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Improving influenza virus backbones by including terminal regions of MDCK-adapted strains on hemagglutinin and neuraminidase gene segments
Vaccine 31 (2013) 4736–4743
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Improving influenza virus backbones by including terminal regions of MDCK-adapted strains on hemagglutinin and neuraminidase gene segments Raúl C. Gomila, Pirada Suphaphiphat, Casey Judge, Terika Spencer, Annette Ferrari, Yingxia Wen, Giuseppe Palladino, Philip R. Dormitzer, Peter W. Mason ∗ Novartis Vaccines and Diagnostics, 45 Sidney Street, Cambridge, MA 02139, United States
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Article history: Received 6 April 2013 Received in revised form 29 July 2013 Accepted 9 August 2013 Available online 20 August 2013 Keywords: Influenza Reverse genetics Hemagglutinin Chimeras Vaccine seeds
a b s t r a c t Reverse genetics approaches can simplify and accelerate the process of vaccine manufacturing by combining the desired genome segments encoding the surface glycoproteins from influenza strains with genome segments (backbone segments) encoding internal and non-structural proteins from high-growth strains. We have developed three optimized high-growth backbones for use in producing vaccine seed viruses for group A influenza strains. Here we show that we can further enhance the productivity of our three optimized backbones by using chimeric hemagglutinin (HA) and neuraminidase (NA) genome segments containing terminal regions (non-coding regions (NCRs) and coding regions for the signal peptide (SP), transmembrane domain (TMD), and cytoplasmic tail (CT)) from two MDCK-adapted high growth strains (PR8x and Hes) and the sequences encoding the ectodomains of the A/Brisbane/10/2010 (H1N1) HA and NA proteins. Viruses in which both the HA and NA genome segments had the high-growth terminal regions produced higher HA yields than viruses that contained one WT and one chimeric HA or NA genome segment. Studies on our best-performing backbone indicated that the increases in HA yield were also reflected in an increase in HA content in partially purified preparations. Our results show that the use of chimeric HA and NA segments with high-growth backbones is a viable strategy that could improve influenza vaccine manufacturing. Possible mechanisms for the enhancement of HA yield are discussed. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Influenza A viruses are enveloped, single-stranded, negativesense RNA viruses that are the prototype members of the Orthomyxoviridae family. The genome of influenza A virus contains eight segments, of which six (backbone segments) encode nonstructural and structural proteins (PB1, PB2, PA, NP, NS1, NS2, M1 and M2), and two encode the virion surface glycoproteins (HA and NA). Seasonal influenza is caused by infection with influenza A or B viruses, which are readily spread from human to human and can cause severe morbidity and mortality especially in the elderly and the very young [1]. Influenza A viruses can infect birds (an important natural host [2]), humans and other mammals (including pigs);
∗ Corresponding author. Tel.: +1 617 871 8292. E-mail addresses: [email protected] (R.C. Gomila), [email protected] (P. Suphaphiphat), [email protected] (C. Judge), [email protected] (T. Spencer), [email protected] (A. Ferrari), [email protected] (Y. Wen), [email protected] (G. Palladino), [email protected] (P.R. Dormitzer), [email protected] (P.W. Mason). 0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.08.026
reassortment can create strains to which the human population is mostly immunologically naive, as was the case for the H1N1(2009) pandemic virus [3,4]. Vaccination can be an effective strategy to protect against seasonal influenza. Much of the seasonal influenza vaccine used today is manufactured from antigen derived from chemically inactivated virus preparations. Virus seeds used for this process consist of either egg isolates derived from clinical samples or high-growth reassortants (HGRs) generated in eggs by co-infection with a WT virus and a high-yield, egg-adapted donor strain (often A/Puerto Rico/8/1934 (PR8)). Frequently, these viruses are 6:2 reassortants that contain the six backbone segments from the egg-adapted strain and the two antigenic segments from the WT strain (HA and NA segments). Other combinations of gene segments are possible, such as 5:3 reassortants (containing the HA, NA, and PB1 segments from the WT virus), which are frequently selected [5]. Although the production of HGRs in eggs has proved effective in the generation of many suitable vaccine viruses, it has proved difficult for some strains [6]. As an alternative approach, vaccine seed viruses can be isolated in qualified cell lines [7]. It is generally found that viruses isolated from mammalian cell culture do not acquire adaptive mutations in HA and NA that can alter antigenicity and
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potentially affect the efficacy of the vaccine, as has been documented for some strains isolated from eggs [8,9]. In this study, we show that three A/Brisbane/10/2010 (H1N1) reassortant viruses with influenza A backbones developed for production of vaccine seeds in MDCK 33016PF cells display superior growth and HA yield compared to a non-reassortant A/Brisbane/10/2010 virus. We show that substitution of the A/Brisbane/10/2010 HA and NA terminal region sequences with sequences from the respective segments of two MDCK-adapted influenza strains further enhances virus growth and HA yield.
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InfiniteTM 200 PRO plate reader (Tecan). Data were analyzed using GraphPad Prism software. 2.4. Hemagglutination inhibition assay The hemagglutination inhibition assay (HAI) was performed as described in the World Health Organization Manual for the Laboratory Diagnosis and Virological Surveillance of Influenza [13]. Ferret antisera FR-359 raised against A/California/07/2009 (IRR) and a 0.75% suspension of chicken erythrocytes (Lampire Biologicals) prepared in phosphate-buffered saline (PBS) were used.
2. Materials and methods 2.1. Cells, viruses and plasmids 293T cells and suspension MDCK 33016PF cells were maintained as previously described [10]. The A/Brisbane/10/2010 virus was obtained from the CDC. The PR8x (H1N1) strain is a derivative of a PR8 strain that was adapted to MDCK 33016PF cells by 5 serial passages [11]. The A/Hessen/105/2007 (Hes) strain is a derivative of a clinical isolate of an A/New Caledonia/20/1999-like virus (H1N1) that was adapted to MDCK 33016PF cells by 30 serial passages [11]. The eight segments from PR8x and Hes, the PB1 segment from A/California/07/2009 and the HA and NA segments from A/Brisbane/10/2010 were cloned in plasmid pKS10 for virus rescue as previously described [10]. HA terminal region chimeras were generated using overlap PCR and cloned into pKS10 as previously described [10]. Overlap PCR and Quikchange (Agilent) mutagenesis were used to generate the NA terminal region chimeras. All plasmids were sequence verified before use in rescue experiments. 2.2. Virus growth in MDCK cells 10 ml suspension cultures of MDCK 33016PF cells (1 × 106 cells/ml) were inoculated with virus at a multiplicity of infection (MOI) of 0.001 and incubated in TubeSpin® Bioreactor 50 (TPP) as previously described [11]. Samples were taken at 0 and 60 h post-infection and frozen at −80 ◦ C until processed. Analogous methods were used for preparations of 60 ml cultures grown in 125 ml shake flasks. Viral titers were determined using a previously described focus-formation assay [12] with slight modifications. Infectious foci were detected using an Alexa Fluor® 488-conjugated goat anti-mouse IgG (Invitrogen), and quantified with a BioSpot® Analyzer (CTL).
2.5. Sucrose density gradient (SDG) separation 40 ml of the harvested medium were concentrated ∼16-fold by centrifugal ultrafiltration (Vivaspin 20 with 300 kDa molecular weight cut-off, Sartorius-Stedim Biotech), and viruses were purified as previously described [11]. A hemagglutination assay with 0.5% guinea pig red blood cells (Cleveland Scientific) was performed to identify the fractions with the highest virion content, which were then pooled. The protein content of the pooled fractions was determined using a BCA assay (Pierce), following the manufacturer’s directions. 2.6. Reversed-phase HPLC (RP-HPLC) Purified virions were analyzed by RP-HPLC as previously described [11]. The concentration of HA1 (a HA maturational cleavage fragment) was quantified using purified HA1 from A/California/07/2009 reagent (NIBSC cat # 09/146 and 09/174) and prepared using identical methods. 2.7. SDS-PAGE and PNGaseF deglycosylation assay Equal volumes from pooled virus-containing sucrose density gradient fractions were deglycosylated following the protocol of Harvey et al. [14] with minor modifications. Samples were separated using 4–12% Nu-PAGE precast gels (Invitrogen), stained overnight by shaking at room temperature using SYPRO-Ruby stain (Sigma) and destained by shaking in 10% methanol for 30 min at room temperature. Gels were scanned using a Chemidoc XRS Imager (BioRad) and analyzed using ImageJ software [15]. 3. Results
2.3. HA ELISA 384w plates (Costar) were coated O/N with Galanthus nivalis (GNA) lectin (Sigma). Plates were washed four times with wash buffer (PBS + 0.05% Tween20) and blocked with 10 mM Tris–HCl + 150 mM NaCl + 3% sucrose + 1% BSA, pH 7.68 (blocking buffer) for 1 h at room temperature. Three-fold serial dilutions of the samples containing a final concentration of 1% Zwittergent 3–14 (Sigma) were prepared, added in duplicate to the plates, and incubated at 37 ◦ C for 30 min in a shaker. Biotinylated-IgG purified from pooled sheep antisera (NIBSC cat# 11/110) raised against A/California/07/2009 (antigenically similar to A/Brisbane/10/2010) were added and further incubated at 37 ◦ C for 30 min in a shaker. Plates were then washed four times with wash buffer and incubated with streptavidin-alkaline phosphatase (KPL) in wash buffer at 37 ◦ C for 30 min in a shaker. Plates were washed four times with wash buffer and developed using 1 mg/ml p-nitrophenyl phosphate (Sigma) in DEA buffer phosphatase substrate (KPL). Plates were read after 40–50 min incubation in the dark at 405 nm using an
3.1. Viruses with optimized backbones outperform the current vaccine seed virus for growth and HA yield in MDCK cell cultures To overcome the limitations of using egg-derived HGRs as seed viruses for manufacturing vaccine in our MDCK 33016PF cell line, we developed three MDCK cell-optimized backbones (Fig 1A; [11]). Fig. 1 shows the data compiled from three independent experiments that compared the HA yield (Fig. 1B and C) and growth (Fig. 1D) of the WT A/Brisbane/10/2010 virus to that of viruses derived from the three optimized backbones (PR8x, #19 and #21) and containing the A/Brisbane/10/2010 HA and NA segments. All viruses with our optimized backbones performed better than the WT A/Brisbane/10/2010 virus. The virus with the #21 backbone (and A/Brisbane/10/2010 HA and NA) produced the highest HA yield increase by ELISA (Fig. 1B, 6-fold more than wild type, P < 0.001) and had the highest hemagglutination (HA) titer (Fig. 1C, ∼10-fold more than WT, P < 0.001) and viral titer (Fig. 1D, ∼50-fold more than WT, P < 0.05).
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Fig. 1. Backbone-derived viruses outperform WT A/Brisbane/10/2010 virus in growth and HA yield. (A) Schematic of the three RG backbones used in this study. PR8x contains six internal segments from the MDCK-adapted PR8x strain. The #19 backbone contains PB2, PB1 and NP from the MDCK-adapted Hes strain and the remaining segments from PR8x. The #21 backbone contains an A/California/07/2009-like PB1 and the remaining segments from PR8x. MDCK 33016 PF cells were infected at an of MOI of 0.001 with RG viruses containing the WT A/Brisbane/10/2010 HA and NA segments and one of our three optimized backbones (PR8x, #19 and #21) and with the WT A/Brisbane/10/2010 virus. (B) HA yield as measured by HA ELISA. (C) HA titers using 0.5% guinea pig RBCs. (D) Viral titers 60 h post-infection as determined by FFA assay. Bars represent the mean plus SEM of three independent experiments. Statistical significance was determined using one-way ANOVA. The mean value of each group was compared to WT virus using Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001.
3.2. Chimeric HA and NA segments with terminal regions from MDCK-adapted strains In addition to using reverse genetics (RG) to combine different backbone gene segments as described above, the method is ideally suited to rationally test changes to the HA and NA gene segments that can improve HA yield [14,16–19]. Based on the highgrowth properties of our MDCK-adapted strains (PR8x and Hes), we constructed chimeric HA and NA segments that combine the nonantigenic terminal regions from HA (non-coding regions (NCRs), signal peptide (SP), transmembrane domain (TMD), and cytoplasmic tail (CT)) and NA (NCRs, CT and TMD) from PR8x and Hes with the ectodomain of A/Brisbane/10/2010 (WT) HA and NA segments, respectively. Fig. 2 shows a diagram of the constructs and a sequence alignment of the terminal regions of HA (panels A and B) and NA (panels C and D). 3.3. PR8x(term) HA and NA constructs significantly enhance HA yield with the PR8x backbone Chimeric HA [14] or NA [19] segments with terminal regions from PR8 can increase HA yield or HA content, respectively, of pdmH1N1(2009) viruses. Therefore, we reasoned that combining chimeric HA and NA segments could further enhance HA yield. To test this hypothesis, we rescued viruses containing different combinations of WT A/Brisbane/10/2010 and chimeric PR8x(term) HA and NA segments with the PR8x backbone and compared the HA yield and growth of the viruses. HA yield (Fig. 3A), as measured
by HA ELISA, was 4-fold higher for the virus with PR8x(term) HA and NA segments than for the virus with WT HA and NA segments (P < 0.01). Virus with the PR8x(term) HA segment and WT NA segment yielded a 3-fold increase in HA compared to the virus with WT HA and NA (P < 0.05). Virus with PR8x(term) HA and NA segments had 2-fold higher HA titers (P < 0.05) and 4-fold higher viral titers than the virus with WT HA and NA segments (Fig. 3B and C). Overall, these data show that viruses with chimeric PR8x(term) HA and NA segments yield more HA than viruses containing only chimeric PR8x(term) HA or NA segments. These findings suggest a strategy of using chimeric HA and NA segments for enhancement of growth and HA yield from H1N1 pdm(2009) strains. 3.4. Chimeric HA and NA constructs enhance HA yield with all three optimized backbones We next tested whether the PR8x(term) or Hes(term) HA and NA segments (Fig. 2) could enhance growth and HA yield of the resulting viruses with any of the three optimized backbones (Fig. 1A). HA yield, as measured by ELISA and normalized to the yield from WT HA and NA segments, increased ∼4-fold with PR8x(term) HA and NA segments and ∼5-fold (P < 0.05) with Hes(term) HA and NA segments using the PR8x backbone (Fig. 4A). HA yield increases correlated with increases in HA titer and viral titers using the PR8x(term) and Hes(term) HA and NA constructs (Fig. 4D and G). With the #19 backbone, HA yield was ∼2.5-fold higher (P < 0.05) with the PR8x(term) HA and NA segments and ∼3-fold higher (P < 0.05) with the Hes(term) HA and NA segments over virus with
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Fig. 2. Schematic diagram and sequence alignment of chimeric HA and NA constructs. (A) HA schematic. The wild type A/Brisbane/10/2010 HA (WT Bris) is shown in white. The terminal regions of HA: NCRs, SP, TMD and CT, from two laboratory adapted strains of H1N1, PR8x (gray) and Hes (slanted lines), were grafted onto the WT A/Brisbane/10/2010 ectodomain to produce the chimeric HA segments shown. (B) Sequence alignment of the terminal regions of WT A/Brisbane/10/2010 (Bris), PR8x and Hes HA. Dashes represent nucleotides conserved among the strains. Nucleotides in bold represent mutations in the region identified by Marsh et al. as important for packaging [26]. The 3 NCR is separated from the signal peptide sequence by the solid bar. For brevity, the ectodomain sequence is omitted (. . ./. . .). The TMD is separated from the CT by the dashed line. The stop codon is underlined and followed by the 5 NCR. (C) NA schematic. The WT A/Brisbane/10/2010 NA (WT Bris) is shown in white. The terminal regions of NA: NCR, CT, and TMD from PR8x (gray) and Hes (slanted lines), were grafted onto the WT A/Brisbane/10/2010 ectodomain to produce the chimeric NA segments shown. (D) Sequence alignment of the terminal regions of A/Brisbane/10/2010 (Bris), PR8x and Hes NA. Dashes represent nucleotides conserved among the strains. Nucleotides in bold represent mutations in the region identified by Fujii et al. as important for packaging [22].The cytoplasmic tail is separated from the 3 NCR by the solid bar and from the TMD by the dashed line. For brevity, the ectodomain sequence is omitted (. . ./. . .). The stop codon is underlined and followed by the 5 NCR.
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R.C. Gomila et al. / Vaccine 31 (2013) 4736–4743 Table 1 Antigenic analysis of viruses with the three optimized backbones. Antigen
Ferret Sera FR-359a (H1N1)
A/Brisbane/10/2010
2560
PR8x + Bris(term) HA/NA PR8x + PR8x(term) HA/NA PR8x + Hes(term) HA/NA
1920 1920 1920
#19 + Bris(term) HA/NA #19 + PR8x(term) HA/NA #19 + Hes(term) HA/NA
1280 1280 2560
#21 + Bris(term) HA/NA #21 + PR8x(term) HA/NA #21 + Hes(term) HA/NA
2560 1920 1280
IVR165 (H3N2)
<10
Values represent the geometric mean of HI titers from duplicate experiments. a Sera raised against A/California/07/2009, which is antigenically similar to A/Brisbane/10/2010.
WT HA and NA segments (Fig. 4B). HA yield increases were not associated with increases in viral titers or HA titers (Fig. 4E and H). We next asked whether we could further improve our #21 backbone, which we have found to be superior (higher HA yield) to both PR8x and #19 with WT A/Brisbane/10/2010 HA and NA segments despite occasional experimental variability in FFU titers (Figs. 1D and Fig. 4G–I). Indeed, we found significant increases with PR8x(term) and Hes(term) HA and NA segments in HA yield, ∼2.5fold (P < 0.01) and ∼3-fold (P < 0.01), respectively, HA titers (2-fold (P < 0.05)) and viral titers over virus containing WT HA and NA segments (Fig. 4C, F and I). Overall, these data show that using chimeric HA and NA segments with terminal regions derived from our MDCK-adapted strains increases HA yield independent of the backbone used. Sequence analyses of the viruses recovered with each backbones and with WT or chimeric HA and NA segments confirmed their sequence identity with the plasmids used in virus rescue (results not shown). To confirm that our viruses with chimeric HA and NA segments maintain their correct antigenicity, a hemagglutination inhibition (HAI) assay was performed using ferret antisera raised against A/California/07/2009, which is antigenically similar to WT A/Brisbane/10/2010. Table 1 shows, as expected, that the viruses with the chimeric HA and NA segments are antigenically indistinguishable (within 2-fold in an HAI assay) from the reference antigen that contains the WT HA and NA segments. 3.5. Increased HA content of viruses containing chimeric HA and NA segments
Fig. 3. PR8x(term) HA and NA segments enhance HA yield over PR8x(term) HA or NA only. MDCK 33016PF cells were infected at an MOI of 0.001 with viruses with the PR8x backbone and the indicated HA and NA gene segment combinations. (A) Fold increase as measured by HA ELISA and compared to the yield using WT A/Brisbane/10/2010 HA and NA segments. (B) HA titer as determined using 0.5% red blood cells from guinea pigs. (C) Virus titers 60 h post infection as determined by FFA assay. Bars represent the mean plus SEM of three independent experiments. Statistical significance was determined using one-way ANOVA. The mean value of each group was compared to Bris HA/NA using Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01.
To further verify that the results observed using unpurified infected cell culture supernatants reflect HA yield from purified viruses, we performed additional characterizations of #21 backbone-derived viruses, which produce the highest amounts of HA (Fig. 5). To this end, two independent larger-scale amplifications (60 ml) of these viruses were performed, and viruses were purified using SDG centrifugation, as described in Section 2. HA1 yield, normalized to the original culture volume of 60 ml, was determined using HPLC. Compared to virus with WT HA and NA segments, virus with the PR8x(term) and Hes(term) HA and NA segments had ∼1.8fold increase (11.3 g/ml vs 6.2 g/ml) and a ∼2.2-fold increase (13.6 g/ml vs 6.2 g/ml) in HA yield, respectively (Fig. 5A). We next determined the HA content in these purified preparations by using either gel densitometry or a combination of HPLC measurement of HA and total protein measurement by BCA assay. For gel densitometry determination, the pooled fractions were treated with PNGaseF, resolved by SDS-PAGE, and then stained with SYPRO-Ruby to permit accurate determination of NP, HA1, M,
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Fig. 4. Chimeric HA and NA segments enhance HA yield with any of the three optimized backbones. MDCK 33016PF cells were infected at an MOI of 0.001 with viruses with one of the three optimized backbones and the indicated HA and NA gene segment combinations with the terminal regions from WT A/Brisbane/10/2010 (Bris), PR8x or Hes. Upper panels (A–C) show the fold increase in HA yield as measured by HA ELISA and compared to the yield using WT HA and NA segments (Bris). Middle panels (D–F) show HA titers 60 h post infection as determined by HA assay. Lower panels (G–I) show virus titers 60 h post infection as determined by FFA assay. Bars represent the mean plus SEM of three independent experiments. Statistical significance was determined using one-way ANOVA. The mean value of each group was compared to Bris HA/NA using Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01.
and HA2 by densitometry [14]. Fig. 5B shows the positions of these bands on the stained gel, and Fig. 5C shows that viruses with the PR8x(term) and Hes(term) HA and NA segments had increases of 14% (P < 0.05) and 32% (P < 0.01), respectively, compared to viruses containing the WT HA and NA segments. To quantitate HA1 content using our HPLC data, we expressed the HA1 values obtained by HPLC (Fig. 5A) as a fraction of the total protein content (measured by the BCA assay) of the pooled fractions, and assigned an arbitrary value of 1 to viruses with Bris(term) HA and NA. The results in Fig. 5C show that viruses with the PR8x(term) and Hes(term) HA and NA segments had increased HA content of 29% and 46%, respectively, compared to WT HA- and NA-containing viruses. 4. Discussion Influenza viruses engineered to encode chimeric HA and NA gene segments with MDCK-adapted terminal regions produced higher yields of HA in cell culture compared to viruses with full-length HA and NA segments from a seasonal strain. The MDCKadapted terminal regions of HA and NA could enhance HA yield by increasing the levels of transcription and/or replication of the
chimeric HA and NA segments. The terminal 13 and 12 nucleotides at the 5 and 3 ends, respectively, of the vRNA are highly conserved among all eight segments and strains of influenza A viruses and are proposed to form a partially double-stranded structure that is recognized by the viral polymerase. Similar “corkscrew” structures are predicted for both the vRNA promoter (to generate mRNAs and cRNAs) and the cRNA promoter (to generate vRNAs) (reviewed in [20]). The nucleotide sequences of the proposed promoter regions of HA are identical for A/Brisbane/10/2010, PR8x and Hes (Fig. 2). However, there are nucleotide differences in the NCRs that are found outside of these conserved regions. It is possible that these differences could enhance the transcriptional and/or replicative activity of the polymerase, conferring an increase in virion or HA yield of viruses containing segments with grafted PR8x or Hes termini. However, experiments using chimeric constructs containing only the NCRs of the MDCK-adapted strains did not produce the enhancement shown by the terminal region chimeras (data not shown), a result also reported by Harvey et al. [14]. Alternatively, our chimeric HA and NA segments could enhance HA yield by acting at the RNA level by increasing the packaging of genome segments into virions. A large body of data favors the selective-incorporation model for the packaging of influenza vRNAs
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Fig. 5. Enhanced HA content of viruses with the #21 backbone and the chimeric HA and NA segments. (A) HA yield of larger scale cultures (60 ml) of MDCK 33016PF cells, infected at an MOI of 0.001 with #21 derived viruses containing the indicated HA and NA pairs. HA yield was measured by HPLC after virus was concentrated and purified by sucrose-gradient density centrifugation. (B) Deglycosylation of HA (dHA) was performed using PNGaseF. Viruses were subsequently separated by SDS-PAGE and viral proteins stained using SYPRO-Ruby. (C) The HA content was calculated from gel densitometry as previously described [34] and from HPLC by dividing values from (A) over the total protein concentration in the fractions, as determined by a BCA assay. HA content values were compared to those of Bris(term) HA and NA (WT control), which were assigned a value of 1. Bars represent the mean plus SEM of two independent experiments. Statistical significance was determined using one-way ANOVA. The mean value of each group was compared to Bris HA/NA using Dunnett’s multiple comparison test. *P < 0.05, **P < 0.001.
[21]. This model states that specific signals in each vRNA segment are involved in RNA-RNA interactions that lead to the incorporation of the eight gene segments into a virion [22]. Although there has been some recent evidence that selective co-packaging is not as efficient as originally predicted [23], the ends of the genome likely play a role in packaging viral segments into virion particles. Specifically, sequences in the terminal regions of HA and NA, as well as coding sequences in all other influenza gene segments have been implicated in vRNA packaging (For reviews, see [21,24]). VRNA–vRNA interactions have been proposed to stabilize interactions between the vRNA–nucleoprotein complexes (vRNPs) inside the virions [24,25]. Marsh et al. identified a region of 15 nucleotides (nt) in the TMD of HA as important for efficient packaging [26]. Introduction of synonymous mutations into the HA segment from an A/WSN/33 and PR8 virus decreased viral titers and HA incorporation. Sequence alignment of the nucleotides encoding the TMD of WT A/Brisbane/10/2010 HA and PR8x and Hes reveals 3 nt differences between them in this 15 nucleotide region (Fig. 2B). Two of these mutations are part of a conservative Val to Leu mutation in the TMD. It is possible that the presence of the packaging signal from the grafted strain allows a more efficient incorporation of the HA genome segment into virions. A 21 nt region in the NA N-terminal region (corresponding to the first 7 amino acids of the TMD) was identified as critical for having a significant level of reporter gene segment incorporation into influenza virions [22]. Sequence alignment of the NA N-terminal regions shows that PR8x and Hes have 3 silent mutations in the 21 nt region compared to the WT A/Brisbane/10/2010 sequence (Fig. 2D). Thus, it is possible that those changes explain why viruses with PR8x(term) HA and NA segments have a higher growth and HA yield that viruses with only PR8x(term) HA segments (Fig. 3).
The terminal region sequences derived from PR8x and Hes HA and NA segments could allow a more efficient interaction with the remaining backbone gene segments (which are also mostly derived from PR8x and Hes). Mutation of the packaging signals of one gene segment has been shown to affect the packaging of other segments [26,27]. One such example is the reduction of PB1 gene segment incorporation into virions after mutation of the HA packaging signals [26,28]. Interestingly, this effect does not appear to be reciprocal, in that mutation of the PB1 packaging signals did not affect HA gene segment incorporation [28]. In addition to more efficiently incorporating HA, potentially changing the packaging sequences of HA could also increase the incorporation of additional segments. The chimeric HA and NA segments could also act at the protein level to increase the incorporation of the glycoproteins into the viral envelope. The majority of the amino acid differences between the WT and chimeric HAs are found in the signal peptide (Fig. 2B). It is possible that the cell-derived signal peptide sequences from MDCKadapted strains allow for more efficient processing and/or secretion of HA. Sequence alignment of NA shows eight amino acid differences between PR8x(term) and Bris(term) and seven amino acid changes between Hes(term) and Bris(term) (Fig. 2D). The majority of the changes are concentrated between residues 14 and 22 in the TMD, which have been shown by alanine-scanning mutagenesis to be important for incorporation of the NA glycoprotein into virions [29]. We analyzed the NA content of virions purified by SDG (Fig. 5) by performing a neuraminidase assay [30], but found it similar among the viruses (data not shown). The CT and TMD of HA and NA contain signals for apical transport of the glycoproteins to sites of virus budding [31], for interactions with lipid rafts [29,32] and for interacting with M1 [33]. It is possible that the cell-derived HA and NA terminal regions mediate a more efficient interaction with M1, helping the virus bud more efficiently and/or increasing
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the HA content of the virions. We have preliminary data suggesting that an A/Victoria/210/2009 (H3N2) strain can also be enhanced by using the MDCK-adapted terminal regions, suggesting that this strategy could be applied to additional influenza strains (RCG, PS, PRD, PWM, unpublished). In conclusion, we show that we can enhance the productivity of our three optimized backbones for virus rescue in MDCK 33016PF cells by replacing the terminal regions of the HA and NA segments with those from MDCK-adapted strains. Contributors RCG, PS, PRD and PWM conceived of the study and designed experiments; RCG cloned the HA/NA chimeras, performed virus rescue and growth analyses; RCG, TS, AF and GP carried out HA ELISA assays; CJ and YW carried out the virus purification and HPLC analysis; RCG, PS and PWM interpreted data; RCG and PWM wrote the manuscript; PS and PRD contributed to the manuscript.
[11]
[12]
[13]
[14]
[15] [16]
[17]
[18]
Conflict of interest [19]
The authors are employees of (or contractors for) Novartis Vaccines and Diagnostics, and some are Novartis shareholders. Novartis Vaccines and Diagnostics manufactures and sells influenza vaccines.
[20]
[21]
Acknowledgements This work was supported in part by BARDA contract HHSO100201000061C. We thank Heidi Trusheim (Novartis Vaccines and Diagnostics, Holly Springs, NC) for helpful discussions and comments. References [1] Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N, Anderson LJ, et al. Mortality associated with influenza and respiratory syncytial virus in the United States. J Am Med Assoc 2003;289(2):179–86. [2] Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y. Evolution and ecology of influenza A viruses. Microbiol Rev 1992;56(1):152–79. [3] Neumann G, Kawaoka Y. The first influenza pandemic of the new millennium. Influenza Other Respir Viruses 2011;5(3):157–66. [4] Garten RJ, Davis CT, Russell CA, Shu B, Lindstrom S, Balish A, et al. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 2009;325(5937):197–201. [5] Fulvini AA, Ramanunninair M, Le J, Pokorny BA, Arroyo JM, Silverman J, et al. Gene constellation of influenza A virus reassortants with high growth phenotype prepared as seed candidates for vaccine production. PLoS One 2011;6(6):e20823. [6] Nobusawa E, Ishihara H, Morishita T, Sato K, Nakajima K. Change in receptorbinding specificity of recent human influenza A viruses (H3N2): a single amino acid change in hemagglutinin altered its recognition of sialyloligosaccharides. Virology 2000;278(2):587–96. [7] Voeten JT, Brands R, Palache AM, van Scharrenburg GJ, Rimmelzwaan GF, Osterhaus AD, et al. Characterization of high-growth reassortant influenza A viruses generated in MDCK cells cultured in serum-free medium. Vaccine 1999;17(15/16):1942–50. [8] Kodihalli S, Justewicz DM, Gubareva LV, Webster RG. Selection of a single amino acid substitution in the hemagglutinin molecule by chicken eggs can render influenza A virus (H3) candidate vaccine ineffective. J Virol 1995;69(8):4888–97. [9] Stevens J, Corper AL, Basler CF, Taubenberger JK, Palese P, Wilson IA. Structure of the uncleaved human H1 hemagglutinin from the extinct 1918 influenza virus. Science (New York, NY) 2004;303(5665):1866–70. [10] Suphaphiphat P, Franti M, Hekele A, Lilja A, Spencer T, Settembre E, et al. Mutations at positions 186 and 194 in the HA gene of the 2009 H1N1
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Improving influenza virus backbones by including terminal regions of MDCK-adapted strains on hemagglutinin and neuraminidase gene segments Raúl C. Gomila, Pirada Suphaphiphat, Casey Judge, Terika Spencer, Annette Ferrari, Yingxia Wen, Giuseppe Palladino, Philip R. Dormitzer, Peter W. Mason ∗ Novartis Vaccines and Diagnostics, 45 Sidney Street, Cambridge, MA 02139, United States
a r t i c l e
i n f o
Article history: Received 6 April 2013 Received in revised form 29 July 2013 Accepted 9 August 2013 Available online 20 August 2013 Keywords: Influenza Reverse genetics Hemagglutinin Chimeras Vaccine seeds
a b s t r a c t Reverse genetics approaches can simplify and accelerate the process of vaccine manufacturing by combining the desired genome segments encoding the surface glycoproteins from influenza strains with genome segments (backbone segments) encoding internal and non-structural proteins from high-growth strains. We have developed three optimized high-growth backbones for use in producing vaccine seed viruses for group A influenza strains. Here we show that we can further enhance the productivity of our three optimized backbones by using chimeric hemagglutinin (HA) and neuraminidase (NA) genome segments containing terminal regions (non-coding regions (NCRs) and coding regions for the signal peptide (SP), transmembrane domain (TMD), and cytoplasmic tail (CT)) from two MDCK-adapted high growth strains (PR8x and Hes) and the sequences encoding the ectodomains of the A/Brisbane/10/2010 (H1N1) HA and NA proteins. Viruses in which both the HA and NA genome segments had the high-growth terminal regions produced higher HA yields than viruses that contained one WT and one chimeric HA or NA genome segment. Studies on our best-performing backbone indicated that the increases in HA yield were also reflected in an increase in HA content in partially purified preparations. Our results show that the use of chimeric HA and NA segments with high-growth backbones is a viable strategy that could improve influenza vaccine manufacturing. Possible mechanisms for the enhancement of HA yield are discussed. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Influenza A viruses are enveloped, single-stranded, negativesense RNA viruses that are the prototype members of the Orthomyxoviridae family. The genome of influenza A virus contains eight segments, of which six (backbone segments) encode nonstructural and structural proteins (PB1, PB2, PA, NP, NS1, NS2, M1 and M2), and two encode the virion surface glycoproteins (HA and NA). Seasonal influenza is caused by infection with influenza A or B viruses, which are readily spread from human to human and can cause severe morbidity and mortality especially in the elderly and the very young [1]. Influenza A viruses can infect birds (an important natural host [2]), humans and other mammals (including pigs);
∗ Corresponding author. Tel.: +1 617 871 8292. E-mail addresses: [email protected] (R.C. Gomila), [email protected] (P. Suphaphiphat), [email protected] (C. Judge), [email protected] (T. Spencer), [email protected] (A. Ferrari), [email protected] (Y. Wen), [email protected] (G. Palladino), [email protected] (P.R. Dormitzer), [email protected] (P.W. Mason). 0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.08.026
reassortment can create strains to which the human population is mostly immunologically naive, as was the case for the H1N1(2009) pandemic virus [3,4]. Vaccination can be an effective strategy to protect against seasonal influenza. Much of the seasonal influenza vaccine used today is manufactured from antigen derived from chemically inactivated virus preparations. Virus seeds used for this process consist of either egg isolates derived from clinical samples or high-growth reassortants (HGRs) generated in eggs by co-infection with a WT virus and a high-yield, egg-adapted donor strain (often A/Puerto Rico/8/1934 (PR8)). Frequently, these viruses are 6:2 reassortants that contain the six backbone segments from the egg-adapted strain and the two antigenic segments from the WT strain (HA and NA segments). Other combinations of gene segments are possible, such as 5:3 reassortants (containing the HA, NA, and PB1 segments from the WT virus), which are frequently selected [5]. Although the production of HGRs in eggs has proved effective in the generation of many suitable vaccine viruses, it has proved difficult for some strains [6]. As an alternative approach, vaccine seed viruses can be isolated in qualified cell lines [7]. It is generally found that viruses isolated from mammalian cell culture do not acquire adaptive mutations in HA and NA that can alter antigenicity and
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potentially affect the efficacy of the vaccine, as has been documented for some strains isolated from eggs [8,9]. In this study, we show that three A/Brisbane/10/2010 (H1N1) reassortant viruses with influenza A backbones developed for production of vaccine seeds in MDCK 33016PF cells display superior growth and HA yield compared to a non-reassortant A/Brisbane/10/2010 virus. We show that substitution of the A/Brisbane/10/2010 HA and NA terminal region sequences with sequences from the respective segments of two MDCK-adapted influenza strains further enhances virus growth and HA yield.
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InfiniteTM 200 PRO plate reader (Tecan). Data were analyzed using GraphPad Prism software. 2.4. Hemagglutination inhibition assay The hemagglutination inhibition assay (HAI) was performed as described in the World Health Organization Manual for the Laboratory Diagnosis and Virological Surveillance of Influenza [13]. Ferret antisera FR-359 raised against A/California/07/2009 (IRR) and a 0.75% suspension of chicken erythrocytes (Lampire Biologicals) prepared in phosphate-buffered saline (PBS) were used.
2. Materials and methods 2.1. Cells, viruses and plasmids 293T cells and suspension MDCK 33016PF cells were maintained as previously described [10]. The A/Brisbane/10/2010 virus was obtained from the CDC. The PR8x (H1N1) strain is a derivative of a PR8 strain that was adapted to MDCK 33016PF cells by 5 serial passages [11]. The A/Hessen/105/2007 (Hes) strain is a derivative of a clinical isolate of an A/New Caledonia/20/1999-like virus (H1N1) that was adapted to MDCK 33016PF cells by 30 serial passages [11]. The eight segments from PR8x and Hes, the PB1 segment from A/California/07/2009 and the HA and NA segments from A/Brisbane/10/2010 were cloned in plasmid pKS10 for virus rescue as previously described [10]. HA terminal region chimeras were generated using overlap PCR and cloned into pKS10 as previously described [10]. Overlap PCR and Quikchange (Agilent) mutagenesis were used to generate the NA terminal region chimeras. All plasmids were sequence verified before use in rescue experiments. 2.2. Virus growth in MDCK cells 10 ml suspension cultures of MDCK 33016PF cells (1 × 106 cells/ml) were inoculated with virus at a multiplicity of infection (MOI) of 0.001 and incubated in TubeSpin® Bioreactor 50 (TPP) as previously described [11]. Samples were taken at 0 and 60 h post-infection and frozen at −80 ◦ C until processed. Analogous methods were used for preparations of 60 ml cultures grown in 125 ml shake flasks. Viral titers were determined using a previously described focus-formation assay [12] with slight modifications. Infectious foci were detected using an Alexa Fluor® 488-conjugated goat anti-mouse IgG (Invitrogen), and quantified with a BioSpot® Analyzer (CTL).
2.5. Sucrose density gradient (SDG) separation 40 ml of the harvested medium were concentrated ∼16-fold by centrifugal ultrafiltration (Vivaspin 20 with 300 kDa molecular weight cut-off, Sartorius-Stedim Biotech), and viruses were purified as previously described [11]. A hemagglutination assay with 0.5% guinea pig red blood cells (Cleveland Scientific) was performed to identify the fractions with the highest virion content, which were then pooled. The protein content of the pooled fractions was determined using a BCA assay (Pierce), following the manufacturer’s directions. 2.6. Reversed-phase HPLC (RP-HPLC) Purified virions were analyzed by RP-HPLC as previously described [11]. The concentration of HA1 (a HA maturational cleavage fragment) was quantified using purified HA1 from A/California/07/2009 reagent (NIBSC cat # 09/146 and 09/174) and prepared using identical methods. 2.7. SDS-PAGE and PNGaseF deglycosylation assay Equal volumes from pooled virus-containing sucrose density gradient fractions were deglycosylated following the protocol of Harvey et al. [14] with minor modifications. Samples were separated using 4–12% Nu-PAGE precast gels (Invitrogen), stained overnight by shaking at room temperature using SYPRO-Ruby stain (Sigma) and destained by shaking in 10% methanol for 30 min at room temperature. Gels were scanned using a Chemidoc XRS Imager (BioRad) and analyzed using ImageJ software [15]. 3. Results
2.3. HA ELISA 384w plates (Costar) were coated O/N with Galanthus nivalis (GNA) lectin (Sigma). Plates were washed four times with wash buffer (PBS + 0.05% Tween20) and blocked with 10 mM Tris–HCl + 150 mM NaCl + 3% sucrose + 1% BSA, pH 7.68 (blocking buffer) for 1 h at room temperature. Three-fold serial dilutions of the samples containing a final concentration of 1% Zwittergent 3–14 (Sigma) were prepared, added in duplicate to the plates, and incubated at 37 ◦ C for 30 min in a shaker. Biotinylated-IgG purified from pooled sheep antisera (NIBSC cat# 11/110) raised against A/California/07/2009 (antigenically similar to A/Brisbane/10/2010) were added and further incubated at 37 ◦ C for 30 min in a shaker. Plates were then washed four times with wash buffer and incubated with streptavidin-alkaline phosphatase (KPL) in wash buffer at 37 ◦ C for 30 min in a shaker. Plates were washed four times with wash buffer and developed using 1 mg/ml p-nitrophenyl phosphate (Sigma) in DEA buffer phosphatase substrate (KPL). Plates were read after 40–50 min incubation in the dark at 405 nm using an
3.1. Viruses with optimized backbones outperform the current vaccine seed virus for growth and HA yield in MDCK cell cultures To overcome the limitations of using egg-derived HGRs as seed viruses for manufacturing vaccine in our MDCK 33016PF cell line, we developed three MDCK cell-optimized backbones (Fig 1A; [11]). Fig. 1 shows the data compiled from three independent experiments that compared the HA yield (Fig. 1B and C) and growth (Fig. 1D) of the WT A/Brisbane/10/2010 virus to that of viruses derived from the three optimized backbones (PR8x, #19 and #21) and containing the A/Brisbane/10/2010 HA and NA segments. All viruses with our optimized backbones performed better than the WT A/Brisbane/10/2010 virus. The virus with the #21 backbone (and A/Brisbane/10/2010 HA and NA) produced the highest HA yield increase by ELISA (Fig. 1B, 6-fold more than wild type, P < 0.001) and had the highest hemagglutination (HA) titer (Fig. 1C, ∼10-fold more than WT, P < 0.001) and viral titer (Fig. 1D, ∼50-fold more than WT, P < 0.05).
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Fig. 1. Backbone-derived viruses outperform WT A/Brisbane/10/2010 virus in growth and HA yield. (A) Schematic of the three RG backbones used in this study. PR8x contains six internal segments from the MDCK-adapted PR8x strain. The #19 backbone contains PB2, PB1 and NP from the MDCK-adapted Hes strain and the remaining segments from PR8x. The #21 backbone contains an A/California/07/2009-like PB1 and the remaining segments from PR8x. MDCK 33016 PF cells were infected at an of MOI of 0.001 with RG viruses containing the WT A/Brisbane/10/2010 HA and NA segments and one of our three optimized backbones (PR8x, #19 and #21) and with the WT A/Brisbane/10/2010 virus. (B) HA yield as measured by HA ELISA. (C) HA titers using 0.5% guinea pig RBCs. (D) Viral titers 60 h post-infection as determined by FFA assay. Bars represent the mean plus SEM of three independent experiments. Statistical significance was determined using one-way ANOVA. The mean value of each group was compared to WT virus using Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001.
3.2. Chimeric HA and NA segments with terminal regions from MDCK-adapted strains In addition to using reverse genetics (RG) to combine different backbone gene segments as described above, the method is ideally suited to rationally test changes to the HA and NA gene segments that can improve HA yield [14,16–19]. Based on the highgrowth properties of our MDCK-adapted strains (PR8x and Hes), we constructed chimeric HA and NA segments that combine the nonantigenic terminal regions from HA (non-coding regions (NCRs), signal peptide (SP), transmembrane domain (TMD), and cytoplasmic tail (CT)) and NA (NCRs, CT and TMD) from PR8x and Hes with the ectodomain of A/Brisbane/10/2010 (WT) HA and NA segments, respectively. Fig. 2 shows a diagram of the constructs and a sequence alignment of the terminal regions of HA (panels A and B) and NA (panels C and D). 3.3. PR8x(term) HA and NA constructs significantly enhance HA yield with the PR8x backbone Chimeric HA [14] or NA [19] segments with terminal regions from PR8 can increase HA yield or HA content, respectively, of pdmH1N1(2009) viruses. Therefore, we reasoned that combining chimeric HA and NA segments could further enhance HA yield. To test this hypothesis, we rescued viruses containing different combinations of WT A/Brisbane/10/2010 and chimeric PR8x(term) HA and NA segments with the PR8x backbone and compared the HA yield and growth of the viruses. HA yield (Fig. 3A), as measured
by HA ELISA, was 4-fold higher for the virus with PR8x(term) HA and NA segments than for the virus with WT HA and NA segments (P < 0.01). Virus with the PR8x(term) HA segment and WT NA segment yielded a 3-fold increase in HA compared to the virus with WT HA and NA (P < 0.05). Virus with PR8x(term) HA and NA segments had 2-fold higher HA titers (P < 0.05) and 4-fold higher viral titers than the virus with WT HA and NA segments (Fig. 3B and C). Overall, these data show that viruses with chimeric PR8x(term) HA and NA segments yield more HA than viruses containing only chimeric PR8x(term) HA or NA segments. These findings suggest a strategy of using chimeric HA and NA segments for enhancement of growth and HA yield from H1N1 pdm(2009) strains. 3.4. Chimeric HA and NA constructs enhance HA yield with all three optimized backbones We next tested whether the PR8x(term) or Hes(term) HA and NA segments (Fig. 2) could enhance growth and HA yield of the resulting viruses with any of the three optimized backbones (Fig. 1A). HA yield, as measured by ELISA and normalized to the yield from WT HA and NA segments, increased ∼4-fold with PR8x(term) HA and NA segments and ∼5-fold (P < 0.05) with Hes(term) HA and NA segments using the PR8x backbone (Fig. 4A). HA yield increases correlated with increases in HA titer and viral titers using the PR8x(term) and Hes(term) HA and NA constructs (Fig. 4D and G). With the #19 backbone, HA yield was ∼2.5-fold higher (P < 0.05) with the PR8x(term) HA and NA segments and ∼3-fold higher (P < 0.05) with the Hes(term) HA and NA segments over virus with
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Fig. 2. Schematic diagram and sequence alignment of chimeric HA and NA constructs. (A) HA schematic. The wild type A/Brisbane/10/2010 HA (WT Bris) is shown in white. The terminal regions of HA: NCRs, SP, TMD and CT, from two laboratory adapted strains of H1N1, PR8x (gray) and Hes (slanted lines), were grafted onto the WT A/Brisbane/10/2010 ectodomain to produce the chimeric HA segments shown. (B) Sequence alignment of the terminal regions of WT A/Brisbane/10/2010 (Bris), PR8x and Hes HA. Dashes represent nucleotides conserved among the strains. Nucleotides in bold represent mutations in the region identified by Marsh et al. as important for packaging [26]. The 3 NCR is separated from the signal peptide sequence by the solid bar. For brevity, the ectodomain sequence is omitted (. . ./. . .). The TMD is separated from the CT by the dashed line. The stop codon is underlined and followed by the 5 NCR. (C) NA schematic. The WT A/Brisbane/10/2010 NA (WT Bris) is shown in white. The terminal regions of NA: NCR, CT, and TMD from PR8x (gray) and Hes (slanted lines), were grafted onto the WT A/Brisbane/10/2010 ectodomain to produce the chimeric NA segments shown. (D) Sequence alignment of the terminal regions of A/Brisbane/10/2010 (Bris), PR8x and Hes NA. Dashes represent nucleotides conserved among the strains. Nucleotides in bold represent mutations in the region identified by Fujii et al. as important for packaging [22].The cytoplasmic tail is separated from the 3 NCR by the solid bar and from the TMD by the dashed line. For brevity, the ectodomain sequence is omitted (. . ./. . .). The stop codon is underlined and followed by the 5 NCR.
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R.C. Gomila et al. / Vaccine 31 (2013) 4736–4743 Table 1 Antigenic analysis of viruses with the three optimized backbones. Antigen
Ferret Sera FR-359a (H1N1)
A/Brisbane/10/2010
2560
PR8x + Bris(term) HA/NA PR8x + PR8x(term) HA/NA PR8x + Hes(term) HA/NA
1920 1920 1920
#19 + Bris(term) HA/NA #19 + PR8x(term) HA/NA #19 + Hes(term) HA/NA
1280 1280 2560
#21 + Bris(term) HA/NA #21 + PR8x(term) HA/NA #21 + Hes(term) HA/NA
2560 1920 1280
IVR165 (H3N2)
<10
Values represent the geometric mean of HI titers from duplicate experiments. a Sera raised against A/California/07/2009, which is antigenically similar to A/Brisbane/10/2010.
WT HA and NA segments (Fig. 4B). HA yield increases were not associated with increases in viral titers or HA titers (Fig. 4E and H). We next asked whether we could further improve our #21 backbone, which we have found to be superior (higher HA yield) to both PR8x and #19 with WT A/Brisbane/10/2010 HA and NA segments despite occasional experimental variability in FFU titers (Figs. 1D and Fig. 4G–I). Indeed, we found significant increases with PR8x(term) and Hes(term) HA and NA segments in HA yield, ∼2.5fold (P < 0.01) and ∼3-fold (P < 0.01), respectively, HA titers (2-fold (P < 0.05)) and viral titers over virus containing WT HA and NA segments (Fig. 4C, F and I). Overall, these data show that using chimeric HA and NA segments with terminal regions derived from our MDCK-adapted strains increases HA yield independent of the backbone used. Sequence analyses of the viruses recovered with each backbones and with WT or chimeric HA and NA segments confirmed their sequence identity with the plasmids used in virus rescue (results not shown). To confirm that our viruses with chimeric HA and NA segments maintain their correct antigenicity, a hemagglutination inhibition (HAI) assay was performed using ferret antisera raised against A/California/07/2009, which is antigenically similar to WT A/Brisbane/10/2010. Table 1 shows, as expected, that the viruses with the chimeric HA and NA segments are antigenically indistinguishable (within 2-fold in an HAI assay) from the reference antigen that contains the WT HA and NA segments. 3.5. Increased HA content of viruses containing chimeric HA and NA segments
Fig. 3. PR8x(term) HA and NA segments enhance HA yield over PR8x(term) HA or NA only. MDCK 33016PF cells were infected at an MOI of 0.001 with viruses with the PR8x backbone and the indicated HA and NA gene segment combinations. (A) Fold increase as measured by HA ELISA and compared to the yield using WT A/Brisbane/10/2010 HA and NA segments. (B) HA titer as determined using 0.5% red blood cells from guinea pigs. (C) Virus titers 60 h post infection as determined by FFA assay. Bars represent the mean plus SEM of three independent experiments. Statistical significance was determined using one-way ANOVA. The mean value of each group was compared to Bris HA/NA using Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01.
To further verify that the results observed using unpurified infected cell culture supernatants reflect HA yield from purified viruses, we performed additional characterizations of #21 backbone-derived viruses, which produce the highest amounts of HA (Fig. 5). To this end, two independent larger-scale amplifications (60 ml) of these viruses were performed, and viruses were purified using SDG centrifugation, as described in Section 2. HA1 yield, normalized to the original culture volume of 60 ml, was determined using HPLC. Compared to virus with WT HA and NA segments, virus with the PR8x(term) and Hes(term) HA and NA segments had ∼1.8fold increase (11.3 g/ml vs 6.2 g/ml) and a ∼2.2-fold increase (13.6 g/ml vs 6.2 g/ml) in HA yield, respectively (Fig. 5A). We next determined the HA content in these purified preparations by using either gel densitometry or a combination of HPLC measurement of HA and total protein measurement by BCA assay. For gel densitometry determination, the pooled fractions were treated with PNGaseF, resolved by SDS-PAGE, and then stained with SYPRO-Ruby to permit accurate determination of NP, HA1, M,
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Fig. 4. Chimeric HA and NA segments enhance HA yield with any of the three optimized backbones. MDCK 33016PF cells were infected at an MOI of 0.001 with viruses with one of the three optimized backbones and the indicated HA and NA gene segment combinations with the terminal regions from WT A/Brisbane/10/2010 (Bris), PR8x or Hes. Upper panels (A–C) show the fold increase in HA yield as measured by HA ELISA and compared to the yield using WT HA and NA segments (Bris). Middle panels (D–F) show HA titers 60 h post infection as determined by HA assay. Lower panels (G–I) show virus titers 60 h post infection as determined by FFA assay. Bars represent the mean plus SEM of three independent experiments. Statistical significance was determined using one-way ANOVA. The mean value of each group was compared to Bris HA/NA using Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01.
and HA2 by densitometry [14]. Fig. 5B shows the positions of these bands on the stained gel, and Fig. 5C shows that viruses with the PR8x(term) and Hes(term) HA and NA segments had increases of 14% (P < 0.05) and 32% (P < 0.01), respectively, compared to viruses containing the WT HA and NA segments. To quantitate HA1 content using our HPLC data, we expressed the HA1 values obtained by HPLC (Fig. 5A) as a fraction of the total protein content (measured by the BCA assay) of the pooled fractions, and assigned an arbitrary value of 1 to viruses with Bris(term) HA and NA. The results in Fig. 5C show that viruses with the PR8x(term) and Hes(term) HA and NA segments had increased HA content of 29% and 46%, respectively, compared to WT HA- and NA-containing viruses. 4. Discussion Influenza viruses engineered to encode chimeric HA and NA gene segments with MDCK-adapted terminal regions produced higher yields of HA in cell culture compared to viruses with full-length HA and NA segments from a seasonal strain. The MDCKadapted terminal regions of HA and NA could enhance HA yield by increasing the levels of transcription and/or replication of the
chimeric HA and NA segments. The terminal 13 and 12 nucleotides at the 5 and 3 ends, respectively, of the vRNA are highly conserved among all eight segments and strains of influenza A viruses and are proposed to form a partially double-stranded structure that is recognized by the viral polymerase. Similar “corkscrew” structures are predicted for both the vRNA promoter (to generate mRNAs and cRNAs) and the cRNA promoter (to generate vRNAs) (reviewed in [20]). The nucleotide sequences of the proposed promoter regions of HA are identical for A/Brisbane/10/2010, PR8x and Hes (Fig. 2). However, there are nucleotide differences in the NCRs that are found outside of these conserved regions. It is possible that these differences could enhance the transcriptional and/or replicative activity of the polymerase, conferring an increase in virion or HA yield of viruses containing segments with grafted PR8x or Hes termini. However, experiments using chimeric constructs containing only the NCRs of the MDCK-adapted strains did not produce the enhancement shown by the terminal region chimeras (data not shown), a result also reported by Harvey et al. [14]. Alternatively, our chimeric HA and NA segments could enhance HA yield by acting at the RNA level by increasing the packaging of genome segments into virions. A large body of data favors the selective-incorporation model for the packaging of influenza vRNAs
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Fig. 5. Enhanced HA content of viruses with the #21 backbone and the chimeric HA and NA segments. (A) HA yield of larger scale cultures (60 ml) of MDCK 33016PF cells, infected at an MOI of 0.001 with #21 derived viruses containing the indicated HA and NA pairs. HA yield was measured by HPLC after virus was concentrated and purified by sucrose-gradient density centrifugation. (B) Deglycosylation of HA (dHA) was performed using PNGaseF. Viruses were subsequently separated by SDS-PAGE and viral proteins stained using SYPRO-Ruby. (C) The HA content was calculated from gel densitometry as previously described [34] and from HPLC by dividing values from (A) over the total protein concentration in the fractions, as determined by a BCA assay. HA content values were compared to those of Bris(term) HA and NA (WT control), which were assigned a value of 1. Bars represent the mean plus SEM of two independent experiments. Statistical significance was determined using one-way ANOVA. The mean value of each group was compared to Bris HA/NA using Dunnett’s multiple comparison test. *P < 0.05, **P < 0.001.
[21]. This model states that specific signals in each vRNA segment are involved in RNA-RNA interactions that lead to the incorporation of the eight gene segments into a virion [22]. Although there has been some recent evidence that selective co-packaging is not as efficient as originally predicted [23], the ends of the genome likely play a role in packaging viral segments into virion particles. Specifically, sequences in the terminal regions of HA and NA, as well as coding sequences in all other influenza gene segments have been implicated in vRNA packaging (For reviews, see [21,24]). VRNA–vRNA interactions have been proposed to stabilize interactions between the vRNA–nucleoprotein complexes (vRNPs) inside the virions [24,25]. Marsh et al. identified a region of 15 nucleotides (nt) in the TMD of HA as important for efficient packaging [26]. Introduction of synonymous mutations into the HA segment from an A/WSN/33 and PR8 virus decreased viral titers and HA incorporation. Sequence alignment of the nucleotides encoding the TMD of WT A/Brisbane/10/2010 HA and PR8x and Hes reveals 3 nt differences between them in this 15 nucleotide region (Fig. 2B). Two of these mutations are part of a conservative Val to Leu mutation in the TMD. It is possible that the presence of the packaging signal from the grafted strain allows a more efficient incorporation of the HA genome segment into virions. A 21 nt region in the NA N-terminal region (corresponding to the first 7 amino acids of the TMD) was identified as critical for having a significant level of reporter gene segment incorporation into influenza virions [22]. Sequence alignment of the NA N-terminal regions shows that PR8x and Hes have 3 silent mutations in the 21 nt region compared to the WT A/Brisbane/10/2010 sequence (Fig. 2D). Thus, it is possible that those changes explain why viruses with PR8x(term) HA and NA segments have a higher growth and HA yield that viruses with only PR8x(term) HA segments (Fig. 3).
The terminal region sequences derived from PR8x and Hes HA and NA segments could allow a more efficient interaction with the remaining backbone gene segments (which are also mostly derived from PR8x and Hes). Mutation of the packaging signals of one gene segment has been shown to affect the packaging of other segments [26,27]. One such example is the reduction of PB1 gene segment incorporation into virions after mutation of the HA packaging signals [26,28]. Interestingly, this effect does not appear to be reciprocal, in that mutation of the PB1 packaging signals did not affect HA gene segment incorporation [28]. In addition to more efficiently incorporating HA, potentially changing the packaging sequences of HA could also increase the incorporation of additional segments. The chimeric HA and NA segments could also act at the protein level to increase the incorporation of the glycoproteins into the viral envelope. The majority of the amino acid differences between the WT and chimeric HAs are found in the signal peptide (Fig. 2B). It is possible that the cell-derived signal peptide sequences from MDCKadapted strains allow for more efficient processing and/or secretion of HA. Sequence alignment of NA shows eight amino acid differences between PR8x(term) and Bris(term) and seven amino acid changes between Hes(term) and Bris(term) (Fig. 2D). The majority of the changes are concentrated between residues 14 and 22 in the TMD, which have been shown by alanine-scanning mutagenesis to be important for incorporation of the NA glycoprotein into virions [29]. We analyzed the NA content of virions purified by SDG (Fig. 5) by performing a neuraminidase assay [30], but found it similar among the viruses (data not shown). The CT and TMD of HA and NA contain signals for apical transport of the glycoproteins to sites of virus budding [31], for interactions with lipid rafts [29,32] and for interacting with M1 [33]. It is possible that the cell-derived HA and NA terminal regions mediate a more efficient interaction with M1, helping the virus bud more efficiently and/or increasing
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the HA content of the virions. We have preliminary data suggesting that an A/Victoria/210/2009 (H3N2) strain can also be enhanced by using the MDCK-adapted terminal regions, suggesting that this strategy could be applied to additional influenza strains (RCG, PS, PRD, PWM, unpublished). In conclusion, we show that we can enhance the productivity of our three optimized backbones for virus rescue in MDCK 33016PF cells by replacing the terminal regions of the HA and NA segments with those from MDCK-adapted strains. Contributors RCG, PS, PRD and PWM conceived of the study and designed experiments; RCG cloned the HA/NA chimeras, performed virus rescue and growth analyses; RCG, TS, AF and GP carried out HA ELISA assays; CJ and YW carried out the virus purification and HPLC analysis; RCG, PS and PWM interpreted data; RCG and PWM wrote the manuscript; PS and PRD contributed to the manuscript.
[11]
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[13]
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[17]
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Conflict of interest [19]
The authors are employees of (or contractors for) Novartis Vaccines and Diagnostics, and some are Novartis shareholders. Novartis Vaccines and Diagnostics manufactures and sells influenza vaccines.
[20]
[21]
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