as the purification process uses a detergent extraction step which may liberate the c terminus of the ha from the viral membrane. while both inter- and intra-molecular cross-linking can occur, only the latter results in cross-linked rosette structures that remain immunogenic, but are not accurately measured in the srid assay. as a result, an apparent potency loss is observed with storage of the wid-type rha vaccine. cysteine mutagenesis is one way to overcome protein oxidation without the introduction of reducing agents in the formulation. however, the utility of this strategy is largely limited to the recombinant influenza vaccine since site specific mutagenesis of the virus prior to amplification in chicken cells or mammalian cells would represent a significant challenge.  rha proteins form higher-order, defined oligomers that become covalently cross-linked through cysteine residues in the tm and ct domains. replacement of these cysteine residues prevents disulfide bond formation at the c-terminus, but does not impact the expression, purification, or folding of the molecule based on our work as well as others. moreover, the particle size and morphology, thermal stability, and antigenicity of the molecule are unaffected. application of the cysteine mutagenesis strategy to rha proteins derived from the other seasonal influenza subtypes, and pandemic strains is currently underway, and a clinical trial is being planned. this technology may be extremely important for pandemic preparedness and would greatly enhance the value of the stock pile if the vaccine is not subject to potency loss at room temperature. 
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. the five cysteine residues in the transmembrane and cytoplasmic domains of the rha protein from the h a/perth/16/ human influenza strain have been substituted to alanine and/or serine residues to produce three different site directed variants. the strategy of substituting tm and ct cysteines to prevent potency loss has been successfully applied to another h rha protein further demonstrating the utility of the approach. 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. during purification and storage of the rha, disulfide mediated cross-linking of the trimers within the rosette occurs and results in reduced potency. 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.
 recombinant hemagglutinin is the active component in flublok®; a trivalent influenza vaccine produced using the baculovirus expression vector system. 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 assay to determine the amount of ha that has the correct antigenic form.  the five cysteine residues in the transmembrane and cytoplasmic domains of the rha protein from the h a/perth/16/ human influenza strain have been substituted to alanine and/or serine residues to produce three different site directed variants. 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 h rha protein further demonstrating the utility of the approach.  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. keywords hemagglutinininfluenzavaccinepotencyprotein cross-linkingprotein stabilityantigencysteineissue-copyright-statement© the author 2014 