egg- or cell culture-derived hemagglutinin mutations impair virus stability and antigen content of...

1 Introduction

According to the WHO statistics, influenza annually caus-es three to five million cases of severe illness and 250,000to 500,000 deaths worldwide. The WHO acknowledgesthat the current manufacturing capacity is insufficient formeeting global vaccine needs and requires new effectiveproduction technologies.

The only controlled seed material available today forthe production of influenza vaccines are egg-derived wildtype strains and high growth reassortants (based on theA/Puerto Rico/8/34 (PR8) backbone) provided by theNational Institute of Biological Standards and Controls,

Research Article

Egg- or cell culture-derived hemagglutinin mutations impairvirus stability and antigen content of inactivated influenzavaccines

Sabine Nakowitsch1, Andrea M. Waltenberger1, Nina Wressnigg1, Nicole Ferstl1, Andrea Triendl1, Bettina Kiefmann1, Emanuele Montomoli2, Giulia Lapini2, Maria Sergeeva3, Thomas Muster1 and Julia R. Romanova1,3

1 Avir Green Hills Biotechnology AG, Vienna, Austria2 Dept. of Physiopathology Experimental Medicine, Public Health, University of Siena, Siena, Italy3 Research Institute of Influenza, Saint Petersburg, Russian Federation

Egg-derived viruses are the only available seed material for influenza vaccine production. Vaccinemanufacturing is done in embryonated chicken eggs, MDCK or Vero cells. In order to contributeto efficient production of influenza vaccines, we investigate whether the quality of inactivated vac-cines is influenced by the propagation substrate. We demonstrate that H3N2 egg-derived seedviruses (A/Brisbane/10/07, IVR147, and A/Uruguay/716/07) triggered the hemagglutinin (HA)conformational change under less acidic conditions (0.2–0.6 pH units) than antigenically similarprimary isolates. This phenotype was associated with HA1 (A138S, L194P) and HA2 (D160N) sub-stitutions, and strongly related to decreased virus stability towards acidic pH and elevated tem-perature. The subsequent propagation of H3N2 and H1N1 egg-derived seed viruses in MDCK andVero cells induced HA2 N50K (H1N1) and D160E (H3N2) mutations, improving virus growth incell culture but further impairing virus stability. The prevention of the loss or recovery of stabilitywas possible by cultivation at acidified conditions. Viruses carrying less stable HAs are more sen-sitive for HA conformational change during concentration, purification and storage. This resultsin decreased detectable HA antigen content – the main potency marker for inactivated influenzavaccines. Thus, virus stability can be a useful marker for predicting the manufacturing scope ofseed viruses.

Keywords: Hemagglutinin antigen content · Inactivated vaccines · Influenza virus · pH stability · Temperature stability

Correspondence: Dr. Julia Romanova, Avir Green Hills Biotechnology AG,Forsthausgasse 11, A–1200 Vienna, AustriaE-mail: [email protected]

Abbreviations: CE, embryonated chicken eggs; E5, virus originated after fiveconsecutive passages in CE; HA, hemagglutinin HAU, hemagglutinationunit; MDCK, Madin Darby canine kidney cells; MES, 2–(N-morpholino)ethanesulfonic acid; MOI, multiplicity of infection; M5, virusoriginated after five consecutive passages in MDCK; NIBSC, National Insti-tute of Biological Standards and Controls; NP, nucleoprotein; PR8, A/Puer-to Rico/8/34; SFM, serum-free medium; SRID, single radial immunodiffu-sion; TCID50, tissue–culture infectious dose; V5, virus originated after fiveconsecutive passages in Vero cells; V5_acidic, virus originated after fiveconsecutive passages in Vero cells under acidified conditions.

Biotechnol. J. 2014, 9, 405–414 DOI 10.1002/biot.201300225

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BiotechnologyJournal

© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 405

Received 28 MAY 2013Revised 14 AUG 2013Accepted 08 OCT 2013Accepted article online 23 OCT 2013

Supporting information available online

The copyright line has been changed since first published on 10 December 2013.

UK (NIBSC). The use of embryonated chicken eggs (CE)for virus isolation is considered to be safe for humans asdemonstrated by the established safe track record of thismethod. The majority of existing influenza vaccines areinactivated subunit or split formulations produced in CE.Often some seed viruses, particularly of the H3N2 sub-type, replicate poorly in CE and require adaptation byserial passages. For instance, during the 2003–2004 sea-son, the production of influenza vaccine was delayed dueto the poor growth of the A/Fujian/411/02 (H3N2) virus [1].In 2008, the WHO substituted the recommended virusA/Brisbane/10/07 (H3N2) by A/Uruguay/716/07 becauseof its low production yield. Passages through CE areknown to select mutations in the HA, which mightchange the receptor-binding specificity, antigenicity, andimmunogenicity of vaccine strains [1–4].

The most developed alternatives to CE are Madin Dar-by canine kidney cell (MDCK) and Vero lines, which arealready used for two licensed influenza vaccines (Novar-tis Optaflu©, Baxter Celvapan©) [5, 6]. Previously, wefound that virus propagation in Vero cells induces muta-tions that impair virus stability, leading to decreasedimmunogenicity of live influenza vaccine candidates [7].The purpose of this study was to investigate whetherpropagation in CE and MDCK cells have a similar effect onvirus stability and to estimate the role of virus stability forthe main potency marker of inactivated influenza vac-cines – the HA antigen content.

2 Materials and methods

2.1 Cells and viruses

The Vero cell line (African green monkey kidney cells,ATCC CCL–81, WHO-certified) was adapted to and culti-vated in a serum-free medium (SFM) OptiPRO SFM (Invit-rogen, Austria) supplemented with 4 mM L–glutamine(Invitrogen, Austria) at 37°C and 5% CO2. MDCK (ATCCCCL–34) were maintained in Dulbecco’s Modified EagleMedium (Invitrogen, Austria) containing 2% fetal bovineserum (Invitrogen) and 2 mM L-glutamine at 37°C and5% CO2. Influenza seed viruses used in this study aredescribed in Table 1. The primary isolates A/Nizhniy Nov-gorod/668/08 (GenBank HA Acc.No. JQ655462, 3 pas-sages in Vero) and A/Astrahan/10/07 (GenBank HAAcc.No. JQ655463, 2 passages in MDCK) contained bothno mutations in HA and NA compared to the swab virus-es.

2.2 Virus propagation and titration

Egg-derived viruses were propagated in the allantoic cav-ity of 9–11 days old specific pathogen-free CE. H1N1 andH3N2 viruses were amplified at 37°C for 48 h and B virus-es at 33°C for 72 h.

Vero or MDCK cells were inoculated with viruses dilut-ed in SFM and incubated at 33°C for 30 min. Then inocu-lum was removed and cells were cultivated in SFM sup-plemented with 5 μg/mL trypsin (Sigma–Aldrich, Austria)at either 37°C for 72 h (H1N1 and H3N2) or at 33°C for120 h for influenza B virus with the addition of 250 ng/mLAmphotericin B (Bristo–Myers Squibb, Austria).

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Table 1. List of viruses used in this study

Abbreviation Subtype Strain name Source Description

H3N2 A/Astrahan/10/07 Institute of Influenza, primary isolate, BN10-likeSt. Petersburg, Russia

BN10 H3N2 A/Brisbane/10/07 NIBSC, 07/205

BR59 H1N1 A/Brisbane/59/07 NIBSC, 07/346

H3N2 IVR147 NIBSC, 07/246 high growth reassortant of BN10 with PR8

H3N2 A/Nizhniy Novgorod/ Institute of Influenza, primary isolate, BN10-like668/08 St. Petersburg, Russia

H3N2 NYMC X-161B NIBSC, 06/242 high growth reassortant of WI67 with PR8

UR716 H3N2 A/Uruguay/716/07 NIBSC, 07/354

H3N2 A/Vienna/25/07 Institute of Virology, primary isolate, WI67-likeVienna, Austria

WI67 H3N2 A/Wisconsin/67/05 NIBSC, 06/184

B B/Malaysia/2506/04 NIBSC, 07/184

Passaging at acidified conditions was done accordingto the method described by Nakowitsch et al. [7], i.e. inoc-ulating Vero cells with viruses diluted in 2–(N-morpholi-no)ethanesulfonic acid (MES) infection buffer adjusted topH 5.6 supplemented with 250 ng/mL Amphotericin Band incubated at 33°C for 30 min. After the removal of theinoculum, cells were cultivated in SFM adjusted to pH 6.5.Infectious virus titers were determined in Vero or MDCKcells and expressed as 50% tissue-culture infectious dos-es (TCID50/mL) as mean of three independent titrations[8].

2.3 Hemifusion assay, thermo stability and infectivity of viruses at different pH

The hemifusion, thermo stability and virus infectivityassays at different pH value were performed as describedelsewhere [7]. For the hemifusion assay Vero cells wereinfected with viruses at multiplicity of infection (MOI) 2diluted in SFM. Infected cells were incubated overnight at37°C (influenza A) or one day at 33°C (influenza B). AfterHA activation by trypsin and incubation with 0.1% 2 mMoctadecyl rhodamine B chloride (R18) (Invitrogen, Aus-tria), labeled erythrocytes (Siemens Healthcare, Austria),the resulting cell-erythrocyte complexes were treatedwith MES buffers of different pH. The critical pH of fusionwas assayed microscopically with 200× magnification byestimating the octadecyl rhodamine B chloride-emittedred fluorescence after the transfer of the dye from ery-throcytes to the cells.

For the infectivity assay, Vero cells were inoculated for30 min at 33°C with viruses diluted to MOI 5 in MES infec-tion buffers. After inoculum removal, cells were incubat-ed in SFM at 37°C for 5 h for influenza A and at 33°C for18 h for influenza B. After fixation, permeation, and block-ing, influenza A-infected cells were stained with mouseanti-influenza A Nucleoprotein (NP) antibodies (Millipore,USA) and influenza B-infected cells were stained withmouse anti-influenza B NP antibodies (Millipore, USA).Infected cells were detected by inducing fluorescence ofgoat anti-mouse Alexa Fluor 488 antibodies (Invitrogen,Austria) using a fluorescent microplate reader at 485 nm.Images were taken on an Olympus CKX41 FluorescenceMicroscope with connected Olympus camera systemE330 with 40× magnification.

2.4 Virus sequencing

Viral RNA was isolated using the QIAmpViral RNA MiniKit (Qiagen, Germany) and processed by RT–PCR (Super-script II [Invitrogen], Go Taq Polymerase [Promega, Ger-many] and Pfu Turbo Polymerase [Stratagene, Germany]).The amplified bands were purified using the Qiaex II GelExtraction kit (Qiagen, Germany) and sequenced by VBC-Biotech Service GmbH (Austria). For presentation of themutations in the influenza A HA protein sequences the

H3 (A/Aichi/2/68) numbering system was used through-out the study.

2.5 Inactivated virus preparation and characterization

Virus supernatants were inactivated by the addition offormaldehyde solution (0.0185% final concentration) andincubation for 24 h at 37ºC. Supernatants were purified bycentrifugation through a 20% sucrose cushion in PBS at113,000 g for 90 min at 4°C in an SW28 rotor. The pelletwas resuspended in TBS buffer (50 mM Tris-HCl, 150 mMNaCl, pH 7.4).

The single radial immunodiffusion (SRID) test was per-formed according to the method described in [9]. Virussamples were analyzed by two operators in duplicate. Thetotal protein content of concentrated preparations wasdetermined by the Quick Start Bradford Protein Assay (Bio-rad, Austria). All the measurements were done in triplicate.

3 Results

3.1 Mutations in HA after the propagation of egg-derived seed viruses in CE, MDCK, or Vero cells

To determine the impact of different propagation sub-strates, egg-derived seed influenza A (BR59 and BN10)and influenza B viruses originated from NIBSC were prop-agated in parallel for five consecutive passages in CE(resulting in variant E5), MDCK (resulting in variant M5)and Vero cells (resulting in variant V5). The HA and NAgenes of the resultant variants were sequenced and com-pared to those of the original seed strains.

Propagation of H1N1 and H3N2 viruses in both celllines led to the selection of various mutations (Table 2, Fig. 1A and 1B). Passages of BR59 (H1N1) in MDCK cells(BR59-M5) resulted in two mutations HA1 N190V and HA2S151T, while passages in Vero cells (BR59-V5) inducedthe same HA1 N190V substitution in combination withHA2 N50K. The propagation of BN10 (H3N2) in both celllines led to the selection of the identical single alterationHA2 D160E. Propagation in CE did not induce any muta-tions in the HA of any egg-derived influenza A virus. Nomutations were observed in the NA protein of H1N1 andH3N2 viruses passaged in any system. We also did notdetect mutations in the HA or NA proteins of influenza Bviruses after passages in any substrate. Hence, the acqui-sition of mutations in seed viruses was restricted to theHA of H1N1 and H3N2 subtypes, when propagated inMDCK and Vero cells.

3.2 Passages in cell-culture affect virus stability

Some of the mutations found in the H1N1 and H3N2 virus-es were located in the HA stalk region and could poten-

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Figure 1. Location of mutations on an HA three dimensional structure model. Mutated residues are indicated on (A) a top and (B) a side view of the HAtrimer. (C, D) The effect of substitutions at the HA1 positions 156, 186 and 194 (colored in magenta). RBS – receptor-binding site. Parts (A, B) were gener-ated with the Cn3D Molecule Viewer provided by NCBI, structure 2HMG, Protein Data Bank; structures (C, D) were generated with Phyre2 [33] on the basisof 1HA0 PDB structure and visualised with Chimera 1.5.3 software [34].

Table 2. Properties and HA sequences of NIBSC derived viruses of different origin

Subtype Virus Origin Amino acid substitutions Fusion pH Infectivity pH HA activity thresholda) thresholdb) temp.

thresholdc)

H1N1 BR59 NIBSC 5.7 5.6 67.0°CBR59-V5 Vero, neutral HA1 N190V, HA2 N50K 6.1 6.0 60.0°CBR59-V5_acidic Vero, acidified HA1 N190V 5.7 5.6 67.0°CBR59-M5 MDCK, neutral HA1 N190V, HA2 S151T 5.7 5.6 67.0°CBR59-E5 Egg – 5.7 5.6 67.0°C

H3N2 BN10 NIBSC 6.0 5.8 59.0°CBN10-V5 Vero, neutral HA2 D160E 6.1 6.1 55.5°CBN10-V5_acidic Vero, acidified HA1 H156Q, HA1 G186V, HA1 P194L 5.6 5.4 62.5°CBN10-M5 MDCK, neutral HA2 D160E 6.1 6.1 55.5°CBN10-E5 Egg – 6.0 5.8 59.0°C

B B NIBSC 5.6 5.8 62.5°CB-V5 Vero, neutral – 5.6 5.8 62.5°CB-M5 MDCK, neutral – 5.6 5.8 62.5°CB-E5 Egg – 5.6 5.8 62.5°C

a) The highest pH inducing HA fusion, data shown in Fig. 2Ab) The lowest pH allowing cell-culture infection, data shown in Fig. 2Bc) The lowest temperature destroying the HA activity, data shown in Fig. 2C

tially shift the threshold pH of the HA conformationalchange (pH of fusion or pH of activation) [7, 10-13]. There-fore, this parameter was assessed for all the viruses stud-ied using the hemifusion assay (Fig. 2A, Table 2). Wefound that the threshold pH of HA fusion activation ofBR59-V5 (H1N1) increased by 0.4 units when compared tothe initial BR59 virus. However, no changes wereobserved for the BR59-M5 variant.

Analyzing the H3N2 viruses, we noticed that thethreshold pH value of HA fusion activation found for the

BN10 (pH ≤6.0) was higher than that observed for BR59(pH ≤5.7). Propagation of BN10 in both cell lines (BN10-V5and BN10-M5) induced a further slight increase (pH =6.1)of this value.

Previously we demonstrated that an increased pHthreshold of HA conformational change is strongly relatedto reduced virus stability at low pH and elevated temper-ature [14]. The investigation of virus infectivity at variouspH values showed an increased sensitivity to acidic con-ditions for the BR59-V5 variant by 0.4 pH units (pH ≥6.0)




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3.3 H3N2 seed viruses are less stable than primaryisolates

By analyzing the H3N2 viruses, we discovered that thethreshold pH value of HA fusion activation found for theBN10 egg-derived virus (pH ≤6.0) was higher than thatobserved earlier for H3N2 human primary isolates(pH ≤5.8) [7, 15, 16]. In order to check whether BN10 lostits stability during isolation in CE, we compared the sta-bility of three H3N2 seed viruses of different passage his-tory with antigenically similar primary isolates. Thus, twoisolates, A/Nizhniy Novgorod/668/08 and A/Astra-han/10/07 were compared with BN10, IVR147, and UR716(Table 3). Investigation of the pH threshold of the HA con-formational change disclosed that A/Nizhniy Nov-gorod/668/08 and A/Astrahan/10/07 performed fusion at alower pH (pH ≤5.8 and ≤5.6, respectively) than BN10(pH ≤6.0) or UR716 and IVR147 (pH ≤6.2) (Fig. 3A). Deter-

compared to BR59 (pH ≥5.6) (Table 2, Fig. 2B). The infec-tivity pH threshold of BN10 (pH ≥5.8) increased by 0.3 pHunits (pH ≥6.1) after passages in MDCK (BN10-M5) andVero cells (BN10-V5). BR59-V5, BN10-M5 and BN10-V5also displayed decreased thermo stability, losing the HAactivity at temperatures 3.5°C to 7.0°C lower than the ini-tial BR59 and BN10 viruses (Table 2, Fig. 2C).

Passaging in CE resulted in no changes in stability atlow pH or elevated temperature for any of the virusesstudied. In addition, no phenotypic changes wereobserved after propagation of the influenza B virus in anysubstrate. These data show that the propagation ofinfluenza egg-derived seed viruses in MDCK and Verocells resulted in an increased pH threshold of HA activa-tion and led in turn to impaired virus stability at low pHand elevated temperatures.




Figure 3. Phenotypic evaluation of H3N2 viruses of different origin. (A) Comparison of the HA fusion activity at different pH values. The expressed andcleaved HAs present on the infected cells were bound to red blood cells labeled with 2 mM octadecyl rhodamine B chloride and treated with buffers at theindicated pH. Fusion-based hemifusion of the erythrocytes was assayed by fluorescence microscopy. The presented images are representative picturesfrom three independent experiments (200× magnification, bar represents 100 μm). (B) Comparison of H3N2 virus infectivity in Vero cells at different pHvalues. The viruses diluted in buffers at the indicated pH were used for infection of Vero cells. The infectivity was assayed by influenza virus NP stainingusing fluorescence microscopy. The presented images are representative pictures from three independent experiments (40× magnification, bar represents200 μm). (C) Virus HA activity was measured before and after preincubation at indicated temperatures. The lowest temperature destroying the HA activitywas indicated as stability end point. Mean values of three independent experiments are presented.

mination of the lowest pH of viral infectivity showed sim-ilar results. The primary isolates were infectious atpH ≥5.6 and ≥5.4, respectively, while the border of infec-tivity for BN10 was pH ≥5.8 and that for UR716 and IVR147was even higher (pH ≥6.1) (Fig. 3B). Both primary isolateswere also more stable at elevated temperature, losing HAactivity at higher temperatures (≥62.5°C) than BN10(≥59.0°C) and UR716 or IVR147 (≥57°C) (Fig. 3C).

The HA amino acid sequence analysis (all sequencediffererences are presented in Table 3) revealed that allegg-derived viruses differed from the primary isolates inposition HA1 194 having phenylalanine (P) instead ofleucine (L). The HA sequence of the IVR147 differed fromthat of BN10 in HA2 position D160N, as observed in bothcell-culture adapted variants BN10-V5 and BN10-M5. TheHA sequence of UR716 differed from that of BN10 in posi-tion HA1 A138S (Fig. 1 A and 1B). The substitutions D53N,K173N, and D291T found in A/Nizhniy Novgorod/668/08most likely reflect strain-specific differences.

The same phenomenon was observed for the WI67H3N2 egg-derived seed virus when compared to the cor-responding high growth reassortant NYMC X-161B andthe antigenically similar isolate A/Vienna/25/07 (Support-ing information, Fig. S1). Both egg-derived viruses (WI67and NYMC X-161B) were less stable at low pH and ele-vated temperature than the primary isolate A/Vienna/25/07. This data indicate that the conventional propagationof H3N2 viruses in any substrate (CE, Vero, or MDCK)leads to an increased pH threshold of HA conformationalchange and impaired virus stability.

3.4 Passages under acidified conditions preserve virus stability

Previously we demonstrated that passaging of H3N2viruses in Vero cells at acidified conditions preserved theHA primary structure and stability [7]. Therefore, weapplied these conditions to the propagation of BR59 andBN10 egg-derived seed viruses in Vero cells. The HA andNA genes of the resulting viruses BR59-V5_acidic and

BN10-V5_acidic were sequenced after five consecutivepassages, and the viruses were analyzed for their pH andthermo stability (Table 2).

The HA sequence of BR59-V5_acidic exhibited thesame replacement N190V in the HA1 subunit as found inall other tissue-culture adapted variants, however nomutations in the HA2 subunit occurred. The stability ofBR59-V5_acidic at low pH and elevated temperature wassimilar to that of the BR59 virus.

The resulting virus BN10-V5_acidic gained three HA1substitutions: H156Q; G186V; and P194L (Fig. 1). Exami-nation of the pH threshold of HA conformational changeshowed a decrease by 0.4 pH units (pH ≤5.6) compared tothe virus BN10 (Fig. 2A and 2B). Likewise, the stability ofBN10-V5_acidic at low pH and elevated temperatureincreased (pH ≥5.4 and 62.5°C, respectively) (Fig. 2C).These results show that the propagation of egg-derivedseed viruses in a continuous cell line under acidified con-ditions prevents the loss of or recovers virus stability.

3.5 An increased virus infectious titer does notcorrelate with a higher HA antigen content

Next we investigated the relevance of virus stability forthe HA antigen content in purified inactivated viruspreparations. The most common assay to analyze the HA content in inactivated influenza vaccines is the SRIDtest. This assay measures the immunoprecipitation areacorrelating with the amount of reactive HA antigen pres-ent in the formulation compared to reference HA antigensand antibodies. We determined the virus growth poten-tial, measured by TCID50 titer, and the HA antigen con-tent, estimated as the ratio of reactive HA/total protein[17], for pairs of concentrated purified inactivated viruspreparations (BR59-V5 versus BR59-V5_acidic and BN10-V5 versus BN10-V5_acidic) (Table 4). The detailedresults for virus yield, total protein, and SRID measure-ments are presented in Supporting information, Figs. S2and S3. BR59-V5 (H1N1) replicated to a higher peak infec-tious titer (8.7 log10TCID50/mL) than the more stable




Table 3. Properties and HA sequences of H3N2 BN10-like viruses of different origin

Virus Origin Amino acid substitutions Fusion Infectivity HA activity

HA1 HA2

pH pH temp.

53 138 173 194 291 160thresholda) thresholdb) thresholdc)

BN10 Egg D A K P D D 6.0 5.8 59.0°CUR716 Egg D S K P D D 6.2 6.1 57.0°CIVR147 Egg D A K P D N 6.2 6.1 57.0°CA/Astrahan/10/07 MDCK D A K L D D 5.6 5.4 62.5°CA/Nizhniy Novgorod/668/08 Vero N A N L T D 5.8 5.6 62.5°C

a) The highest pH inducing HA fusion, data shown in Fig. 3Ab) The lowest pH allowing cell-culture infection, data shown in Fig. 3Bc) The lowest temperature destroying the HA activity, data shown in Fig. 3C

BR59-V5_acidic virus (8.0 log10TCID50/mL). However, thecalculated HA antigen content was almost two timeshigher for BR59-V5_acidic (0.29 vs. 0.16). Interestingly,both viruses were found to have a maximal titre of128 hemaglutination units (HAU) in 50 μL. Similar resultswere obtained for the corresponding H3N2 virus pair. The less stable virus BN10-V5 replicated to a 1.0 log10higher titer (8.6 log10TCID50/mL) than BN10-V5_acidic (7.6 log10TCID50/mL), while the HA antigen content wastwo times higher for the BN10-V5_acidic virus (0.43 vs.0.21). Thus, we found that inactivated purified viruspreparations made from better growing but less stableviruses had lower HA antigen content than that of stableviruses replicating to lower infectious titers.

4 Discussion

We found that the propagation of egg-derived seed virus-es in MDCK (BN10) as well as in Vero cells (BN10 andBR59) resulted in viruses with reduced stability at acidicpH and elevated temperature. This phenotypic changewas associated with HA mutations increasing the pHthreshold of HA conformational change. For the BR59_V5H1N1 virus the mutation was located in the HA stalkregion (HA2 N50K) that is known to affect the trimer asso-ciation by means of interactions between the long and theouter HA helixes [10]. The occurrence of this mutation,and the corresponding phenotypic change, could beavoided by passaging in acidified conditions. MutationHA1 N190V, observed in both BR59 cell line adapted vari-ants, was found not to be related to virus stability. Posi-tion 190, being part of the HA receptor-binding site (RBS),has been described as the key determinant for effectivebinding to human-like receptors [18, 19]. Propagation ofthe BN10 H3N2 virus in both cell lines led to similar phe-notypic changes, and was associated with the HA2 D160Emutation located in the stem region in close proximity tothe viral membrane. Subsequent passages of egg-derivedviruses in CE did not induce any mutations. No pheno-

typic changes or mutations in HA or NA proteins wereobserved for the influenza B virus.

It is noteworthy that all the investigated H3N2 seedviruses (BN10, IVR147 and UR716) were less stable thanantigenically similar primary isolates. The reversion of theBN10 stability to the level of primary isolates wasachieved by virus propagation under acidified conditionsin Vero cells and was associated with the occurrence ofthe HA1 mutations: H156Q; G186V; and P194L. The sub-stitution P194L can be considered as the direct reversionof an egg-adapted mutation, because the MDCK isolatedprecursor of BN10 (isolated by the Australian WHOinfluenza center, GenBank Accession number EU199250)had 194L, while the egg-derived BN10 virus (sequencedby the Centers for Disease Control and Prevention,Atlanta USA, GenBank Accession number EU199466)already had 194P. By analyzing GenBank amino acidsequences we noticed that 156Q was often present inH3N2 viruses when sequenced directly from a nasal swabof influenza patients, while 156H was found more often inegg-derived sequences [20]. The amino acids G186 andP194 are known to be selected in conjunction with effi-cient virus growth in CE and MDCK cells [2, 7, 21]. There-fore, although the HA sequence of the primary isolate ofBN10 is not available, one can assume that the substitu-tions HA1: H156Q; G186V; and P194L, are related to virusisolation and adaptation to CE.

Although it was described previously that mutationsat position 186 and 194 might increase the pH of fusion by0.4 and 0.5 pH units, respectively [15], it remains unclearas to how the mutations located on the top of the HA1globular part in the vicinity of the RBS (156, 186 and 194)influence virus stability. The amino acid 156H is likely toform a positively charged cluster together with L158 andL160, which might enhance the electrostatic repulsionbetween the monomers of the HA1 globular head domainsand lower the trimer stability (Fig. 1C and 1D) [22]. Posi-tion 186 lies close to the RBS at the monomer interface,and the substitution G→V could potentially modulate themonomer interactions. The mutation L194P could break




Table 4. Comparison of the growth and HA yield of viruses varying in stability

Subtype Namea) max titre max HAU/ HA yield total protein HA contentlog10TCID50/mLb) 50 μLc) [μg/mL]d) [μg/mL]e) (HA yield/

total protein)

H1N1 BR59-V5 8.7 ± 0.4 128 32.8 ± 0.1 200 ± 4.7 0.16BR59-V5_acidic 8.0 ± 0.4 128 26.1 ± 0.3 90 ± 8.9 0.29

H3N2 BN10-V5 8.6 ± 0.1 128 17.1 ± 0.3 83 ± 12.3 0.21BN10-V5_acidic 7.6 ± 0.1 128 34.3 ± 0.4 80 ± 3.4 0.43

a) Designation of viruses after 5 passages in Vero cells at neutral (V5, unstable) or acidified (V5_acidic, stable) conditions; characterization of these viruses is shownin Table 2

b) Maximal infectious titers are shown in Supporting information, Fig. S2A and S2Bc) Maximal HA titre (HAU/50μL), data shown in Supporting information, Fig. S2C and S2Dd) HA yield at the peak infectious titre time point determined by SRID test, data shown in Supporting information, Fig. S3e) Total protein content at the peak infectious titre time point determined by Bradford assay, data shown in Supporting information, Fig. S3

the last α-helix coil, exposing the non-regular structure inthe inter-monomer space inducing destabilization [23].

Better growth of the seed viruses IVR147 and UR716(isolated on primary chicken kidney cells) in CE was asso-ciated with a decrease of virus stability by means of thesubstitutions HA2 D160N (described above) and HA1A138S. The HA1 position 138 is located at a right angle toamino acid 226 in the RBS, and was shown to be the dom-inant factor determining binding efficiency to sialic acid(Fig. 1A) [24, 25]. It remains unclear as to how a change atthis position might influence virus stability, however itseems that passages in cultured kidney cells had an evenmore dramatic effect than CE.

An HA-containing destabilizing alterations wouldconvert much easier to its low pH form when exposed tomanufacture stress factors such as temperature, pH shiftor denaturing reagents, resulting in virus degradationduring production, purification, and storage [26]. Thepotency SRID test is based on antibody binding to thenative conformation of the HA and cannot detect HAs intheir low pH form [27]. This effect can explain our obser-vation that more stable viruses provided higher HA con-tent in purified virus preparations in spite of having low-er maximal infectious titers.

The HA is the immunologically most important sur-face glycoprotein of the influenza virus and its structure,as determined by the primary protein structure and thenative trimeric conformation, might be crucial for vaccinequality. Stability was shown to be important for the infec-tivity, immunogenicity, and transmissibility of influenzaviruses [7, 14, 28]. In the context of inactivated influenzavaccines, preparations containing many HAs in the lowpH form would have decreased efficiency because theytrigger antibodies to epitopes that are not accessible dur-ing a natural influenza infection [29]. In addition, theseHAs have a tendency to form aggregates that can inducesevere adverse effects [30, 31]. The HA stability is morecrucial for vaccine preparations comprising separatedantigens and demanding intensive purification. There-fore producers of subunit, split, or recombinant vaccines(e.g. Flublok) should pay more attention to the seed mate-rial used in manufacturing. The assessment of virus sta-bility could be a useful tool to estimate the potential of aseed virus for vaccine production.

Our data confirm the recent observation of Murakamiet al. [32], demonstrating that the introduction of muta-tions into the HA2 subunit led to improved influenza virusgrowth in Vero cells. The authors suggested this methodas a good perspective for improved vaccine productiontechnology. In contrast, our data clearly show that muta-tions increasing virus growth are strongly related toimpaired virus stability and decreased HA antigen con-tent of purified virus preparations, demonstrating a nega-tive effect of passaging in Vero cells on vaccine quality.

The preservation or reversion of the stable virus phe-notype, by acidifying the cultivation conditions, enables

an important improvement that can be made to influenzavaccines that are produced in continuous cell lines.Another way to avoid adaptation mutations would be theuse of reverse genetics for the construction of influenzaseed virus reassortants. In this case, human primaryinfectious material could be used directly as a source forthe generation of plasmids encoding influenza surfaceglycoproteins. This approach has already been used suc-cessfully for the production of vaccine candidates againstH5N1, the highly pathogenic avian influenza viruses.

This work has been partially funded by the EuropeanCommission’s 6-th Framework Program projects “Intra -nasal H5 Vaccine” (SP5B-CT-2007-044512) and “FLUVACC”(LSHB-CT-2005-518281).

Conflict of Interest: The authors declare that during thetime this work was performed all authors affiliated withAVIR Green Hills Biotechnology were paid employees.The authors E. Montomoli and G. Lapini performed theSRID assay as contract work. M. Sergeeva prepared thedoctoral thesis under the supervision of Dr. Romanova andhad no commercial conflict of interest.

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