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Development of high-yield influenza A virus vaccine viruses

Development of high-yield influenza A virus vaccine viruses ARTICLE Received 3 Feb 2015 | Accepted 23 Jul 2015 | Published 2 Sep 2015 DOI: 10.1038/ncomms9148 OPEN Development of high-yield influenza A virus vaccine viruses 1 1,2 3 4,5 6 5 Jihui Ping , Tiago J.S. Lopes , Chairul A. Nidom , Elodie Ghedin , Catherine A. Macken , Adam Fitch , 1 1 1 1,2 Masaki Imai , Eileen A. Maher , Gabriele Neumann & Yoshihiro Kawaoka Vaccination is one of the most cost-effective ways to prevent infection. Influenza vaccines propagated in cultured cells are approved for use in humans, but their yields are often suboptimal. Here, we screened A/Puerto Rico/8/34 (PR8) virus mutant libraries to develop vaccine backbones (defined here as the six viral RNA segments not encoding haemagglutinin and neuraminidase) that support high yield in cell culture. We also tested mutations in the coding and regulatory regions of the virus, and chimeric haemagglutinin and neuraminidase genes. A combination of high-yield mutations from these screens led to a PR8 backbone that improved the titres of H1N1, H3N2, H5N1 and H7N9 vaccine viruses in African green monkey kidney and Madin–Darby canine kidney cells. This PR8 backbone also improves titres in embryonated chicken eggs, a common propagation system for influenza viruses. This PR8 vaccine backbone thus represents an advance in seasonal and pandemic influenza vaccine development. Department of Pathobiological Sciences, School of Veterinary Medicine, Influenza Research Institute, University of Wisconsin-Madison, Madison, Wisconsin 53711, USA. Division of Virology, Department of Microbiology and Immunology and International Research Center for Infectious Diseases, The Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. Avian Influenza-Zoonosis Research Center, Airlangga University, Surabaya 60115, Indonesia. 4 5 Department of Biology, New York University, New York, New York 10003 USA. Department of Computational & Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 USA. Bioinformatics Institute, University of Auckland, Auckland 1010, New Zealand. Correspondence and requests for materials should be addressed to Y.K. (email: kawaokay@svm.vetmed.wisc.edu). NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 nfluenza A viruses belong to the family Orthomyxoviridae with reassortant viruses or viruses encoding chimeric or mutated HA 22–27 a genome composed of eight single-stranded, negative-sense and/or NA proteins ; to date, none of these studies has Iviral RNA segments. Based on the antigenicity of the two resulted in commercially available, improved influenza vaccine major surface antigens, haemagglutinin (HA) and neuraminidase production. An alternative strategy for boosting vaccine (NA), influenza A viruses are currently categorized into 18 HA production is to modify the virus backbone (that is, PR8) for subtypes (H1–H18) and 11 NA subtypes (N1–N11). Each year, increased replication efficiency. This approach has the potential seasonal influenza A viruses of the H1N1 and H3N2 subtypes and to work in a strain-independent manner, avoiding time required influenza B viruses cause several hundred million human for preliminary adaptation of a new strain to the culture system. infections and 250,000 to 500,000 deaths worldwide (http:// We therefore carried out a comprehensive study to develop a PR8 www.who.int/mediacentre/factsheets/fs211/en/). Occasionally, vaccine backbone that significantly improves the virus yield of reassortment of viral RNA segments results in novel strains to various seasonal and pandemic influenza vaccines strains in cell which most humans are naı¨ve, such as during the pandemics of culture. As we show, the same backbone can also improve yield in 1918, 1957, 1968 and 2009 (reviewed in ref. 1). In addition, egg culture systems. sporadic infections of humans with avian influenza A viruses of the H5N1 or H7N9 subtypes (resulting in case fatality rates of B60 and B30%, respectively) (http://www.who.int/influenza/ Results human_animal_interface/EN_GIP_20140124CumulativeNumber Virus library screens for high-yield variants. Currently, it is not 2,3 H5N1cases.pdf?ua=1) have been a major public health concern. known which mutations in PR8 increase the viruses’ replicative Due to frequent ‘antigenic drift’ (that is, the accumulation of ability in cultured cells. Therefore, we used a high-throughput point mutations in the antigenic epitopes of HA) and occasional mutagenesis and screening approach to identify mutations asso- ‘antigenic shift’ (that is, the introduction into human populations ciated with high yield of PR8. Specifically, we used error-prone of an HA to which humans are immunologically naive), new PCR to generate large sets (that is, ‘libraries’) of viral cDNAs with influenza A vaccine strains are recommended every B1–5 years random mutations (see Methods for details). Using reverse by the World Health Organization (WHO). For the generation of genetics approaches, these mutant cDNA libraries were used to inactivated vaccines, the HA and NA viral RNA segments of the generate virus libraries composed of tens of thousands of variants recommended strains are typically combined with the remaining possessing random amino-acid changes in one or several viral six viral RNA segments of A/Puerto Rico/8/34 (PR8; H1N1) virus proteins. Based on UW-PR8 (a high-growth variant of PR8 28,29 by using reassortment or reverse genetics approaches. Most maintained at the University of Wisconsin-Madison ), we vaccine viruses are then propagated in embryonated chicken eggs. generated the following nine virus libraries: six libraries However, this widely used influenza vaccine production platform possessing random mutations in each of the ‘internal’ genes has several vulnerabilities, including the potential for the egg (that is, PB2, PB1, PA, NP, M and NS); one library possessing supply to become a limiting factor for rapid large-scale vaccine random mutations in the genes encoding the PB2, PB1 and PA production in the event of an influenza pandemic. In addition, proteins, which together form the viral polymerase complex; one virus propagation in embryonated chicken eggs frequently results library possessing random mutations in the PB2 and NS genes, in egg-adapting mutations in HA that can affect the antigenicity which code for the major virulence factors PB2 and NS1, 4–11 1,30 of the virus and may be responsible for the reduced efficacy of respectively (reviewed in ); and one library possessing random 12,13 some influenza vaccines . mutations in the NS and M genes since the M1 protein (encoded Mammalian cell culture-based vaccine production has distinct by the M gene) is associated with high-growth properties advantages over the currently prevalent egg-based production (Fig. 1). Since high-yield vaccine backbones are urgently needed system: it is more easily scaled up and has reduced risk for the for vaccines to pandemic H5N1 viruses, we combined the HA and emergence of mutations that result in antigenic changes .Asan NA genes of an H5N1 virus, A/chicken/Indonesia/NC/2009 added advantage, the absence of egg proteins in cell culture-based (Indo09; H5N1; subclade 2.1.3.2) with the UW-PR8 backbone for vaccines eliminates potential complications for individuals with our library generation and screening procedure. We replaced the 15,16 egg allergies . To date, influenza vaccines produced in African multibasic sequence at the Indo09 HA cleavage site with a single green monkey kidney (Vero) and Madin–Darby canine kidney basic amino acid so that the viruses generated with the PR8 17,18 (MDCK) cells have been approved for human use in the backbone were of low pathogenicity, and therefore exempt from United States and/or some European countries. Select Agent status by the United States Animal and Plant Health Vaccine virus yield is a critical parameter in the vaccine Inspection Service (APHIS) and approved for studies at BSL-2. manufacturing process. Although the PR8 backbone generally To select high-yield variants, each of the nine libraries of confers efficient replication in embryonated chicken eggs, viruses composed of a mutant UW-PR8 backbone with HA and occasionally PR8-based vaccines have low yields, leading to NA from Indo09 was passaged 12 times in MDCK cells; in delays in vaccine manufacturing. Examples are the original A/ parallel, these nine libraries were combined after the second 19 20,21 Fujian/411/2002 (H3N2) and 2009 pandemic H1N1 passage, and then passaged a further 10 times in MDCK cells vaccine candidates, which replicated poorly in embryonated (Supplementary Fig. 1). From these experiments, we randomly chicken eggs. In addition, lower virus yields in cultured cells selectedB100–150 plaques per library, resulting in a total of 1434 compared with embryonated chicken eggs may have hampered individual, plaque-purified viruses (Fig. 1). The yield of each the wider use of cell culture-based influenza vaccine production plaque-purified virus was compared in haemagglutination (HA) platforms . Therefore, efforts have been undertaken to optimize assays with the yield of a control virus composed of an unmutated the production parameters and the vaccine viruses. Sequential UW-PR8 backbone and the Indo09 HA/NA genes (UW- passages of vaccine viruses in embryonated chicken eggs can PR8_Indo09). We identified 36 viruses with HA titres that were improve virus yield in an egg-based vaccine production system at least twofold higher than that of the control virus. These high- (reviewed in refs 20,21). However, passaging in eggs adds to the yield candidates were ‘purified’ through an additional round of production time, must be repeated for any low-producing strain, plaque assays, followed by virus amplification in MDCK cells. To and frequently leads to mutations in HA that may affect viral identify the mutations responsible for the increased replicative antigenicity, as stated earlier. Some studies have attempted to ability of the 36 high-yield candidates in MDCK cells, we improve the replication efficiency of vaccine viruses by using sequenced their entire genomes. Twenty-nine different 2 NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 ARTICLE Plasmid libraries possessing random mutations Selected potentially yield-enhancing mutations in the PB2, PB1, PA, NP, M and/or NS genes; described in the literature, and tested them in and A/chicken/Indonesia/NC/2009 (H5N1; the backgroud of UW-PR8 internal genes and Indo09) NA and low-pathogenic HA genes Indo09 NA and low-pathogenic HA genes Nine virus libraries possessing random 36 recombinant viruses possessing single or mutations in one or several PR8 viral genes double mutations described in the literature 12 consecutive passages in MDCK cells Plaque purification and HA titre assessment Assess virus growth in MDCK cells of 1,434 viral plaques Identified 36 high-yield viruses representing Identified 10 mutations that confer high-yield 29 genotypes in MDCK cells Mix plasmids encoding 34 (combinations of) mutations identified above Reverse genetics, followed by five consecutive passages in Vero cells Plaque purification and HA titer assessment of 216 viral plaques Identified 16 high-yield viruses (Re)created 7 potential high-yield viral backbones by reverse genetics (HY#1–7) C4U promoter mutation in PB2, Assessment of virus and HA titers in PB1 and PA segments Vero cells: lead candidate - HY#1 High-yield backbone (PR8-HY): PB2-C4U, -I504V; PB1-C4U, -M40L/G180W; PA-C4U, -R401K; NP-I116L; NS1-A30P/R118K (nucleotide changes are shown in italics) Construct chimeric HA and NA segments Test PR8-HY with wild-type or chimeric HA and NA segments of H5N1, H7N9 or human H1N1 and H3N2 viruses Evaluate growth Evaluate growth Evaluate growth characteristics in characteristics in characteristics in embryonated chicken eggs Vero cells MDCK cells Evaluate total protein Evaluate total protein and HA yield and HA yield Figure 1 | Flow chart summarizing the selection and testing of PR8-HY. Details are described in the text. combinations of mutations were detected (Fig. 1 and overlapping PB1-F2 protein). Although the HA and NA genes Supplementary Table 1). The most frequently observed amino- were not targeted by PCR-mediated mutagenesis, several acid changes localized to the polymerase subunits PB2 (namely, mutations were detected in these proteins, likely reflecting PB2-M202L/F323L and PB2-I504V) and PB1 (namely, PB1- adaptation of the Indo09 HA and NA proteins to MDCK cells. V644A and PB1-E112G; note that the nucleotide change causing None of the mutations in HA restored the multibasic sequence at the PB1-E112G mutation also causes an R81G mutation in the the HA cleavage site. NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications 3 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 0 0 Testing of mutations identified in the literature. Multiple nucleotides at the 3 and 5 termini of the vRNAs, respectively, are amino-acid changes have been associated with altered replication highly conserved among the eight viral RNA segments as well as kinetics of a variety of viruses in mammalian systems among viruses. Mutations in these regions can significantly affect 33,34 (Supplementary Table 2). The effect of most of these mutations the viral polymerase activity in reporter gene assays . We, on UW-PR8 in mammalian systems is unknown. We, therefore, therefore, tested 20 UW-PR8_Indo09 virus libraries that used site-directed mutagenesis to generate 36 mutant UW- possessed various mutations at several positions in the PR8_Indo09 viruses, each carrying a mutation(s) selected from promoter region of the HA gene; however, we were unable to Supplementary Table 2, and assessed their growth kinetics in select mutants with increased replicative ability in MDCK cells. MDCK cells (Fig. 1 and Supplementary Fig. 2). Ten mutants The viral promoters are separated from the start and stop exhibited significant increases in virus yield compared with the codons of the viral open reading frames by a variable number of control virus UW-PR8_Indo09 (Supplementary Fig. 2 and nucleotides, depending on the viral RNA segment. These so- Supplementary Table 3). One of these mutants (carrying PB2- called ‘non-coding regions’ differ among the viral RNA segments I504V and PA-I550L) was also isolated from our randomly and among virus strains. Here, we tested 11 virus libraries in mutated virus libraries (see Supplementary Table 1), suggesting which portions of the non-coding region of the UW-PR8_Indo09 that it confers high-yield properties to UW-PR8_Indo09. HA segment were randomized; these mutations did not increase the replicative ability of UW-PR8_Indo09 virus in MDCK cells. Potential combinatorial effects of mutations. On the basis of Our random mutations of the promoter region did not affect our library screens and the systematic testing of mutations virus yield in MDCK cells. Nevertheless, potentially significant identified through literature searches, we generated 34 mutants variability in the fourth position from the 3 end of the viral for further study. Thirty-two of these carried single or double RNAs, where a C or U (C4 or U4) residue is found, has been mutations selected from those that caused the greatest increases reported . Specifically, in MDCK cells, the titre (assessed by in yield and/or were found most frequently (Fig. 1 and using plaque assays) of a A/WSN/33 virus encoding U4 in all Supplementary Table 4). In addition, the NP-R422K mutation eight viral RNA segments was more than two log units higher was selected because of its role in NP oligomer formation , than that of a control A/WSN/33 virus encoding C4 (ref. 35). We, although it did not confer a statistically significant increase in therefore, converted the C4 residues found in the three poly- virus titres (Supplementary Fig. 2 and Supplementary Table 3). merase genes of HY#1 to U4, resulting in HY#1 þ C4U_Indo09, Similarly, the NS1-K55E mutation was selected for its which replicated to slightly higher titres in Vero cells compared demonstrated contribution to the high-growth properties of with HY#1_Indo09 (Fig. 2b and Supplementary Table 7); UW-PR8 virus , although it did not increase virus titres here HY#1 þ C4U_Indo09 was therefore selected as the final high- (Supplementary Fig. 2 and Supplementary Table 3). To test yield backbone in Vero cells, and was designated PR8-HY (Fig. 1). whether combinations of these mutations would further increase Compared with the UW-PR8 backbone, the PR8-HY backbone the growth properties of UW-PR8_Indo09, we transfected 293T possesses the following mutations: PB2-C4U (nucleotide changes cells with eight RNA polymerase I plasmids encoding the wild- are shown in italics), PB2-I504V, PB1-C4U, PB1-M40L/G180W, type viral RNA segments, and with 34 RNA polymerase I PA-C4U, PA-R401K, NP-I116L and NS1-A30P/R118K (Fig. 1). plasmids encoding the mutations shown in Supplementary Table 4. To initiate viral RNA transcription and replication, Chimeric HA and NA genes increase virus yield. In parallel to these cells were also transfected with four plasmids encoding the the development of a UW-PR8-based high-yield backbone, we A/WSN/33 (H1N1) polymerase and NP proteins. In total, the also tested chimeric HA and NA genes/proteins, a strategy that cells were co-transfected with 46 different plasmids. This strategy 22–27 has been shown to increase virus yield . In these chimeras, the should result in viruses encoding all candidate high-yield extracellular domains of HA and NA are derived from the mutations shown in Supplementary Table 4. recommended vaccine strain, whereas the transmembrane and Ideally, a backbone for cell-based vaccine production will intracellular domains originate from PR8 viruses to ensure confer increased growth properties in both MDCK and Vero cells. optimal compatibility with the internal, PR8 virus-derived viral We therefore passaged the above-described virus mixture five times genes and proteins. We, therefore, generated a UW- in Vero cells and isolated 216 individual viruses, which were PR8_Indo09 virus in which only the extracellular domains Chim assayed for HA titres (Fig. 1). Sixteen viruses exhibited HA titres of of the HA and NA proteins were derived from Indo09 virus (see 9–9.5 6.5 2 , compared with an HA titre of 2 for the control UW- Supplementary Fig. 3 and Supplementary Table 8). In Vero cells, PR8_Indo09 virus (Fig. 1 and Supplementary Table 5). Sequence the virus and HA titres of this virus were significantly higher than analysis of these high-yield candidates revealed dominant muta- those of the UW-PR8_Indo09 virus, which encodes the authentic tions in several viral proteins (Supplementary Table 5). Indo09 HA and NA genes (Fig. 3a and Supplementary Table 9). To confirm the yield-enhancing effects of the amino-acid Therefore, we next tested the chimeric Indo09 HA and NA genes changes in these 16 selected viruses, we used reverse genetics to with the high-yield backbone (PR8-HY), resulting in PR8- make seven different mutant UW-PR8_Indo09 viruses, referred HY_Indo09 . We detected a small, but statistically signifi- Chim to as HY#1–7_Indo09 (Supplementary Table 6). Each of these cant increase in virus titres for PR8-HY_Indo09 relative to Chim mutant viruses carries a different combination of amino-acid those for PR8-HY_Indo09 (Fig. 3b and Supplementary Table 9). changes selected from those carried by the 16 high-yield viruses. Six of these mutants exhibited significantly higher HA and/or virus titres in Vero cells than the control UW-PR8_Indo09 virus Evaluation of candidate vaccine viruses in Vero cells. Ideally, (Fig. 2a and Supplementary Table 7). The highest titres were the virus backbone used for influenza vaccine production should detected for HY#1_Indo09, which carries the PB2-I504V, PB1- yield high virus and HA titres with the HA and NA genes of M40L/G180W, PA-R401K, NP-I116L and NS1-A30P/R118K varied seasonal and pandemic influenza viruses. Accordingly, we mutations; the HY#1 vaccine backbone was therefore selected first tested the PR8-HY backbone with wild-type or chimeric HA for further studies. and NA genes of different WHO-recommended H5N1 vaccine viruses, namely, A/Vietnam/1203/2004 (Clade 1; VN04), A/ Mutations in the influenza promoter and non-coding regions. Hubei/1/2010 (Clade 2.3.2.1a; Hubei10), A/Egypt/N03072/2010 The influenza A viral promoter sequences, that is, the 12 and 13 (Clade 2.2.1; Egypt10), and A/Indonesia/05/2005 (Clade 2.1.3.2; 4 NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 ARTICLE a Viral titre HA titre 9 10 UW-PR8_Indo09 HY#1_Indo09 HY#2_Indo09 HY#3_Indo09 HY#4_Indo09 HY#5_Indo09 HY#6_Indo09 HY#7_Indo09 3 0 0 24 48 72 96 0 24 48 72 96 9 10 UW-PR8_Indo09 HY#1_Indo09 HY#1+C4U_Indo09 (PR8-HY_Indo09) 2 0 0 12 24 36 48 60 72 0 12 24 36 48 60 72 Hours post infection Hours post infection Figure 2 | Growth kinetics and HA titres of HY#1–7 high-yield candidates in Vero cells. (a) Growth kinetics and HA titres of high-yield candidates in Vero cells. Vero cells were infected in triplicate with the indicated viruses at a multiplicity of infection (MOI) of 0.005 and incubated at 37 C. Supernatants were collected at the indicated time points, and the virus titres were determined by plaque assays in MDCK cells. In parallel, we determined the HA titres of the collected supernatants by performing HA assays. (b) Effect of the C4U promoter mutation in the viral polymerase genes on viral growth kinetics and HA titres. Shown is the comparison of viruses possessing the parental UW-PR8 backbone (UW-PR8_Indo09), the HY#1 backbone (HY#1_Indo09), or the HY#1 backbone with C4U mutations in the PB2, PB1 and PA genes (HY#1 þ C4U_Indo09). Experiments were carried out as described in a. The values presented are the average of three independent experiments s.d. Viral titre HA titre 9 9 8 8 7 7 6 6 5 5 UW-PR8_Indo09 4 UW-PR8_Indo09 Chim 3 3 1 1 0 122436486072 0 122436486072 6 5 UW-PR8_Indo09 5 PR8-HY_Indo09 PR8-HY_Indo09 chim 2 0 0 122436486072 0 122436486072 Hours post infection Hours post infection Figure 3 | Growth kinetics and HA titres of HA/NA chimeric viruses in Vero cells. (a) Growth kinetics and HA titres of UW-PR8-based viruses with wild- type or chimeric Indo09 HA and NA genes. (b) Growth kinetics and HA titres of viruses possessing the UW-PR8 backbone in combination with Indo09 HA and NA genes, or the PR8-HY backbone in combination with wild-type or chimeric Indo09 HA and NA genes. Experiments were carried out as described in the legend to Fig. 2. The values presented are the average of three independent experiments s.d. Indo05); all H5 HA genes were mutated to encode an HA clea- replicating Egypt10 virus, which was also tested at 96 h post vage sequence characteristic of low pathogenic viruses. We tested infection (Fig. 4a–d). The PR8-HY backbone increased virus and titres up to 72 h post infection, except for the inefficiently HA titres significantly compared with the respective control NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications 5 & 2015 Macmillan Publishers Limited. All rights reserved. –1 Virus yield (Log PFU ml ) –1 –1 –1 Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) 10 10 HA titre (Log ) HA titre (Log ) HA titre (Log ) 2 HA titre (Log ) 2 2 2 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 a b Viral titre HA titre Viral titre HA titre 10 10 10 10 9 9 8 8 7 7 8 8 6 6 7 5 7 5 4 4 6 6 3 3 2 2 5 5 1 1 4 0 4 0 0 122436486072 0 122436486072 0 122436486072 0 122436486072 UW-PR8_Hubei10 UW-PR8_VN04 PR8-HY_Hubei10 PR8-HY_VN04 PR8-HY_Hubei10 PR8-HY_VN04 Chim Chim c d 10 11 10 11 10 10 9 9 8 8 7 7 6 6 5 5 4 4 3 3 2 2 1 1 3 0 5 0 0 1224364860728496 0 1224364860728496 0 122436486072 0 122436486072 UW-PR8_Egypt10 UW-PR8_Indo05 PR8-HY_Egypt10 PR8-HY_Indo05 PR8-HY_Indo05 PR8-HY_Egypt10 Chim Chim e f 10 9 9 8 8 7 7 6 7 6 5 5 5 6 4 4 3 5 3 1 1 0 0 3 0 0 1224364860728496 0 1224364860728496 0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 168 UW-PR8_Anhui13 Hours post infection Hours post infection PR8-HY_Anhui13 X-181 PR8-HY_Anhui13 PR8-HY_X-181 Chim PR8-HY_X-181 Chim 9 10 8 8 7 6 6 4 5 2 4 0 0 1224364860728496 0 1224364860728496 Hours post infection Hours post infection X-223A PR8-HY_X-223A Figure 4 | Evaluation of PR8-HY backbone vaccine candidates propagated in Vero cells. Growth kinetics and HA titres of UW-PR8- and PR8-HY-based viruses encoding the wild type, or wild-type and chimeric HA and NA segments of the A/Vietnam/1203/2004 (VN04, H5N1) (a), A/Hubei/1/2010 (Hubei10, H5N1) (b), A/Egypt/N03072/2010 (Egypt10, H5N1) (c), A/Indonesia/5/2005 (Indo05, H5N1) (d) or A/Anhui/1/2013 (Anhui13, H7N9) (e) viruses. Panels (f) and (g) show a comparison of current seasonal H1N1 and H3N2 vaccine viruses (X-181 and X-223A, respectively) with PR8-HY backbone viruses possessing wild-type or chimeric HA and NA segments derived from X-181 or X-223A viruses. Experiments were carried out as described in the legend to Fig. 2, with the exception of those involving X-181 and X-223A viruses, which were inoculated at an MOI of 0.1. The values presented are the average of three independent experiments s.d. viruses possessing the UW-PR8 backbone (Fig. 4a–d and titres for Hubei10, Egypt10 and Indo05, but not for VN04 viruses Supplementary Table 10). The use of chimeric HA and NA genes (Fig. 4a–d and Supplementary Table 10). Together, these provided a statistically significant increase in virus and/or HA improvements increased virus titres B5  220-fold compared 6 NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. –1 –1 –1 Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) –1 10 10 10 Virus yield (Log PFU ml ) HA titre (Log ) HA titre (Log ) HA titre (Log ) 2 2 2 HA titre (Log ) –1 –1 –1 Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) 10 10 Virus yield (Log PFU ml ) HA titre (Log ) HA titre (Log ) 2 2 HA titre (Log ) 2 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 ARTICLE Table 1 | Comparison of growth characteristics of PR8-HY viruses possessing wild-type or chimeric HA and NA genes. Virus Growth Peak virus titre Peak HA titre Total viral protein yield HA content substrate –1 n PFU ml Statistical Fold- 2 Statistical Fold- Statistical Fold- Statistical Fold- significance increase significance increase significance increase significance increase compared versus compared versus compared versus compared versus with UW- UW-PR8 with UW- UW-PR8 with UW- UW-PR8 with UW- UW-PR8 PR8 PR8 PR8 PR8 PR8-HY_ Vero 1.0  10 Po0.005 28.5 9.5–10 Po0.005 5.7 Po0.005 9.8 Po0.005 10.8 Indo09 MDCK 1.0  10 Po0.005 4.6 9.5 Po0.005 3 ND* ND ND ND Chim (H5N1) Egg 7.0  10 Po0.005 5.9 11–11.5 Po0.005 2.9 Po0.005 4.4 Po0.005 5.3 PR8-HY_ Vero 8.8  10 Po0.005 5.1 8.5 Po0.005 2.2 Po0.005 1.8 Po0.005 1.9 VN04 Chim MDCK 5.8  10 Po0.01 1.4 9 Po0.005 1.3 ND ND ND ND 9 w (H5N1) Egg 2.7  10 Po0.005 4.6 10 — 2 NDNDND ND PR8-HY_ Vero 7.6  10 Po0.005 9.5 8.5–9 Po0.005 3.5 Po0.005 2.1 Po0.005 2.3 Hubei10 Chim MDCK 2.2  10 Po0.01 1.8 8.5 Po0.005 1.3 ND ND ND ND (H5N1) Egg 4.0  10 Po0.005 29 9.5–10 Po0.005 4.9 Po0.005 6.9 Po0.005 7.3 PR8-HY_ Vero 1.3  10 Po0.005 221 10.5–11 Po0.005 76 Po0.005 9.7 Po0.005 11.3 Egypt10 Chim MDCK 6.1  10 Po0.005 3.6 9–9.5 Po0.005 1.7 ND ND ND ND (H5N1) Egg 4.7  10 Po0.005 7.5 12.5 — 2.7 ND ND ND ND PR8-HY_ Vero 8.5  10 Po0.005 28.6 9 Po0.005 4.5 Po0.005 4.9 Po0.005 5.5 Indo05 Chim MDCK 7.1  10 Po0.005 2.4 9.5 Po0.005 3 ND ND ND ND (H5N1) Egg 7.3  10 Po0.005 12.5 12.5– Po0.005 4.6 Po0.005 2.6 Po0.005 3.1 PR8-HY_ Vero 1.3  10 Po0.005 173 8–8.5 Po0.005 3.6 Po0.005 9.9 Po0.005 12.4 Anhui13 Chim MDCK 1.5  10 Po0.005 29 9.5–10 Po0.005 5.5 ND ND ND ND (H7N9) Egg 3.6  10 Po0.005 172 11.5 Po0.005 17.7 Po0.005 14.8 Po0.005 16.9 8 y y y y y y y PR8-HY_X- Vero 1.3  10 Po0.005 269 8 Po0.005 134 Po0.005 3.3 Po0.005 3.9 8 y y y 181 or Chim MDCK 3.3  10 Po0.005 4.7 9.5 — 1.6 ND ND ND ND 9 y y y y y y y PR8-HY_X- Egg 7.2  10 Po0.005 5 11.5–12 Po0.05 2.7 Po0.005 1.8 Po0.005 2.1 (H1N1) 8 y y y y y y y PR8-HY_ Vero 3.7  10 Po0.005 9.3 9–9.5 Po0.005 2.3 Po0.005 2.1 Po0.005 2.2 8 y y y y X-223A MDCK 6.7  10 Po0.005 5.3 9.5 Po0.05 2 ND ND ND ND 9 y y y y y y (H3N2) Egg 3.8  10 Po0.05 6 11–11.5 — 1.3 Po0.005 1.3 Po0.005 1.3 *ND: not determined. wNo significant difference. zSince PR8-HY_X-181 does not grow in Vero cells, PR8-HY_X-181 was used to evaluate total viral protein and HA content in Vero cells. Chim yComparisons were carried out between high-growth X-181 and wild-type X-181 viruses, or between high-growth and wild-type X-223A viruses, respectively. with the respective UW-PR8 viruses (Table 1), demonstrating the influenza vaccines. Interestingly, chimeric HA and NA genes had potential of the PR8-HY backbone as vaccine vector. a strongly attenuating effect on this vaccine candidate (Fig. 4f). Next, we used the same strategy to test the PR8-HY backbone The current human seasonal H3N2 vaccine virus is based on the with the wild-type or chimeric HA and NA genes of A/Anhui/1/ A/Texas/50/2012 HA and NA genes combined with the 2013 (H7N9) virus, a representative of the recently emerged remaining genes of the WHO-recommended PR8 isolate. This H7N9 viruses that have caused severe respiratory infections in virus replicates to moderate titres in Vero cells, which can be 2,3 humans with high-case fatality rates . The Vero cell titres of improved significantly with the use of the PR8-HY backbone PR8-HY viruses expressing wild-type or chimeric A/Anhui/1/ (Fig. 4g and Supplementary Table 10). Notably, we were unable to 2013 HA and NA genes were 85-fold and 173-fold higher, generate a PR8-HY virus possessing chimeric X-223A-derived respectively, than those of the UW-PR8-based control virus HA and NA genes. Thus, for both seasonal human vaccine viruses (Table 1 and Fig. 4e). A significant increase in HA titres was tested here, chimeric HA and NA genes were attenuating, rather found in conjunction with this significant increase in virus titres than titre enhancing. (Fig. 4e and Supplementary Table 10). Finally, we tested the PR8-HY backbone with the wild-type and chimeric HA and NA genes of the current human H1N1 and Evaluation of candidate vaccine viruses in MDCK cells. After H3N2 vaccine viruses, New York Medical Center (NYMC) X-181 demonstrating that the PR8-HY backbone confers high yield to (X-181) and NYMC X-223A (X-223A), respectively. X-181, pandemic and seasonal influenza vaccine candidates in Vero cells, which possesses the A/California/07/2009 (H1N1; CA09) we performed similar studies in MDCK cells. Based on the effi- HA, NA and PB1 genes and the remaining genes from of the cient growth kinetics of the tested viruses in MDCK cells, titres WHO-recommended PR8 virus, did not replicate to high titres, were determined for up to 48 h post infection (Fig. 5). For all even after an extended period of replication (Fig. 4f); by contrast, viruses tested, the PR8-HY backbone conferred higher virus and the PR8-HY backbone with the CA09 HA and NA genes HA titres compared with the UW-PR8 backbone, although not all increased virus titres 4250-fold (Fig. 4f, Table 1 and differences were statistically significant (Fig. 5 and Supplementary Supplementary Table 10); hence, our high-growth vaccine Table 11). Chimeric HA and NA genes further improved the candidate confers significantly higher virus titres than the NYMC virus and/or HA titres of some viruses, namely, PR8-HY_An- PR8 backbone currently used for the generation of seasonal hui and PR8-HY_Hubei10 (Fig. 5 and Supplementary Chim Chim NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications 7 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 Table 11). In combination, the PR8-HY backbone and chimeric Mice infected with PR8-HY-backbone-based viruses also lost HA and NA genes increased virus titres by 1.4  29-fold, and HA more weight (Supplementary Fig. 5b) and had higher lung virus titres by 1.3  5.5-fold (Table 1). titres on days 3 and 5 post infection than did mice infected with the UW-PR8-based virus (Supplementary Fig. 5c), suggesting that the higher replicative ability of PR8-HY-based viruses may cause Evaluation of candidate vaccine viruses in embryonated eggs. slightly higher mouse virulence. However, these increases in Although Vero and MDCK cell lines are now available to pro- virulence were small and PR8-HY-based vaccines would be pagate influenza vaccines for human use, embryonated chicken administered as inactivated vaccines; therefore, the slightly higher eggs remain the most commonly used propagation system, mouse virulence of the PR8-HY backbone compared with the prompting us to test our PR8-HY-based vaccine candidates in UW-PR8 backbone should not be a major issue for vaccine eggs. As shown in Fig. 6 and Supplementary Table 12, the PR8- production, although additional studies may be needed in the HY backbone significantly increased virus and/or HA titres of the future to address this issue. candidate vaccines, and the use of chimeric HA and NA genes further increased the titres of some viruses, such as Anhui13. The Genetic stability of the PR8-HY vaccine backbone. Vaccine combined effect of the PR8-HY backbone and chimeric HA and viruses need to have high genetic stability to minimize the risk of NA genes increased virus titres by 4.6  172-fold, resulting in 9  1 emergence of variants with unwanted properties. To evaluate the titres of 410 plaque-forming units (PFU) ml (Table 1); HA genetic stability of our novel vaccine candidate, PR8-HY_Indo05 titres increased by 1.3  17.7-fold (Table 1). Relatively small and PR8-HY_Indo05 were each passaged 10 times in Vero Chim increases in HA titres were detected for the human seasonal PR8- cells and embryonated chicken eggs, and their whole genome HY_X-181 and PR8-HY_X-223A viruses compared with the Chim sequences were then analysed. None of the yield-enhancing parental X-181 and X-223A viruses. X-181 and X-223A were mutations converted to wild-type sequence during passages in selected as vaccine viruses because of their efficient replication in 11 cultured cells or embryonated chicken eggs, and no additional embryonated chicken eggs (resulting in HA titres of up to 2 mutations were detected in any of the viral genes. In addition, we Log ), so further increases may be difficult to achieve. Collec- inoculated the viruses shown in Table 1 into MDCK cells (at an tively, however, our data demonstrate that the Vero cell-opti- MOI of 0.001), Vero cells (at an MOI of 0.005) or embryonated mized PR8-HY vaccine backbone increases vaccine virus titres in chicken eggs (2  10 PFU per egg). Forty-eight hours later, we embryonated chicken eggs. collected cell culture supernatants and allantoic fluids and sequenced the HA and NA genes of the amplified viruses; no Evaluation of total viral protein yield and HA content. Inac- mutations were found in these genes. These findings further tivated influenza vaccines are standardized by HA content; the support the vaccine potential of the PR8-HY backbone. most commonly used vaccine preparations contain 15mg each of H1 HA, H3 HA and type B HA. Total viral protein yield and HA Discussion content are therefore important parameters in vaccine optimi- Human infections with avian H5N1 or H7N9 viruses have raised zation. Here, we compared the total viral protein yield and HA concerns about a looming pandemic and the ability of standard content of our selected vaccine candidates propagated in Vero influenza vaccine production methods to supply sufficient cells or embryonated chicken eggs. Viruses collected from cell amounts of an effective vaccine in a limited time. Vaccine viruses culture supernatants or egg allantoic fluid were concentrated and that replicate to higher titres than current vaccine viruses would purified by use of sucrose gradient centrifugation. Total viral lessen these concerns and also avoid production delays with protein yield was determined using the Pierce BCA Protein Assay seasonal vaccines in the event that the original vaccine virus Kit (Thermo Scientific, Rochester, NY, USA). In comparison with candidates grow to low titres. Through a comprehensive study the UW-PR8-based control viruses, the PR8-HY backbone yiel- that combined random and site-directed mutagenesis of different ded significantly higher amounts of total viral protein (Fig. 7a,d). functional regions of the viral genome (that is, coding, To evaluate the HA content, the purified virus samples were non-coding and promoter regions), we generated a high-yield deglycosylated with PNGase F (thus allowing the detection of PR8-based vaccine backbone that significantly increased the yield HA2, which in its glycosylated form is similar in size to M1) and of pandemic H5N1 and H7N9 vaccine candidates, and of then analysed by using SDS–polyacrylamide gel electrophoresis seasonal H1N1 and H3N2 vaccine viruses. Although this vaccine (SDS–PAGE) (Fig. 7b,e and Supplementary Fig. 4). Based on a backbone was selected for its high-yield properties in Vero and densitometry analysis, the amounts of HA1, HA2, NP and M1 MDCK cells, it also improved vaccine virus yields in embryonated were determined. To calculate the HA content, we divided the chicken eggs. HA amount (calculated by summing the amounts of HA1 and Vaccine production in cell culture has a number of possible HA2) by the sum of the amounts of HA1, HA2, NP and M1, and benefits over production in embryonated eggs. The biggest multiplied this value by the amount of total viral protein in the drawback of influenza virus amplification in embryonated 4–11 samples analysed via gel electrophoresis. The results showed that chicken eggs is the emergence of antigenic variants . Such PR8-HY-based vaccine candidates displayed a significantly higher unwanted adaptation may have contributed to the reduced 12,13 HA content than those possessing the PR8-UW backbone efficacy of some influenza vaccines . In contrast, the HAs of (Fig. 7c,f). We have not yet performed single radial immuno- cell culture-propagated viruses are typically identical to those of 4–7,12,36–45 diffusion assays or assessed the ratio of infectious to physical the original isolates . Moreover, influenza viruses particles. isolated from humans often replicate more efficiently in cultured mammalian cells than in embryonated chicken 46,47 Virulence of PR8-HY-based vaccine viruses in mice. To rule out eggs . Several studies have also suggested that human the possibility that the high-yield-conferring mutations in the influenza vaccines prepared from viruses propagated in cultured PR8-HY backbone render the virus highly virulent in mammals, cells induce better protective immunity than those grown in 9,48–52 we compared the virulence in mice of UW-PR8_Indo05, PR8- embryonated chicken eggs . HY_Indo05 and PR8-HY_Indo05 . The doses required to kill Despite the advantages of cell culture-based influenza vaccines Chim 50% of infected animals (mouse lethal dose 50, MLD ) were compared with egg-grown products, the former represent only a 2.5 2 2.25 10 ,10 and 10 PFU, respectively (Supplementary Fig. 5a). small percentage of the influenza vaccine market. Vero cells have 8 NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 ARTICLE a b Viral titre HA titre Viral titre HA titre 10 10 9 9 8 8 8 6 6 7 4 6 6 3 2 5 4 0 4 0 0 12243648 0 12243648 0 12243648 0 12243648 UW-PR8_VN04 UW-PR8_Indo09 PR8-HY_VN04 PR8-HY_Indo09 PR8-HY_Indo09 PR8-HY_VN04 Chim Chim 9 9 9 10 7 7 6 7 5 5 6 5 3 3 5 1 1 0 3 0 0 12 24 36 48 0 12 24 36 48 0 12 24 36 48 0 12 24 36 48 UW-PR8_Egypt10 UW-PR8_Hubei10 PR8-HY_Egypt10 PR8-HY_Hubei10 PR8-HY_Egypt10 PR8-HY_Hubei10 Chim Chim e f 9 10 10 11 8 8 6 4 5 2 4 0 1 0 0 12 24 36 48 0 12 24 36 48 0 12 24 36 48 0 12 24 36 48 UW-PR8_Anhui13 UW-PR8_Indo05 PR8-HY_Anhui13 PR8-HY_Indo05 PR8-HY_Anhui13 PR8-HY_Indo05 Chim Chim 9 10 9 11 6 5 3 0 2 0 0 12243648 0 12243648 012 24 36 48 012 24 36 48 Hours post infection Hours post infection Hours post infection Hours post infection X-181 X-223A PR8-HY_X-181 PR8-HY_X-223A PR8-HY_X-181 Chim Figure 5 | Evaluation of PR8-HY vaccine candidate viruses propagated in MDCK cells. Growth kinetics and HA titres of UW-PR8- and PR8-HY-based viruses encoding the wild-type, or wild-type and chimeric HA and NA segments of the A/chicken/Indonesia/NC/2009 (Indo09, H5N1) (a), A/Vietnam/ 1203/2004 (VN04, H5N1) (b), A/Hubei/1/2010 (Hubei10, H5N1) (c), A/Egypt/N03072/2010 (Egypt10, H5N1) (d), A/Indonesia/5/2005 (Indo05, H5N1) (e), or A/Anhui/1/2013 (Anhui13, H7N9) (f) viruses. Panels (g) and (h) show a comparison of current seasonal H1N1 and H3N2 vaccine viruses (X- 181 and X-223A, respectively) with PR8-HY backbone viruses possessing wild-type or chimeric HA and NA segments derived from X-181 or X-223A viruses. MDCK cells were infected in triplicate with the indicated viruses at an MOI of 0.001 and incubated at 37 C; otherwise, experiments were carried out as described in the legend to Fig. 2. The values presented are the average of three independent experiments s.d. NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications 9 & 2015 Macmillan Publishers Limited. All rights reserved. –1 –1 –1 –1 Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) 10 Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) 10 10 HA titre (Log ) HA titre (Log ) HA titre (Log ) 2 2 HA titre (Log ) 2 –1 Virus yield (Log PFU ml ) –1 –1 –1 Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) 10 10 HA titre (Log ) HA titre (Log ) HA titre (Log ) 2 2 2 HA titre (Log ) 2 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 a b Viral titre HA titre Viral titre HA titre 10 12 10 11 11 10 9 10 8 8 7 7 6 4 5 2 4 0 3 0 0 1224364860 0 1224364860 012 24 36 48 60 012 24 36 48 60 UW-PR8_VN04 UW-PR8_Indo09 PR8-HY_Indo09 PR8-HY_VN04 PR8-HY_Indo09 PR8-HY_VN04 Chim Chim 10 11 10 14 9 12 8 10 6 6 5 4 4 2 3 0 4 0 012 24 36 48 60 012 24 36 48 60 0 1224364860 0 1224364860 UW-PR8_Hubei10 UW-PR8_Egypt10 PR8-HY_Hubei10 PR8-HY_Egypt10 PR8-HY_Hubei10 PR8-HY_Egypt10 Chim Chim e f 10 14 10 12 9 8 8 6 5 6 7 4 4 3 6 2 5 0 0 0 0 1224364860 0 1224364860 0 1224364860 0 1224364860 UW-PR8_Anhui13 UW-PR8_Indo05 PR8-HY_Indo05 PR8-HY_Anhui13 PR8-HY_Indo05 PR8-HY_Anhui13 Chim Chim 11 13 10 12 12 11 9 10 8 8 7 6 6 4 2 5 2 1 1 4 0 4 0 0 1224364860 0 1224364860 0 12 24 36 48 60 0 12 24 36 48 60 Hours post-infection Hours post-infection Hours post-infection Hours post-infection X-181 X-223A PR8-HY_X-181 PR8-HY_X-223A PR8-HY_X-181 Chim Figure 6 | Evaluation of PR8-HY vaccine candidate viruses propagated in embryonated chicken eggs. Growth kinetics and HA titres of UW-PR8- and PR8-HY-based viruses encoding the wild-type, or wild-type and chimeric HA and NA segments of the A/chicken/Indonesia/NC/2009 (Indo09, H5N1) (a), A/Vietnam/1203/2004 (VN04, H5N1) (b), A/Hubei/1/2010 (Hubei10, H5N1) (c), A/Egypt/N03072/2010 (Egypt10, H5N1) (d), A/Indonesia/5/2005 (Indo05, H5N1) (e), or A/Anhui/1/2013 (Anhui13, H7N9) (f) viruses. Panels (g) and (h) show a comparison of current seasonal H1N1 and H3N2 vaccine viruses (X-181 and X-223A, respectively) with PR8-HY backbone viruses possessing wild-type or chimeric HA and NA segments derived from X-181 or X- 223A viruses. Ten-day-old embryonated chicken eggs (four per virus) were inoculated with 2  10 PFU of the respective viruses and incubated at 35 C for the indicated periods of time. The values presented are the average of three independent experiments s.d. 53 54 been used for the production of inactivated poliovirus , rabies virus was developed and licensed in Europe (http://www.baxter. and live smallpox vaccines , resulting in an extensive track com/press_room/press_releases/2009/10_07_09-celvapan.html). record for Vero cell-derived vaccines for human use. In October One MDCK cell-derived vaccine has been available in Europe since 2009, a Vero cell-based vaccine to the novel pandemic H1N1 2001 (Influvac, Solvay Pharmaceuticals) and a second since 2007 10 NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. –1 –1 –1 –1 Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) 10 10 10 10 HA titre (Log ) HA titre (Log ) HA titre (Log ) HA titre (Log ) 2 2 2 –1 –1 –1 –1 Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) 10 10 10 10 HA titre (Log ) HA titre (Log ) HA titre (Log ) HA titre (Log ) 2 2 2 2 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 ARTICLE a c *** *** *** *** *** *** 4 *** 7 *** *** *** *** *** 0 0 PNGaseF: + + + + ++ + + + + ++ –– – – – – – –– – – – 250 kDa 150 kDa 100 kDa 75 kDa NP 50 kDa HA1 Deglycosylated HA1 37 kDa M1 25 kDa HA2 20 kDa Deglycosylated HA2 15 kDa d f 22 *** 12 *** *** *** *** *** *** *** *** *** *** *** 0 0 PNGaseF: ++ + + ++ + + + + + + –– – – – – – –– – – – 250 kDa 150 kDa 100 kDa 75 kDa HA1 NP 50 kDa Deglycosylated HA1 37 kDa M1 25 kDa HA2 20 kDa Deglycosylated HA2 15 kDa Figure 7 | Evaluation of the total viral protein and HA content of PR8-HY candidate vaccine viruses. Total viral protein yield of Vero cell- (a) or egg- grown (d), sucrose gradient-purified virus samples. The total protein content of virus concentrates was measured by using the Pierce BCA assay kit (Thermo Fisher) according to the manufacturer’s instructions. SDS–PAGE analyses of virus samples from Vero cells (b) or embryonated chicken eggs (e). Virus concentrates were deglycosylated with PNGase (PNFase F þ ) or left untreated (PNGase F  ). HA contents of Vero-; (c) and egg-grown (f) viruses. The HA contents were calculated based on the total viral protein amounts (Fig. 7a,d) and the relative amounts of HA (Fig. 7b,e); for details, see Study Design. The HA contents are expressed in mg l (for Vero cell-grown viruses) or mg per 100 eggs (for viruses grown in embryonated chicken eggs). Asterisks indicate a significant difference. The values presented are the average of three independent experiments s.d. P-values were calculated by using Tukey’s post-hoc test, comparing the total viral protein yield and HA content of wild-type viruses with that of recombinant high-yield vaccine viruses; ***Po0.005. NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications 11 & 2015 Macmillan Publishers Limited. All rights reserved. UW-PR8_Indo09 PR8-HY_Indo09 Chim UW-PR8_Hubei10 PR8-HY_Hubei10 Chim UW-PR8_Indo05 PR8-HY_Indo05 Chim UW-PR8_Anhui13 PR8-HY_Anhui13 Chim X-181 PR8-HY_X-181 X-223A PR8-HY_X-223A UW-PR8_Indo09 PR8-HY_Indo09 Chim UW-PR8_Hubei10 PR8-HY_Hubei10 Chim UW-PR8_Indo05 PR8-HY_Indo05 Chim UW-PR8_Anhui13 PR8-HY_Anhui13 Chim X-181 PR8-HY_X-181 Chim X-223A PR8-HY_X-223A UW-PR8_Indo09 PR8-HY_Indo09 chim UW-PR8_Hubei10 UW-PR8_Indo09 PR8-HY_Hubei10 chim PR8-HY_Indo09 Chim UW-PR8_Indo05 UW-PR8_Hubei10 PR8-HY_Hubei10 Chim PR8-HY_Indo05 chim UW-PR8_Indo05 PR8-HY_Indo05 Chim UW-PR8_Anhui13 UW-PR8_Anhui13 PR8-HY_Anhui13 PR8-HY_Anhui13 Chim chim X-181 X-181 PR8-HY_X-181 X-223A PR8-HY_X-181 PR8-HY_X-223A UW-PR8_Indo09 PR8-HY_Indo09 X-223A Chim UW-PR8_Hubei10 PR8-HY_X-223A PR8-HY_Hubei10 Chim UW-PR8_Indo05 PR8-HY_Indo05 Chim UW-PR8_Anhui13 PR8-HY_Anhui13 Chim X-181 PR8-HY_X-181 Chim UW-PR8_Indo09 X-223A PR8-HY_X-223A PR8-HY_Indo09 chim UW-PR8_Hubei10 PR8-HY_Hubei10 chim UW-PR8_Indo05 PR8-HY_Indo05 chim UW-PR8_Anhui13 PR8-HY_Anhui13 chim X-181 PR8-HY_X-181 chim X-223A PR8-HY_X-223A Total viral protein –1 (mg per 100 eggs) Total viral protein (mg l ) –1 Total viral protein HA content (mg l ) (mg per 100 eggs) ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 0 0 (Optaflu, Novartis), but such vaccines were not available in the The influenza virus promoter sequences at the 5 and 3 ends of United States until 2012 (Flucelvax, Novartis) (http://www.fda.gov/ the negative-sense viral RNA and the positive-sense complemen- newsevents/newsroom/pressannouncements/ucm328982.htm). tary RNA are highly conserved, with the exception of the fourth Our high-yield vaccine backbone, PR8-HY, in some instances positions from the 3 end of the viral RNA. At this position, the in combination with chimeric HA and NA viral RNA segments, polymerase genes have been reported to encode a C residue, increased peak virus titres in Vero cells 4.6  269-fold compared whereas the remaining five RNA segment encode a U residue. with PR8-UW-based vaccines (Table 1). In MDCK cells and Consistent with the earlier studies , we found that C4U embryonated chicken eggs, virus titres increased 1.4  29-fold replacement increased viral replicative ability. By contrast, and 4.6–172-fold, respectively (Table 1). The increases in titre modifications at other positions of the promoter region of the conferred by the PR8-HY backbone for pandemic H5N1 and HA segment did not confer growth advantages. H7N9 vaccine candidates were substantial; it is not yet clear why Several studies have found that chimeric HA and NA proteins smaller improvements in titre were found for seasonal human (in which the intracellular and transmembrane domains are vaccine viruses in MDCK and Vero cells. It should be kept in derived from PR8 virus, while the extracellular domains are mind that most comparisons in Table 1 were made against the derived from a (candidate) vaccine virus) confer a growth control strain UW-PR8, which replicates to higher titres than advantage over viruses that possess the full-length HA and NA 28,29 22–27 other PR8 strains . genes/proteins of the respective vaccine virus . The The high-yield vaccine backbone PR8-HY carries seven amino- underlying mechanism is not fully understood but may reflect acid changes across five different viral proteins compared with the incompatibilities among the proteins and/or viral packaging UW-PR8 strain: PB2-I504V, PB1-M40L/G180W, PA-R401K, signals of the viral components derived from the different viruses. NP-I116L and NS1-A30P/R118K. A number of growth-enhan- Incompatibility among viral proteins and/or packaging signals cing mutations described in the literature did not increase the may also explain why we did not detect a consistent pattern; while replicative ability of UW-PR8, most likely because many chimeric HA and NA segments provided clear advantages with mutations in influenza viral proteins have context-specific effects some vaccine candidates (for example, A/Hubei/1/2010 and (that is, their effect depends on the virus used for testing). The A/Anhui/1/2013), no significant differences were found for other context-specific effects of many mutations, and the fact that candidates. In the event of a pandemic, both versions of candidate random mutagenesis may create mutants that do not frequently vaccines could be generated and compared. Nowadays, this can emerge in nature (for example, those that require two mutations be achieved conveniently through the use of chemically in one codon) most likely also explain why the PB2-I504V synthesized HA and NA genes. mutation was the only amino-acid change identified both through In conclusion, we here present a high-yield PR8 vaccine virus screens of random libraries and through the testing of potentially backbone that could improve the titres of pandemic and seasonal yield-enhancing mutations previously described in the literature. influenza vaccines in both cultured cells and embryonated Interestingly, Rolling et al. detected this mutation in three chicken eggs. independent studies of adaptation of PR8 virus to Mx1- expressing mice, suggesting that PB2-V504 increases the Methods replicative ability of PR8 virus. Consistent with this hypothesis, Study design. Our studies were designed to develop influenza vaccine viruses with increased yield in mammalian cells and/or embryonated chicken eggs. The details database searches revealed that 499% of all human and avian of the study design are described below. PB2 proteins encode valine at position 504 (Supplementary Table 13); in fact, we found only 39 human and 4 avian viruses Viruses and cells. 293T human embryonic kidney cells (obtained from American that encode PB2-I504. Hence, our high-yield vaccine virus Type Culture Collection (ATCC)) were maintained in DMEM supplemented with acquired the amino acid commonly found at this position. The 10% fetal bovine serum. MDCK cells (obtained from ATCC) were grown in MEM biological significance of the PB2-I504V mutation, which is containing 5% new born calf serum. Vero cells (obtained from ATCC) were grown located at the surface of PB2 (ref. 57), is yet not known. in MEM containing 10% fetal bovine serum. Vaccine virus NYMC X-181 (derived The significance of the PB1-M40L and PB1-G180W mutations from A/California/07/2009 (pandemic H1N1)) and NYMC X-223A (derived from A/Texas/50/2012 (seasonal H3N2)) were kindly provided by the National Institute is also currently not known. At position 40, methionine is highly for Biological Standards and Control (NIBSC; Potters Bar, UK). All other viruses conserved among human and avian PB1 proteins. By contrast, at were generated by use of reverse genetics. position 180 of PB1, 499% of human and avian PB1 proteins encode glutamic acid, whereas glycine (as encoded by PR8 Construction of plasmids. The detoxified HA and NA for VN04 were generated viruses) is found very rarely (Supplementary Table 13). Interest- previously . The wild-type sequences for the HA and NA genes of A/Hubei/1/ ingly, an analysis of the PB1 proteins of recent seasonal influenza 2010 (H5N1), A/Egypt/N03072/2010 (H5N1), A/Indonesia/05/2005 (H5N1) and vaccines found a PB1-G180E mutation in 11 of 21 vaccine A/Anhui/1/2013 (H7N9) were obtained from GISAID.org (Supplementary Table 14). The H5N1 gene segments (NA and detoxified HA) were engineered by viruses . Tryptophan (as found in our study) may have been site-directed mutagenesis of related sequences or were chemically synthesized selected from our mutant virus library because its length is similar (Genescript, USA), then amplified by PCR and inserted into the RNA polymerase I to that of glutamic acid, although these amino acids differ in vector pHH21 (ref. 60). The Anhui13 HA and NA sequences were chemically ‘bulkiness’, hydrophobicity and charge. synthesized and cloned into pHH21 as above. The HA and NA genes of X-181 and X-223A were amplified by PCR with reverse transcription (RT–PCR) from the The PA-R401 and NP-I116 residues are highly conserved respective vaccine virus and cloned into the pHH21 vector. among human and avian influenza A virus proteins, while the To rapidly construct chimeric HA and NA segments, universal chimeric HA lysine and leucine residues found at these positions of the PR8- and NA vectors were constructed. Briefly, the pHH21-PR8-HA and pHH21-PR8- HY backbone are very rare (Supplementary Table 13). No NA plasmids were modified to contain two BsmBI restriction enzyme sites that were then used to replace the HA and NA ecto-domain sequences, respectively. biological functions have been identified for these residues. This cloning strategy does not introduce unwanted amino-acid changes into the Likewise, no functions have been identified for the amino acids at HA and NA sequences. positions 30 and 118 of NS1. The NS1-A30P mutation detected in PR8-HY has not been observed in nature (Supplementary Construction of plasmid libraries. Random mutations were introduced into the Table 13). The NS1-K118 residue encoded by the PR8-HY six internal genes of UW-PR8 virus by error-prone PCR using GeneMorph II backbone is expressed by roughly one-third of avian NS1 Random Mutagenesis Kit. Briefly, PCR reaction conditions and target DNA tem- proteins, while most avian and almost all human NS1 proteins plate amounts were optimized to generate 1–4 amino acids substitutions per possess arginine at this position (Supplementary Table 13). protein. The randomly mutated cDNAs were then inserted into RNA polymerase 12 NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 ARTICLE I vector to generate randomly mutated plasmid libraries. The diversity of the SDS–PAGE electrophoresis. One microlitre of virus concentrate was mixed with plasmid libraries was confirmed by sequence analysis: we found that 75–92% of the water to a total volume of 10ml. Loading dye (2.5ml) with 2% (v/v) b-mercap- sequenced clones retained the start codon, lacked premature stop codons, and toethanol as the reducing agent was added to each sample. Samples were heated to possessed, on average, 1.95–3.9 amino acid changes per PCR product. 95 C for 5 min before loading onto the NuPage 4–12% Bris-Tris precast gel (Life technology). Gels were run at 150 V for 120 min using 1  MES buffer (Bio-Rad) and then stained with SYPRO-Ruby (Sigma). Quantification of protein amounts was carried out by using ImageJ software (National Institutes of Health). To cal- Virus rescue and virus library generation. All viruses and virus libraries used in this study were generated by means of reverse genetics, using eight pHH21-based culate the HA content, we divided the HA amount (calculated by summing the amounts of HA1 and HA2) by the sum of the amounts of HA1, HA2, NP and M1, RNA polymerase I plasmids for viral RNA synthesis and four protein-expressing plasmids to synthesize the viral replication complex as described by Neumann and multiplied this value by the amount of total viral protein in the samples analysed by use of gel electrophoresis. et al. Virus stocks were generated by infecting MDCK cells, Vero cells or embryonated chicken eggs with 100ml of virus-containing supernatant derived from plasmid-transfected 293T cells. The titres of all virus stocks were determined Virulence studies in mice. To determine the MLD , three mice/group (a sample by means of plaque assays in MDCK cells. To generate virus libraries, cells were size adequate to detect large effects between groups) of 6-week-old female BALB/c transfected with a mutant plasmid library instead of the wild-type construct. At mice (Jackson Laboratory, Bar Harbor, ME, USA) were anaesthetized with iso- 48 h post transfection, supernatants derived from plasmid-transfected 293T cells 1 6 flurane and inoculated intranasally with 10-fold dilutions of virus (from 10 to 10 were passaged in MDCK and Vero cells by infecting them at a MOI of 0.01. All PFU) in a volume of 50ml. For these experiments, mice were randomized and experiments involving wild type or reassortant A/Anhui/1/2013 (H7N9) virus were investigators were not blinded. Body weight changes and survival were recorded carried out in biosafety level 3 containment. Experiments with H5N1 viruses that daily until day 14, and the MLD was calculated by the method of Reed and were exempt from Select Agent status by APHIS and approved by the University of Muench. To assess virus replication in mice, 10 PFU of recombinant viruses were Wisconsin-Madison Institutional Biosafety Committee for work at BSL-2 were used to infect six additional mice. At days 3 and 5 post infection, three mice in each used at that containment level. group were euthanized and their lungs were collected and homogenized. Virus titres were determined by plaque assays in MDCK cells. Evaluation of viral growth kinetics. To analyse the growth characteristics of viruses, Vero or MDCK cells were infected in triplicate (a sample size adequate to Genetic stability testing. To evaluate the genetic stability of the high-yield detect large effects between groups) with recombinant viruses at a MOI of 0.001 backbone, the recombinant viruses were consecutively passaged 10 times in Vero (MDCK cell infection) or 0.005 (Vero cell infection). One hour after incubation at cells at an MOI of 0.1. In parallel, these viruses were also consecutively passaged 10 37 C, the cells were washed once with phosphate-buffered saline (PBS), and fresh times in embryonated chicken eggs at an inoculation dosage of 2  10 PFU per MEM/BSA medium with 0.5mgml tosyl phenylalanyl chloromethyl ketone egg. Viruses sampled after the 10th passage in the supernatants of Vero cells and (TPCK) trypsin was added. Supernatants were collected at the indicated time the allantoic fluid of embryonated chicken eggs were sequenced by means of Sanger points and the virus titres in the supernatants were determined by plaque assays in sequencing. MDCK cells. For Vero cell infections, fresh TPCK trypsin was added to the supernatants every day (0.5mgml ). To analyse viral growth kinetics in Virus genome sequencing. To determine the mutations present in the 36 high- embryonated chicken eggs, 2  10 PFU of virus was inoculated into 10-day-old yield candidate viruses isolated from the MDCK passages, we performed whole embryonated chicken eggs. The allantoic fluids of four eggs each were harvested genome sequencing on the Ion Torrent PGM System (Life Technologies). Briefly, at the indicated time points. Virus titres were determined by plaque assays in 5ml of total RNA extracted from the supernatant of virus-infected cells was used to MDCK cells. amplify all eight viral segments in a multi-segment RT–PCR reaction . Barcoded, The haemagglutination (HA) titres of supernatants derived from infected 200-base insert libraries were then produced using the Ion Xpress Plus Fragment MDCK and Vero cells or allantoic fluid derived from inoculated eggs were Library Kit (Life Technologies), with an eight-cycle limited PCR to increase the determined by using an HA assay. Briefly, 50ml of virus sample was serially diluted numbers of fragments with adaptors. Final library pools were constructed 2-fold in 96-well U-bottom microtitre plates (Thermo Scientific) that contained containing equimolar amounts of each barcoded component, and the pools were 50ml of PBS per well, and then 50ml of 0.5% turkey red blood cells was added to run on Ion Torrent 314 chips. each well. After a 45-min incubation at room temperature, the highest agglutinating well was read as the virus HA titre. Statistical analysis. The data were analysed by using the R software (www.r- project.org), version 3.1. For comparisons of multiple groups with measurements Virus concentration and purification. Two 4-Layers Easy-Fill Cell Factories collected independently at different time points (that is, viral growth curves in (Thermo Scientific) of Vero cells were infected with recombinant viruses, and fresh embryonated chicken eggs), we used two-way analysis of variance followed by –1 TPCK trypsin was added every day (0.5mgml ). Alternatively, viruses were grown Tukey’s post-hoc test. For comparisons of measurements from multiple groups in 10-day-old embryonated chicken eggs. Infected cell culture supernatants were collected at a single time point, we used one-way analysis of variance also followed collected at 72 h post infection, whereas allantoic fluids were collected 48 h after egg by Tukey’s post-hoc test. For comparisons of multiple groups with dependent inoculation. Cell culture supernatants or allantoic fluids were clarified by cen- measurements (that is, viral growth curves in cell culture for which aliquots were trifugation (3,500 r.p.m., 15 min, 4 C). Viruses were then pelleted by centrifugation collected from the same culture at different time points), we fitted a linear mixed- (18,500 r.p.m., 90 min at 4 C in a Beckman Type19 rotor), resuspended in 5 ml of effects model to the data using the R package NLME, and the time, the virus PBS and loaded onto 30 ml, 20–50% continuous sucrose gradients that were cen- strains, and the interaction between these two factors were considered. Next we trifuged at 25,000 r.p.m. for 90 min at 4 C in a Beckman SW32 rotor. The virus built a contrast matrix to compare the strains in a pairwise fashion at the same time band was collected, diluted in PBS, pelleted by centrifugation (25,000 r.p.m., points (for example, group_1 versus group_2 at 24 h post infection, group_1 versus 90 min, 4 C in a Beckman SW32 rotor), and the final virus pellet was resuspended group_3 at 24 h post infection, group_2 versus group_3 at 24 h post infection), in 400ml of PBS (including 0.1% b-propiolactone (BPL)) overnight at 4 Cto using the R package PHIA. Because the comparisons were performed individually, inactivate the virus particles. Then, samples were incubated at 37 C for 45 min to the final P-values were adjusted using Holm’s method to account for multiple inactivate the BPL, aliquoted, and stored at  80 C. comparisons. Finally, for comparisons involving only two groups with measure- A/Anhui/1/2013 (H7N9) recombinant viruses were amplified in BSL-3 ments at single time points, we used two-tailed, unpaired t-tests; if multiple containment and inactivated by treatment with 0.1% BPL overnight. Virus comparisons were performed, the P-values were adjusted using Holm’s method. inactivation was confirmed by negative HA assays after two consecutive passages In all cases, except for the data shown in Fig. 7, raw data were converted to the in embryonated chicken eggs (the method and validation of virus inactivation logarithmical scale before the analysis and the results were considered statistically were approved by the Select Agent Program of the University of Wisconsin- significant if we obtained P-values (or adjusted P-values)o0.05, and the variance Madison). between groups was assessed using Levene’s test (it was similar for the groups being compared, with P-value40.05). Total protein assay. Total protein content of virus concentrates was determined by using the Pierce BCA protein assay kit (Thermo Scientific) according to the Ethics and biosafety. Our experiments in mice followed the University of Wis- manufacturer’s instructions. consin-Madison’s Animal Care and Use Protocol. All experiments were approved by the Animal Care and Use Committee of the University of Wisconsin-Madison (protocol number V00806), which acknowledged and accepted both the legal and Deglycosylation of viral proteins using PNGase F. Virus proteins were degly- ethical responsibility for the animals, as specified in the Fundamental Guidelines cosylated by using PNGase F (New England Biolabs). Specifically, 10ml of virus for Proper Conduct of Animal Experiment and Related Activities in the Animal concentrates were denatured according to the manufacturer’s instructions in a total Welfare Act and associated Animal Welfare Regulations and Public Health Service reaction volume of 60ml, followed by incubation at 37 C for 20 h with 2mlofa1/ Policy (USA). 10 dilution of PNGase F enzyme in the buffer provided by the manufacturer and All experiments were completed before the US Government announced a with NP40 at a final concentration of 1%. research pause on certain gain-of-function studies on 17 October 2014. NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications 13 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 References 28. Horimoto, T. et al. Enhanced growth of seed viruses for H5N1 influenza 1. Wright, P. 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J.P. and G.N. wrote the contribute to high virulence of influenza A virus in mice. J. Virol. 83, manuscript. 6673–6680 (2009). 57. Pflug, A., Guilligay, D., Reich, S. & Cusack, S. Structure of influenza A polymerase bound to the viral RNA promoter. Nature 516, 355–360 (2014). 58. Plant, E. P., Liu, T. M., Xie, H. & Ye, Z. Mutations to A/Puerto Rico/8/34 PB1 Additional information gene improves seasonal reassortant influenza A virus growth kinetics. Vaccine Accession codes. The sequences of the six internal viral RNA segments of the high-yield 31, 207–212 (2012). PR8 backbone have been deposited in the GenBank under accession codes KT314334 to 59. Imai, M. et al. Experimental adaptation of an influenza H5 HA confers KT314339. respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Supplementary information accompanies this paper at http://www.nature.com/ Nature 486, 420–428 (2012). naturecommunications 60. Neumann, G. et al. Generation of influenza A viruses entirely from cloned cDNAs. Proc. Natl Acad. Sci. USA 96, 9345–9350 (1999). Competing financial interests: J.P., C.A.N., E.G., C.A.M., A.F., M.I, E.A.M, and T.J.S.L. 61. Zhou, B. et al. Single-reaction genomic amplification accelerates sequencing have no competing interests. G.N. and Y.K. are Co-founders of FluGen. Y.K. is also a paid and vaccine production for classical and Swine origin human influenza a consultant of Crucell. viruses. J. Virol. 83, 10309–10313 (2009). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Acknowledgements We thank Susan Watson for scientific editing. We thank the National Institute for How to cite this article: Ping, J. et al. Development of high-yield influenza A virus Biological Standards and Control (NIBSC, Potters Bar, UK) for the vaccine viruses vaccine viruses. Nat. Commun. 6:8148 doi: 10.1038/ncomms9148 (2015). NYMC X-181 and NYMC X-223A. This work was supported by the NIAID-funded Center for Research on Influenza Pathogenesis (CRIP, HHSN266200700010C), by the This work is licensed under a Creative Commons Attribution 4.0 Wisconsin Alumni Research Foundation, by the Japan Initiative for Global Research International License. The images or other third party material in this Network on Infectious Diseases from the Ministry of Education, Culture, Sports, Science, article are included in the article’s Creative Commons license, unless indicated otherwise and Technology, Japan, by ERATO, Japan, Strategic Basic Research Programs of Japan in the credit line; if the material is not included under the Creative Commons license, Science and Technology Agency, by the Advanced Research & Development Programs for Medical Innovation from Japan Agency for Medical Research and development, users will need to obtain permission from the license holder to reproduce the material. AMED, and by the Bill & Melinda Gates Foundation (OPPGH5383). 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Abstract

ARTICLE Received 3 Feb 2015 | Accepted 23 Jul 2015 | Published 2 Sep 2015 DOI: 10.1038/ncomms9148 OPEN Development of high-yield influenza A virus vaccine viruses 1 1,2 3 4,5 6 5 Jihui Ping , Tiago J.S. Lopes , Chairul A. Nidom , Elodie Ghedin , Catherine A. Macken , Adam Fitch , 1 1 1 1,2 Masaki Imai , Eileen A. Maher , Gabriele Neumann & Yoshihiro Kawaoka Vaccination is one of the most cost-effective ways to prevent infection. Influenza vaccines propagated in cultured cells are approved for use in humans, but their yields are often suboptimal. Here, we screened A/Puerto Rico/8/34 (PR8) virus mutant libraries to develop vaccine backbones (defined here as the six viral RNA segments not encoding haemagglutinin and neuraminidase) that support high yield in cell culture. We also tested mutations in the coding and regulatory regions of the virus, and chimeric haemagglutinin and neuraminidase genes. A combination of high-yield mutations from these screens led to a PR8 backbone that improved the titres of H1N1, H3N2, H5N1 and H7N9 vaccine viruses in African green monkey kidney and Madin–Darby canine kidney cells. This PR8 backbone also improves titres in embryonated chicken eggs, a common propagation system for influenza viruses. This PR8 vaccine backbone thus represents an advance in seasonal and pandemic influenza vaccine development. Department of Pathobiological Sciences, School of Veterinary Medicine, Influenza Research Institute, University of Wisconsin-Madison, Madison, Wisconsin 53711, USA. Division of Virology, Department of Microbiology and Immunology and International Research Center for Infectious Diseases, The Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. Avian Influenza-Zoonosis Research Center, Airlangga University, Surabaya 60115, Indonesia. 4 5 Department of Biology, New York University, New York, New York 10003 USA. Department of Computational & Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 USA. Bioinformatics Institute, University of Auckland, Auckland 1010, New Zealand. Correspondence and requests for materials should be addressed to Y.K. (email: kawaokay@svm.vetmed.wisc.edu). NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 nfluenza A viruses belong to the family Orthomyxoviridae with reassortant viruses or viruses encoding chimeric or mutated HA 22–27 a genome composed of eight single-stranded, negative-sense and/or NA proteins ; to date, none of these studies has Iviral RNA segments. Based on the antigenicity of the two resulted in commercially available, improved influenza vaccine major surface antigens, haemagglutinin (HA) and neuraminidase production. An alternative strategy for boosting vaccine (NA), influenza A viruses are currently categorized into 18 HA production is to modify the virus backbone (that is, PR8) for subtypes (H1–H18) and 11 NA subtypes (N1–N11). Each year, increased replication efficiency. This approach has the potential seasonal influenza A viruses of the H1N1 and H3N2 subtypes and to work in a strain-independent manner, avoiding time required influenza B viruses cause several hundred million human for preliminary adaptation of a new strain to the culture system. infections and 250,000 to 500,000 deaths worldwide (http:// We therefore carried out a comprehensive study to develop a PR8 www.who.int/mediacentre/factsheets/fs211/en/). Occasionally, vaccine backbone that significantly improves the virus yield of reassortment of viral RNA segments results in novel strains to various seasonal and pandemic influenza vaccines strains in cell which most humans are naı¨ve, such as during the pandemics of culture. As we show, the same backbone can also improve yield in 1918, 1957, 1968 and 2009 (reviewed in ref. 1). In addition, egg culture systems. sporadic infections of humans with avian influenza A viruses of the H5N1 or H7N9 subtypes (resulting in case fatality rates of B60 and B30%, respectively) (http://www.who.int/influenza/ Results human_animal_interface/EN_GIP_20140124CumulativeNumber Virus library screens for high-yield variants. Currently, it is not 2,3 H5N1cases.pdf?ua=1) have been a major public health concern. known which mutations in PR8 increase the viruses’ replicative Due to frequent ‘antigenic drift’ (that is, the accumulation of ability in cultured cells. Therefore, we used a high-throughput point mutations in the antigenic epitopes of HA) and occasional mutagenesis and screening approach to identify mutations asso- ‘antigenic shift’ (that is, the introduction into human populations ciated with high yield of PR8. Specifically, we used error-prone of an HA to which humans are immunologically naive), new PCR to generate large sets (that is, ‘libraries’) of viral cDNAs with influenza A vaccine strains are recommended every B1–5 years random mutations (see Methods for details). Using reverse by the World Health Organization (WHO). For the generation of genetics approaches, these mutant cDNA libraries were used to inactivated vaccines, the HA and NA viral RNA segments of the generate virus libraries composed of tens of thousands of variants recommended strains are typically combined with the remaining possessing random amino-acid changes in one or several viral six viral RNA segments of A/Puerto Rico/8/34 (PR8; H1N1) virus proteins. Based on UW-PR8 (a high-growth variant of PR8 28,29 by using reassortment or reverse genetics approaches. Most maintained at the University of Wisconsin-Madison ), we vaccine viruses are then propagated in embryonated chicken eggs. generated the following nine virus libraries: six libraries However, this widely used influenza vaccine production platform possessing random mutations in each of the ‘internal’ genes has several vulnerabilities, including the potential for the egg (that is, PB2, PB1, PA, NP, M and NS); one library possessing supply to become a limiting factor for rapid large-scale vaccine random mutations in the genes encoding the PB2, PB1 and PA production in the event of an influenza pandemic. In addition, proteins, which together form the viral polymerase complex; one virus propagation in embryonated chicken eggs frequently results library possessing random mutations in the PB2 and NS genes, in egg-adapting mutations in HA that can affect the antigenicity which code for the major virulence factors PB2 and NS1, 4–11 1,30 of the virus and may be responsible for the reduced efficacy of respectively (reviewed in ); and one library possessing random 12,13 some influenza vaccines . mutations in the NS and M genes since the M1 protein (encoded Mammalian cell culture-based vaccine production has distinct by the M gene) is associated with high-growth properties advantages over the currently prevalent egg-based production (Fig. 1). Since high-yield vaccine backbones are urgently needed system: it is more easily scaled up and has reduced risk for the for vaccines to pandemic H5N1 viruses, we combined the HA and emergence of mutations that result in antigenic changes .Asan NA genes of an H5N1 virus, A/chicken/Indonesia/NC/2009 added advantage, the absence of egg proteins in cell culture-based (Indo09; H5N1; subclade 2.1.3.2) with the UW-PR8 backbone for vaccines eliminates potential complications for individuals with our library generation and screening procedure. We replaced the 15,16 egg allergies . To date, influenza vaccines produced in African multibasic sequence at the Indo09 HA cleavage site with a single green monkey kidney (Vero) and Madin–Darby canine kidney basic amino acid so that the viruses generated with the PR8 17,18 (MDCK) cells have been approved for human use in the backbone were of low pathogenicity, and therefore exempt from United States and/or some European countries. Select Agent status by the United States Animal and Plant Health Vaccine virus yield is a critical parameter in the vaccine Inspection Service (APHIS) and approved for studies at BSL-2. manufacturing process. Although the PR8 backbone generally To select high-yield variants, each of the nine libraries of confers efficient replication in embryonated chicken eggs, viruses composed of a mutant UW-PR8 backbone with HA and occasionally PR8-based vaccines have low yields, leading to NA from Indo09 was passaged 12 times in MDCK cells; in delays in vaccine manufacturing. Examples are the original A/ parallel, these nine libraries were combined after the second 19 20,21 Fujian/411/2002 (H3N2) and 2009 pandemic H1N1 passage, and then passaged a further 10 times in MDCK cells vaccine candidates, which replicated poorly in embryonated (Supplementary Fig. 1). From these experiments, we randomly chicken eggs. In addition, lower virus yields in cultured cells selectedB100–150 plaques per library, resulting in a total of 1434 compared with embryonated chicken eggs may have hampered individual, plaque-purified viruses (Fig. 1). The yield of each the wider use of cell culture-based influenza vaccine production plaque-purified virus was compared in haemagglutination (HA) platforms . Therefore, efforts have been undertaken to optimize assays with the yield of a control virus composed of an unmutated the production parameters and the vaccine viruses. Sequential UW-PR8 backbone and the Indo09 HA/NA genes (UW- passages of vaccine viruses in embryonated chicken eggs can PR8_Indo09). We identified 36 viruses with HA titres that were improve virus yield in an egg-based vaccine production system at least twofold higher than that of the control virus. These high- (reviewed in refs 20,21). However, passaging in eggs adds to the yield candidates were ‘purified’ through an additional round of production time, must be repeated for any low-producing strain, plaque assays, followed by virus amplification in MDCK cells. To and frequently leads to mutations in HA that may affect viral identify the mutations responsible for the increased replicative antigenicity, as stated earlier. Some studies have attempted to ability of the 36 high-yield candidates in MDCK cells, we improve the replication efficiency of vaccine viruses by using sequenced their entire genomes. Twenty-nine different 2 NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 ARTICLE Plasmid libraries possessing random mutations Selected potentially yield-enhancing mutations in the PB2, PB1, PA, NP, M and/or NS genes; described in the literature, and tested them in and A/chicken/Indonesia/NC/2009 (H5N1; the backgroud of UW-PR8 internal genes and Indo09) NA and low-pathogenic HA genes Indo09 NA and low-pathogenic HA genes Nine virus libraries possessing random 36 recombinant viruses possessing single or mutations in one or several PR8 viral genes double mutations described in the literature 12 consecutive passages in MDCK cells Plaque purification and HA titre assessment Assess virus growth in MDCK cells of 1,434 viral plaques Identified 36 high-yield viruses representing Identified 10 mutations that confer high-yield 29 genotypes in MDCK cells Mix plasmids encoding 34 (combinations of) mutations identified above Reverse genetics, followed by five consecutive passages in Vero cells Plaque purification and HA titer assessment of 216 viral plaques Identified 16 high-yield viruses (Re)created 7 potential high-yield viral backbones by reverse genetics (HY#1–7) C4U promoter mutation in PB2, Assessment of virus and HA titers in PB1 and PA segments Vero cells: lead candidate - HY#1 High-yield backbone (PR8-HY): PB2-C4U, -I504V; PB1-C4U, -M40L/G180W; PA-C4U, -R401K; NP-I116L; NS1-A30P/R118K (nucleotide changes are shown in italics) Construct chimeric HA and NA segments Test PR8-HY with wild-type or chimeric HA and NA segments of H5N1, H7N9 or human H1N1 and H3N2 viruses Evaluate growth Evaluate growth Evaluate growth characteristics in characteristics in characteristics in embryonated chicken eggs Vero cells MDCK cells Evaluate total protein Evaluate total protein and HA yield and HA yield Figure 1 | Flow chart summarizing the selection and testing of PR8-HY. Details are described in the text. combinations of mutations were detected (Fig. 1 and overlapping PB1-F2 protein). Although the HA and NA genes Supplementary Table 1). The most frequently observed amino- were not targeted by PCR-mediated mutagenesis, several acid changes localized to the polymerase subunits PB2 (namely, mutations were detected in these proteins, likely reflecting PB2-M202L/F323L and PB2-I504V) and PB1 (namely, PB1- adaptation of the Indo09 HA and NA proteins to MDCK cells. V644A and PB1-E112G; note that the nucleotide change causing None of the mutations in HA restored the multibasic sequence at the PB1-E112G mutation also causes an R81G mutation in the the HA cleavage site. NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications 3 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 0 0 Testing of mutations identified in the literature. Multiple nucleotides at the 3 and 5 termini of the vRNAs, respectively, are amino-acid changes have been associated with altered replication highly conserved among the eight viral RNA segments as well as kinetics of a variety of viruses in mammalian systems among viruses. Mutations in these regions can significantly affect 33,34 (Supplementary Table 2). The effect of most of these mutations the viral polymerase activity in reporter gene assays . We, on UW-PR8 in mammalian systems is unknown. We, therefore, therefore, tested 20 UW-PR8_Indo09 virus libraries that used site-directed mutagenesis to generate 36 mutant UW- possessed various mutations at several positions in the PR8_Indo09 viruses, each carrying a mutation(s) selected from promoter region of the HA gene; however, we were unable to Supplementary Table 2, and assessed their growth kinetics in select mutants with increased replicative ability in MDCK cells. MDCK cells (Fig. 1 and Supplementary Fig. 2). Ten mutants The viral promoters are separated from the start and stop exhibited significant increases in virus yield compared with the codons of the viral open reading frames by a variable number of control virus UW-PR8_Indo09 (Supplementary Fig. 2 and nucleotides, depending on the viral RNA segment. These so- Supplementary Table 3). One of these mutants (carrying PB2- called ‘non-coding regions’ differ among the viral RNA segments I504V and PA-I550L) was also isolated from our randomly and among virus strains. Here, we tested 11 virus libraries in mutated virus libraries (see Supplementary Table 1), suggesting which portions of the non-coding region of the UW-PR8_Indo09 that it confers high-yield properties to UW-PR8_Indo09. HA segment were randomized; these mutations did not increase the replicative ability of UW-PR8_Indo09 virus in MDCK cells. Potential combinatorial effects of mutations. On the basis of Our random mutations of the promoter region did not affect our library screens and the systematic testing of mutations virus yield in MDCK cells. Nevertheless, potentially significant identified through literature searches, we generated 34 mutants variability in the fourth position from the 3 end of the viral for further study. Thirty-two of these carried single or double RNAs, where a C or U (C4 or U4) residue is found, has been mutations selected from those that caused the greatest increases reported . Specifically, in MDCK cells, the titre (assessed by in yield and/or were found most frequently (Fig. 1 and using plaque assays) of a A/WSN/33 virus encoding U4 in all Supplementary Table 4). In addition, the NP-R422K mutation eight viral RNA segments was more than two log units higher was selected because of its role in NP oligomer formation , than that of a control A/WSN/33 virus encoding C4 (ref. 35). We, although it did not confer a statistically significant increase in therefore, converted the C4 residues found in the three poly- virus titres (Supplementary Fig. 2 and Supplementary Table 3). merase genes of HY#1 to U4, resulting in HY#1 þ C4U_Indo09, Similarly, the NS1-K55E mutation was selected for its which replicated to slightly higher titres in Vero cells compared demonstrated contribution to the high-growth properties of with HY#1_Indo09 (Fig. 2b and Supplementary Table 7); UW-PR8 virus , although it did not increase virus titres here HY#1 þ C4U_Indo09 was therefore selected as the final high- (Supplementary Fig. 2 and Supplementary Table 3). To test yield backbone in Vero cells, and was designated PR8-HY (Fig. 1). whether combinations of these mutations would further increase Compared with the UW-PR8 backbone, the PR8-HY backbone the growth properties of UW-PR8_Indo09, we transfected 293T possesses the following mutations: PB2-C4U (nucleotide changes cells with eight RNA polymerase I plasmids encoding the wild- are shown in italics), PB2-I504V, PB1-C4U, PB1-M40L/G180W, type viral RNA segments, and with 34 RNA polymerase I PA-C4U, PA-R401K, NP-I116L and NS1-A30P/R118K (Fig. 1). plasmids encoding the mutations shown in Supplementary Table 4. To initiate viral RNA transcription and replication, Chimeric HA and NA genes increase virus yield. In parallel to these cells were also transfected with four plasmids encoding the the development of a UW-PR8-based high-yield backbone, we A/WSN/33 (H1N1) polymerase and NP proteins. In total, the also tested chimeric HA and NA genes/proteins, a strategy that cells were co-transfected with 46 different plasmids. This strategy 22–27 has been shown to increase virus yield . In these chimeras, the should result in viruses encoding all candidate high-yield extracellular domains of HA and NA are derived from the mutations shown in Supplementary Table 4. recommended vaccine strain, whereas the transmembrane and Ideally, a backbone for cell-based vaccine production will intracellular domains originate from PR8 viruses to ensure confer increased growth properties in both MDCK and Vero cells. optimal compatibility with the internal, PR8 virus-derived viral We therefore passaged the above-described virus mixture five times genes and proteins. We, therefore, generated a UW- in Vero cells and isolated 216 individual viruses, which were PR8_Indo09 virus in which only the extracellular domains Chim assayed for HA titres (Fig. 1). Sixteen viruses exhibited HA titres of of the HA and NA proteins were derived from Indo09 virus (see 9–9.5 6.5 2 , compared with an HA titre of 2 for the control UW- Supplementary Fig. 3 and Supplementary Table 8). In Vero cells, PR8_Indo09 virus (Fig. 1 and Supplementary Table 5). Sequence the virus and HA titres of this virus were significantly higher than analysis of these high-yield candidates revealed dominant muta- those of the UW-PR8_Indo09 virus, which encodes the authentic tions in several viral proteins (Supplementary Table 5). Indo09 HA and NA genes (Fig. 3a and Supplementary Table 9). To confirm the yield-enhancing effects of the amino-acid Therefore, we next tested the chimeric Indo09 HA and NA genes changes in these 16 selected viruses, we used reverse genetics to with the high-yield backbone (PR8-HY), resulting in PR8- make seven different mutant UW-PR8_Indo09 viruses, referred HY_Indo09 . We detected a small, but statistically signifi- Chim to as HY#1–7_Indo09 (Supplementary Table 6). Each of these cant increase in virus titres for PR8-HY_Indo09 relative to Chim mutant viruses carries a different combination of amino-acid those for PR8-HY_Indo09 (Fig. 3b and Supplementary Table 9). changes selected from those carried by the 16 high-yield viruses. Six of these mutants exhibited significantly higher HA and/or virus titres in Vero cells than the control UW-PR8_Indo09 virus Evaluation of candidate vaccine viruses in Vero cells. Ideally, (Fig. 2a and Supplementary Table 7). The highest titres were the virus backbone used for influenza vaccine production should detected for HY#1_Indo09, which carries the PB2-I504V, PB1- yield high virus and HA titres with the HA and NA genes of M40L/G180W, PA-R401K, NP-I116L and NS1-A30P/R118K varied seasonal and pandemic influenza viruses. Accordingly, we mutations; the HY#1 vaccine backbone was therefore selected first tested the PR8-HY backbone with wild-type or chimeric HA for further studies. and NA genes of different WHO-recommended H5N1 vaccine viruses, namely, A/Vietnam/1203/2004 (Clade 1; VN04), A/ Mutations in the influenza promoter and non-coding regions. Hubei/1/2010 (Clade 2.3.2.1a; Hubei10), A/Egypt/N03072/2010 The influenza A viral promoter sequences, that is, the 12 and 13 (Clade 2.2.1; Egypt10), and A/Indonesia/05/2005 (Clade 2.1.3.2; 4 NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 ARTICLE a Viral titre HA titre 9 10 UW-PR8_Indo09 HY#1_Indo09 HY#2_Indo09 HY#3_Indo09 HY#4_Indo09 HY#5_Indo09 HY#6_Indo09 HY#7_Indo09 3 0 0 24 48 72 96 0 24 48 72 96 9 10 UW-PR8_Indo09 HY#1_Indo09 HY#1+C4U_Indo09 (PR8-HY_Indo09) 2 0 0 12 24 36 48 60 72 0 12 24 36 48 60 72 Hours post infection Hours post infection Figure 2 | Growth kinetics and HA titres of HY#1–7 high-yield candidates in Vero cells. (a) Growth kinetics and HA titres of high-yield candidates in Vero cells. Vero cells were infected in triplicate with the indicated viruses at a multiplicity of infection (MOI) of 0.005 and incubated at 37 C. Supernatants were collected at the indicated time points, and the virus titres were determined by plaque assays in MDCK cells. In parallel, we determined the HA titres of the collected supernatants by performing HA assays. (b) Effect of the C4U promoter mutation in the viral polymerase genes on viral growth kinetics and HA titres. Shown is the comparison of viruses possessing the parental UW-PR8 backbone (UW-PR8_Indo09), the HY#1 backbone (HY#1_Indo09), or the HY#1 backbone with C4U mutations in the PB2, PB1 and PA genes (HY#1 þ C4U_Indo09). Experiments were carried out as described in a. The values presented are the average of three independent experiments s.d. Viral titre HA titre 9 9 8 8 7 7 6 6 5 5 UW-PR8_Indo09 4 UW-PR8_Indo09 Chim 3 3 1 1 0 122436486072 0 122436486072 6 5 UW-PR8_Indo09 5 PR8-HY_Indo09 PR8-HY_Indo09 chim 2 0 0 122436486072 0 122436486072 Hours post infection Hours post infection Figure 3 | Growth kinetics and HA titres of HA/NA chimeric viruses in Vero cells. (a) Growth kinetics and HA titres of UW-PR8-based viruses with wild- type or chimeric Indo09 HA and NA genes. (b) Growth kinetics and HA titres of viruses possessing the UW-PR8 backbone in combination with Indo09 HA and NA genes, or the PR8-HY backbone in combination with wild-type or chimeric Indo09 HA and NA genes. Experiments were carried out as described in the legend to Fig. 2. The values presented are the average of three independent experiments s.d. Indo05); all H5 HA genes were mutated to encode an HA clea- replicating Egypt10 virus, which was also tested at 96 h post vage sequence characteristic of low pathogenic viruses. We tested infection (Fig. 4a–d). The PR8-HY backbone increased virus and titres up to 72 h post infection, except for the inefficiently HA titres significantly compared with the respective control NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications 5 & 2015 Macmillan Publishers Limited. All rights reserved. –1 Virus yield (Log PFU ml ) –1 –1 –1 Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) 10 10 HA titre (Log ) HA titre (Log ) HA titre (Log ) 2 HA titre (Log ) 2 2 2 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 a b Viral titre HA titre Viral titre HA titre 10 10 10 10 9 9 8 8 7 7 8 8 6 6 7 5 7 5 4 4 6 6 3 3 2 2 5 5 1 1 4 0 4 0 0 122436486072 0 122436486072 0 122436486072 0 122436486072 UW-PR8_Hubei10 UW-PR8_VN04 PR8-HY_Hubei10 PR8-HY_VN04 PR8-HY_Hubei10 PR8-HY_VN04 Chim Chim c d 10 11 10 11 10 10 9 9 8 8 7 7 6 6 5 5 4 4 3 3 2 2 1 1 3 0 5 0 0 1224364860728496 0 1224364860728496 0 122436486072 0 122436486072 UW-PR8_Egypt10 UW-PR8_Indo05 PR8-HY_Egypt10 PR8-HY_Indo05 PR8-HY_Indo05 PR8-HY_Egypt10 Chim Chim e f 10 9 9 8 8 7 7 6 7 6 5 5 5 6 4 4 3 5 3 1 1 0 0 3 0 0 1224364860728496 0 1224364860728496 0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 168 UW-PR8_Anhui13 Hours post infection Hours post infection PR8-HY_Anhui13 X-181 PR8-HY_Anhui13 PR8-HY_X-181 Chim PR8-HY_X-181 Chim 9 10 8 8 7 6 6 4 5 2 4 0 0 1224364860728496 0 1224364860728496 Hours post infection Hours post infection X-223A PR8-HY_X-223A Figure 4 | Evaluation of PR8-HY backbone vaccine candidates propagated in Vero cells. Growth kinetics and HA titres of UW-PR8- and PR8-HY-based viruses encoding the wild type, or wild-type and chimeric HA and NA segments of the A/Vietnam/1203/2004 (VN04, H5N1) (a), A/Hubei/1/2010 (Hubei10, H5N1) (b), A/Egypt/N03072/2010 (Egypt10, H5N1) (c), A/Indonesia/5/2005 (Indo05, H5N1) (d) or A/Anhui/1/2013 (Anhui13, H7N9) (e) viruses. Panels (f) and (g) show a comparison of current seasonal H1N1 and H3N2 vaccine viruses (X-181 and X-223A, respectively) with PR8-HY backbone viruses possessing wild-type or chimeric HA and NA segments derived from X-181 or X-223A viruses. Experiments were carried out as described in the legend to Fig. 2, with the exception of those involving X-181 and X-223A viruses, which were inoculated at an MOI of 0.1. The values presented are the average of three independent experiments s.d. viruses possessing the UW-PR8 backbone (Fig. 4a–d and titres for Hubei10, Egypt10 and Indo05, but not for VN04 viruses Supplementary Table 10). The use of chimeric HA and NA genes (Fig. 4a–d and Supplementary Table 10). Together, these provided a statistically significant increase in virus and/or HA improvements increased virus titres B5  220-fold compared 6 NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. –1 –1 –1 Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) –1 10 10 10 Virus yield (Log PFU ml ) HA titre (Log ) HA titre (Log ) HA titre (Log ) 2 2 2 HA titre (Log ) –1 –1 –1 Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) 10 10 Virus yield (Log PFU ml ) HA titre (Log ) HA titre (Log ) 2 2 HA titre (Log ) 2 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 ARTICLE Table 1 | Comparison of growth characteristics of PR8-HY viruses possessing wild-type or chimeric HA and NA genes. Virus Growth Peak virus titre Peak HA titre Total viral protein yield HA content substrate –1 n PFU ml Statistical Fold- 2 Statistical Fold- Statistical Fold- Statistical Fold- significance increase significance increase significance increase significance increase compared versus compared versus compared versus compared versus with UW- UW-PR8 with UW- UW-PR8 with UW- UW-PR8 with UW- UW-PR8 PR8 PR8 PR8 PR8 PR8-HY_ Vero 1.0  10 Po0.005 28.5 9.5–10 Po0.005 5.7 Po0.005 9.8 Po0.005 10.8 Indo09 MDCK 1.0  10 Po0.005 4.6 9.5 Po0.005 3 ND* ND ND ND Chim (H5N1) Egg 7.0  10 Po0.005 5.9 11–11.5 Po0.005 2.9 Po0.005 4.4 Po0.005 5.3 PR8-HY_ Vero 8.8  10 Po0.005 5.1 8.5 Po0.005 2.2 Po0.005 1.8 Po0.005 1.9 VN04 Chim MDCK 5.8  10 Po0.01 1.4 9 Po0.005 1.3 ND ND ND ND 9 w (H5N1) Egg 2.7  10 Po0.005 4.6 10 — 2 NDNDND ND PR8-HY_ Vero 7.6  10 Po0.005 9.5 8.5–9 Po0.005 3.5 Po0.005 2.1 Po0.005 2.3 Hubei10 Chim MDCK 2.2  10 Po0.01 1.8 8.5 Po0.005 1.3 ND ND ND ND (H5N1) Egg 4.0  10 Po0.005 29 9.5–10 Po0.005 4.9 Po0.005 6.9 Po0.005 7.3 PR8-HY_ Vero 1.3  10 Po0.005 221 10.5–11 Po0.005 76 Po0.005 9.7 Po0.005 11.3 Egypt10 Chim MDCK 6.1  10 Po0.005 3.6 9–9.5 Po0.005 1.7 ND ND ND ND (H5N1) Egg 4.7  10 Po0.005 7.5 12.5 — 2.7 ND ND ND ND PR8-HY_ Vero 8.5  10 Po0.005 28.6 9 Po0.005 4.5 Po0.005 4.9 Po0.005 5.5 Indo05 Chim MDCK 7.1  10 Po0.005 2.4 9.5 Po0.005 3 ND ND ND ND (H5N1) Egg 7.3  10 Po0.005 12.5 12.5– Po0.005 4.6 Po0.005 2.6 Po0.005 3.1 PR8-HY_ Vero 1.3  10 Po0.005 173 8–8.5 Po0.005 3.6 Po0.005 9.9 Po0.005 12.4 Anhui13 Chim MDCK 1.5  10 Po0.005 29 9.5–10 Po0.005 5.5 ND ND ND ND (H7N9) Egg 3.6  10 Po0.005 172 11.5 Po0.005 17.7 Po0.005 14.8 Po0.005 16.9 8 y y y y y y y PR8-HY_X- Vero 1.3  10 Po0.005 269 8 Po0.005 134 Po0.005 3.3 Po0.005 3.9 8 y y y 181 or Chim MDCK 3.3  10 Po0.005 4.7 9.5 — 1.6 ND ND ND ND 9 y y y y y y y PR8-HY_X- Egg 7.2  10 Po0.005 5 11.5–12 Po0.05 2.7 Po0.005 1.8 Po0.005 2.1 (H1N1) 8 y y y y y y y PR8-HY_ Vero 3.7  10 Po0.005 9.3 9–9.5 Po0.005 2.3 Po0.005 2.1 Po0.005 2.2 8 y y y y X-223A MDCK 6.7  10 Po0.005 5.3 9.5 Po0.05 2 ND ND ND ND 9 y y y y y y (H3N2) Egg 3.8  10 Po0.05 6 11–11.5 — 1.3 Po0.005 1.3 Po0.005 1.3 *ND: not determined. wNo significant difference. zSince PR8-HY_X-181 does not grow in Vero cells, PR8-HY_X-181 was used to evaluate total viral protein and HA content in Vero cells. Chim yComparisons were carried out between high-growth X-181 and wild-type X-181 viruses, or between high-growth and wild-type X-223A viruses, respectively. with the respective UW-PR8 viruses (Table 1), demonstrating the influenza vaccines. Interestingly, chimeric HA and NA genes had potential of the PR8-HY backbone as vaccine vector. a strongly attenuating effect on this vaccine candidate (Fig. 4f). Next, we used the same strategy to test the PR8-HY backbone The current human seasonal H3N2 vaccine virus is based on the with the wild-type or chimeric HA and NA genes of A/Anhui/1/ A/Texas/50/2012 HA and NA genes combined with the 2013 (H7N9) virus, a representative of the recently emerged remaining genes of the WHO-recommended PR8 isolate. This H7N9 viruses that have caused severe respiratory infections in virus replicates to moderate titres in Vero cells, which can be 2,3 humans with high-case fatality rates . The Vero cell titres of improved significantly with the use of the PR8-HY backbone PR8-HY viruses expressing wild-type or chimeric A/Anhui/1/ (Fig. 4g and Supplementary Table 10). Notably, we were unable to 2013 HA and NA genes were 85-fold and 173-fold higher, generate a PR8-HY virus possessing chimeric X-223A-derived respectively, than those of the UW-PR8-based control virus HA and NA genes. Thus, for both seasonal human vaccine viruses (Table 1 and Fig. 4e). A significant increase in HA titres was tested here, chimeric HA and NA genes were attenuating, rather found in conjunction with this significant increase in virus titres than titre enhancing. (Fig. 4e and Supplementary Table 10). Finally, we tested the PR8-HY backbone with the wild-type and chimeric HA and NA genes of the current human H1N1 and Evaluation of candidate vaccine viruses in MDCK cells. After H3N2 vaccine viruses, New York Medical Center (NYMC) X-181 demonstrating that the PR8-HY backbone confers high yield to (X-181) and NYMC X-223A (X-223A), respectively. X-181, pandemic and seasonal influenza vaccine candidates in Vero cells, which possesses the A/California/07/2009 (H1N1; CA09) we performed similar studies in MDCK cells. Based on the effi- HA, NA and PB1 genes and the remaining genes from of the cient growth kinetics of the tested viruses in MDCK cells, titres WHO-recommended PR8 virus, did not replicate to high titres, were determined for up to 48 h post infection (Fig. 5). For all even after an extended period of replication (Fig. 4f); by contrast, viruses tested, the PR8-HY backbone conferred higher virus and the PR8-HY backbone with the CA09 HA and NA genes HA titres compared with the UW-PR8 backbone, although not all increased virus titres 4250-fold (Fig. 4f, Table 1 and differences were statistically significant (Fig. 5 and Supplementary Supplementary Table 10); hence, our high-growth vaccine Table 11). Chimeric HA and NA genes further improved the candidate confers significantly higher virus titres than the NYMC virus and/or HA titres of some viruses, namely, PR8-HY_An- PR8 backbone currently used for the generation of seasonal hui and PR8-HY_Hubei10 (Fig. 5 and Supplementary Chim Chim NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications 7 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 Table 11). In combination, the PR8-HY backbone and chimeric Mice infected with PR8-HY-backbone-based viruses also lost HA and NA genes increased virus titres by 1.4  29-fold, and HA more weight (Supplementary Fig. 5b) and had higher lung virus titres by 1.3  5.5-fold (Table 1). titres on days 3 and 5 post infection than did mice infected with the UW-PR8-based virus (Supplementary Fig. 5c), suggesting that the higher replicative ability of PR8-HY-based viruses may cause Evaluation of candidate vaccine viruses in embryonated eggs. slightly higher mouse virulence. However, these increases in Although Vero and MDCK cell lines are now available to pro- virulence were small and PR8-HY-based vaccines would be pagate influenza vaccines for human use, embryonated chicken administered as inactivated vaccines; therefore, the slightly higher eggs remain the most commonly used propagation system, mouse virulence of the PR8-HY backbone compared with the prompting us to test our PR8-HY-based vaccine candidates in UW-PR8 backbone should not be a major issue for vaccine eggs. As shown in Fig. 6 and Supplementary Table 12, the PR8- production, although additional studies may be needed in the HY backbone significantly increased virus and/or HA titres of the future to address this issue. candidate vaccines, and the use of chimeric HA and NA genes further increased the titres of some viruses, such as Anhui13. The Genetic stability of the PR8-HY vaccine backbone. Vaccine combined effect of the PR8-HY backbone and chimeric HA and viruses need to have high genetic stability to minimize the risk of NA genes increased virus titres by 4.6  172-fold, resulting in 9  1 emergence of variants with unwanted properties. To evaluate the titres of 410 plaque-forming units (PFU) ml (Table 1); HA genetic stability of our novel vaccine candidate, PR8-HY_Indo05 titres increased by 1.3  17.7-fold (Table 1). Relatively small and PR8-HY_Indo05 were each passaged 10 times in Vero Chim increases in HA titres were detected for the human seasonal PR8- cells and embryonated chicken eggs, and their whole genome HY_X-181 and PR8-HY_X-223A viruses compared with the Chim sequences were then analysed. None of the yield-enhancing parental X-181 and X-223A viruses. X-181 and X-223A were mutations converted to wild-type sequence during passages in selected as vaccine viruses because of their efficient replication in 11 cultured cells or embryonated chicken eggs, and no additional embryonated chicken eggs (resulting in HA titres of up to 2 mutations were detected in any of the viral genes. In addition, we Log ), so further increases may be difficult to achieve. Collec- inoculated the viruses shown in Table 1 into MDCK cells (at an tively, however, our data demonstrate that the Vero cell-opti- MOI of 0.001), Vero cells (at an MOI of 0.005) or embryonated mized PR8-HY vaccine backbone increases vaccine virus titres in chicken eggs (2  10 PFU per egg). Forty-eight hours later, we embryonated chicken eggs. collected cell culture supernatants and allantoic fluids and sequenced the HA and NA genes of the amplified viruses; no Evaluation of total viral protein yield and HA content. Inac- mutations were found in these genes. These findings further tivated influenza vaccines are standardized by HA content; the support the vaccine potential of the PR8-HY backbone. most commonly used vaccine preparations contain 15mg each of H1 HA, H3 HA and type B HA. Total viral protein yield and HA Discussion content are therefore important parameters in vaccine optimi- Human infections with avian H5N1 or H7N9 viruses have raised zation. Here, we compared the total viral protein yield and HA concerns about a looming pandemic and the ability of standard content of our selected vaccine candidates propagated in Vero influenza vaccine production methods to supply sufficient cells or embryonated chicken eggs. Viruses collected from cell amounts of an effective vaccine in a limited time. Vaccine viruses culture supernatants or egg allantoic fluid were concentrated and that replicate to higher titres than current vaccine viruses would purified by use of sucrose gradient centrifugation. Total viral lessen these concerns and also avoid production delays with protein yield was determined using the Pierce BCA Protein Assay seasonal vaccines in the event that the original vaccine virus Kit (Thermo Scientific, Rochester, NY, USA). In comparison with candidates grow to low titres. Through a comprehensive study the UW-PR8-based control viruses, the PR8-HY backbone yiel- that combined random and site-directed mutagenesis of different ded significantly higher amounts of total viral protein (Fig. 7a,d). functional regions of the viral genome (that is, coding, To evaluate the HA content, the purified virus samples were non-coding and promoter regions), we generated a high-yield deglycosylated with PNGase F (thus allowing the detection of PR8-based vaccine backbone that significantly increased the yield HA2, which in its glycosylated form is similar in size to M1) and of pandemic H5N1 and H7N9 vaccine candidates, and of then analysed by using SDS–polyacrylamide gel electrophoresis seasonal H1N1 and H3N2 vaccine viruses. Although this vaccine (SDS–PAGE) (Fig. 7b,e and Supplementary Fig. 4). Based on a backbone was selected for its high-yield properties in Vero and densitometry analysis, the amounts of HA1, HA2, NP and M1 MDCK cells, it also improved vaccine virus yields in embryonated were determined. To calculate the HA content, we divided the chicken eggs. HA amount (calculated by summing the amounts of HA1 and Vaccine production in cell culture has a number of possible HA2) by the sum of the amounts of HA1, HA2, NP and M1, and benefits over production in embryonated eggs. The biggest multiplied this value by the amount of total viral protein in the drawback of influenza virus amplification in embryonated 4–11 samples analysed via gel electrophoresis. The results showed that chicken eggs is the emergence of antigenic variants . Such PR8-HY-based vaccine candidates displayed a significantly higher unwanted adaptation may have contributed to the reduced 12,13 HA content than those possessing the PR8-UW backbone efficacy of some influenza vaccines . In contrast, the HAs of (Fig. 7c,f). We have not yet performed single radial immuno- cell culture-propagated viruses are typically identical to those of 4–7,12,36–45 diffusion assays or assessed the ratio of infectious to physical the original isolates . Moreover, influenza viruses particles. isolated from humans often replicate more efficiently in cultured mammalian cells than in embryonated chicken 46,47 Virulence of PR8-HY-based vaccine viruses in mice. To rule out eggs . Several studies have also suggested that human the possibility that the high-yield-conferring mutations in the influenza vaccines prepared from viruses propagated in cultured PR8-HY backbone render the virus highly virulent in mammals, cells induce better protective immunity than those grown in 9,48–52 we compared the virulence in mice of UW-PR8_Indo05, PR8- embryonated chicken eggs . HY_Indo05 and PR8-HY_Indo05 . The doses required to kill Despite the advantages of cell culture-based influenza vaccines Chim 50% of infected animals (mouse lethal dose 50, MLD ) were compared with egg-grown products, the former represent only a 2.5 2 2.25 10 ,10 and 10 PFU, respectively (Supplementary Fig. 5a). small percentage of the influenza vaccine market. Vero cells have 8 NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 ARTICLE a b Viral titre HA titre Viral titre HA titre 10 10 9 9 8 8 8 6 6 7 4 6 6 3 2 5 4 0 4 0 0 12243648 0 12243648 0 12243648 0 12243648 UW-PR8_VN04 UW-PR8_Indo09 PR8-HY_VN04 PR8-HY_Indo09 PR8-HY_Indo09 PR8-HY_VN04 Chim Chim 9 9 9 10 7 7 6 7 5 5 6 5 3 3 5 1 1 0 3 0 0 12 24 36 48 0 12 24 36 48 0 12 24 36 48 0 12 24 36 48 UW-PR8_Egypt10 UW-PR8_Hubei10 PR8-HY_Egypt10 PR8-HY_Hubei10 PR8-HY_Egypt10 PR8-HY_Hubei10 Chim Chim e f 9 10 10 11 8 8 6 4 5 2 4 0 1 0 0 12 24 36 48 0 12 24 36 48 0 12 24 36 48 0 12 24 36 48 UW-PR8_Anhui13 UW-PR8_Indo05 PR8-HY_Anhui13 PR8-HY_Indo05 PR8-HY_Anhui13 PR8-HY_Indo05 Chim Chim 9 10 9 11 6 5 3 0 2 0 0 12243648 0 12243648 012 24 36 48 012 24 36 48 Hours post infection Hours post infection Hours post infection Hours post infection X-181 X-223A PR8-HY_X-181 PR8-HY_X-223A PR8-HY_X-181 Chim Figure 5 | Evaluation of PR8-HY vaccine candidate viruses propagated in MDCK cells. Growth kinetics and HA titres of UW-PR8- and PR8-HY-based viruses encoding the wild-type, or wild-type and chimeric HA and NA segments of the A/chicken/Indonesia/NC/2009 (Indo09, H5N1) (a), A/Vietnam/ 1203/2004 (VN04, H5N1) (b), A/Hubei/1/2010 (Hubei10, H5N1) (c), A/Egypt/N03072/2010 (Egypt10, H5N1) (d), A/Indonesia/5/2005 (Indo05, H5N1) (e), or A/Anhui/1/2013 (Anhui13, H7N9) (f) viruses. Panels (g) and (h) show a comparison of current seasonal H1N1 and H3N2 vaccine viruses (X- 181 and X-223A, respectively) with PR8-HY backbone viruses possessing wild-type or chimeric HA and NA segments derived from X-181 or X-223A viruses. MDCK cells were infected in triplicate with the indicated viruses at an MOI of 0.001 and incubated at 37 C; otherwise, experiments were carried out as described in the legend to Fig. 2. The values presented are the average of three independent experiments s.d. NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications 9 & 2015 Macmillan Publishers Limited. All rights reserved. –1 –1 –1 –1 Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) 10 Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) 10 10 HA titre (Log ) HA titre (Log ) HA titre (Log ) 2 2 HA titre (Log ) 2 –1 Virus yield (Log PFU ml ) –1 –1 –1 Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) 10 10 HA titre (Log ) HA titre (Log ) HA titre (Log ) 2 2 2 HA titre (Log ) 2 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 a b Viral titre HA titre Viral titre HA titre 10 12 10 11 11 10 9 10 8 8 7 7 6 4 5 2 4 0 3 0 0 1224364860 0 1224364860 012 24 36 48 60 012 24 36 48 60 UW-PR8_VN04 UW-PR8_Indo09 PR8-HY_Indo09 PR8-HY_VN04 PR8-HY_Indo09 PR8-HY_VN04 Chim Chim 10 11 10 14 9 12 8 10 6 6 5 4 4 2 3 0 4 0 012 24 36 48 60 012 24 36 48 60 0 1224364860 0 1224364860 UW-PR8_Hubei10 UW-PR8_Egypt10 PR8-HY_Hubei10 PR8-HY_Egypt10 PR8-HY_Hubei10 PR8-HY_Egypt10 Chim Chim e f 10 14 10 12 9 8 8 6 5 6 7 4 4 3 6 2 5 0 0 0 0 1224364860 0 1224364860 0 1224364860 0 1224364860 UW-PR8_Anhui13 UW-PR8_Indo05 PR8-HY_Indo05 PR8-HY_Anhui13 PR8-HY_Indo05 PR8-HY_Anhui13 Chim Chim 11 13 10 12 12 11 9 10 8 8 7 6 6 4 2 5 2 1 1 4 0 4 0 0 1224364860 0 1224364860 0 12 24 36 48 60 0 12 24 36 48 60 Hours post-infection Hours post-infection Hours post-infection Hours post-infection X-181 X-223A PR8-HY_X-181 PR8-HY_X-223A PR8-HY_X-181 Chim Figure 6 | Evaluation of PR8-HY vaccine candidate viruses propagated in embryonated chicken eggs. Growth kinetics and HA titres of UW-PR8- and PR8-HY-based viruses encoding the wild-type, or wild-type and chimeric HA and NA segments of the A/chicken/Indonesia/NC/2009 (Indo09, H5N1) (a), A/Vietnam/1203/2004 (VN04, H5N1) (b), A/Hubei/1/2010 (Hubei10, H5N1) (c), A/Egypt/N03072/2010 (Egypt10, H5N1) (d), A/Indonesia/5/2005 (Indo05, H5N1) (e), or A/Anhui/1/2013 (Anhui13, H7N9) (f) viruses. Panels (g) and (h) show a comparison of current seasonal H1N1 and H3N2 vaccine viruses (X-181 and X-223A, respectively) with PR8-HY backbone viruses possessing wild-type or chimeric HA and NA segments derived from X-181 or X- 223A viruses. Ten-day-old embryonated chicken eggs (four per virus) were inoculated with 2  10 PFU of the respective viruses and incubated at 35 C for the indicated periods of time. The values presented are the average of three independent experiments s.d. 53 54 been used for the production of inactivated poliovirus , rabies virus was developed and licensed in Europe (http://www.baxter. and live smallpox vaccines , resulting in an extensive track com/press_room/press_releases/2009/10_07_09-celvapan.html). record for Vero cell-derived vaccines for human use. In October One MDCK cell-derived vaccine has been available in Europe since 2009, a Vero cell-based vaccine to the novel pandemic H1N1 2001 (Influvac, Solvay Pharmaceuticals) and a second since 2007 10 NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. –1 –1 –1 –1 Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) 10 10 10 10 HA titre (Log ) HA titre (Log ) HA titre (Log ) HA titre (Log ) 2 2 2 –1 –1 –1 –1 Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) Virus yield (Log PFU ml ) 10 10 10 10 HA titre (Log ) HA titre (Log ) HA titre (Log ) HA titre (Log ) 2 2 2 2 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 ARTICLE a c *** *** *** *** *** *** 4 *** 7 *** *** *** *** *** 0 0 PNGaseF: + + + + ++ + + + + ++ –– – – – – – –– – – – 250 kDa 150 kDa 100 kDa 75 kDa NP 50 kDa HA1 Deglycosylated HA1 37 kDa M1 25 kDa HA2 20 kDa Deglycosylated HA2 15 kDa d f 22 *** 12 *** *** *** *** *** *** *** *** *** *** *** 0 0 PNGaseF: ++ + + ++ + + + + + + –– – – – – – –– – – – 250 kDa 150 kDa 100 kDa 75 kDa HA1 NP 50 kDa Deglycosylated HA1 37 kDa M1 25 kDa HA2 20 kDa Deglycosylated HA2 15 kDa Figure 7 | Evaluation of the total viral protein and HA content of PR8-HY candidate vaccine viruses. Total viral protein yield of Vero cell- (a) or egg- grown (d), sucrose gradient-purified virus samples. The total protein content of virus concentrates was measured by using the Pierce BCA assay kit (Thermo Fisher) according to the manufacturer’s instructions. SDS–PAGE analyses of virus samples from Vero cells (b) or embryonated chicken eggs (e). Virus concentrates were deglycosylated with PNGase (PNFase F þ ) or left untreated (PNGase F  ). HA contents of Vero-; (c) and egg-grown (f) viruses. The HA contents were calculated based on the total viral protein amounts (Fig. 7a,d) and the relative amounts of HA (Fig. 7b,e); for details, see Study Design. The HA contents are expressed in mg l (for Vero cell-grown viruses) or mg per 100 eggs (for viruses grown in embryonated chicken eggs). Asterisks indicate a significant difference. The values presented are the average of three independent experiments s.d. P-values were calculated by using Tukey’s post-hoc test, comparing the total viral protein yield and HA content of wild-type viruses with that of recombinant high-yield vaccine viruses; ***Po0.005. NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications 11 & 2015 Macmillan Publishers Limited. All rights reserved. UW-PR8_Indo09 PR8-HY_Indo09 Chim UW-PR8_Hubei10 PR8-HY_Hubei10 Chim UW-PR8_Indo05 PR8-HY_Indo05 Chim UW-PR8_Anhui13 PR8-HY_Anhui13 Chim X-181 PR8-HY_X-181 X-223A PR8-HY_X-223A UW-PR8_Indo09 PR8-HY_Indo09 Chim UW-PR8_Hubei10 PR8-HY_Hubei10 Chim UW-PR8_Indo05 PR8-HY_Indo05 Chim UW-PR8_Anhui13 PR8-HY_Anhui13 Chim X-181 PR8-HY_X-181 Chim X-223A PR8-HY_X-223A UW-PR8_Indo09 PR8-HY_Indo09 chim UW-PR8_Hubei10 UW-PR8_Indo09 PR8-HY_Hubei10 chim PR8-HY_Indo09 Chim UW-PR8_Indo05 UW-PR8_Hubei10 PR8-HY_Hubei10 Chim PR8-HY_Indo05 chim UW-PR8_Indo05 PR8-HY_Indo05 Chim UW-PR8_Anhui13 UW-PR8_Anhui13 PR8-HY_Anhui13 PR8-HY_Anhui13 Chim chim X-181 X-181 PR8-HY_X-181 X-223A PR8-HY_X-181 PR8-HY_X-223A UW-PR8_Indo09 PR8-HY_Indo09 X-223A Chim UW-PR8_Hubei10 PR8-HY_X-223A PR8-HY_Hubei10 Chim UW-PR8_Indo05 PR8-HY_Indo05 Chim UW-PR8_Anhui13 PR8-HY_Anhui13 Chim X-181 PR8-HY_X-181 Chim UW-PR8_Indo09 X-223A PR8-HY_X-223A PR8-HY_Indo09 chim UW-PR8_Hubei10 PR8-HY_Hubei10 chim UW-PR8_Indo05 PR8-HY_Indo05 chim UW-PR8_Anhui13 PR8-HY_Anhui13 chim X-181 PR8-HY_X-181 chim X-223A PR8-HY_X-223A Total viral protein –1 (mg per 100 eggs) Total viral protein (mg l ) –1 Total viral protein HA content (mg l ) (mg per 100 eggs) ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 0 0 (Optaflu, Novartis), but such vaccines were not available in the The influenza virus promoter sequences at the 5 and 3 ends of United States until 2012 (Flucelvax, Novartis) (http://www.fda.gov/ the negative-sense viral RNA and the positive-sense complemen- newsevents/newsroom/pressannouncements/ucm328982.htm). tary RNA are highly conserved, with the exception of the fourth Our high-yield vaccine backbone, PR8-HY, in some instances positions from the 3 end of the viral RNA. At this position, the in combination with chimeric HA and NA viral RNA segments, polymerase genes have been reported to encode a C residue, increased peak virus titres in Vero cells 4.6  269-fold compared whereas the remaining five RNA segment encode a U residue. with PR8-UW-based vaccines (Table 1). In MDCK cells and Consistent with the earlier studies , we found that C4U embryonated chicken eggs, virus titres increased 1.4  29-fold replacement increased viral replicative ability. By contrast, and 4.6–172-fold, respectively (Table 1). The increases in titre modifications at other positions of the promoter region of the conferred by the PR8-HY backbone for pandemic H5N1 and HA segment did not confer growth advantages. H7N9 vaccine candidates were substantial; it is not yet clear why Several studies have found that chimeric HA and NA proteins smaller improvements in titre were found for seasonal human (in which the intracellular and transmembrane domains are vaccine viruses in MDCK and Vero cells. It should be kept in derived from PR8 virus, while the extracellular domains are mind that most comparisons in Table 1 were made against the derived from a (candidate) vaccine virus) confer a growth control strain UW-PR8, which replicates to higher titres than advantage over viruses that possess the full-length HA and NA 28,29 22–27 other PR8 strains . genes/proteins of the respective vaccine virus . The The high-yield vaccine backbone PR8-HY carries seven amino- underlying mechanism is not fully understood but may reflect acid changes across five different viral proteins compared with the incompatibilities among the proteins and/or viral packaging UW-PR8 strain: PB2-I504V, PB1-M40L/G180W, PA-R401K, signals of the viral components derived from the different viruses. NP-I116L and NS1-A30P/R118K. A number of growth-enhan- Incompatibility among viral proteins and/or packaging signals cing mutations described in the literature did not increase the may also explain why we did not detect a consistent pattern; while replicative ability of UW-PR8, most likely because many chimeric HA and NA segments provided clear advantages with mutations in influenza viral proteins have context-specific effects some vaccine candidates (for example, A/Hubei/1/2010 and (that is, their effect depends on the virus used for testing). The A/Anhui/1/2013), no significant differences were found for other context-specific effects of many mutations, and the fact that candidates. In the event of a pandemic, both versions of candidate random mutagenesis may create mutants that do not frequently vaccines could be generated and compared. Nowadays, this can emerge in nature (for example, those that require two mutations be achieved conveniently through the use of chemically in one codon) most likely also explain why the PB2-I504V synthesized HA and NA genes. mutation was the only amino-acid change identified both through In conclusion, we here present a high-yield PR8 vaccine virus screens of random libraries and through the testing of potentially backbone that could improve the titres of pandemic and seasonal yield-enhancing mutations previously described in the literature. influenza vaccines in both cultured cells and embryonated Interestingly, Rolling et al. detected this mutation in three chicken eggs. independent studies of adaptation of PR8 virus to Mx1- expressing mice, suggesting that PB2-V504 increases the Methods replicative ability of PR8 virus. Consistent with this hypothesis, Study design. Our studies were designed to develop influenza vaccine viruses with increased yield in mammalian cells and/or embryonated chicken eggs. The details database searches revealed that 499% of all human and avian of the study design are described below. PB2 proteins encode valine at position 504 (Supplementary Table 13); in fact, we found only 39 human and 4 avian viruses Viruses and cells. 293T human embryonic kidney cells (obtained from American that encode PB2-I504. Hence, our high-yield vaccine virus Type Culture Collection (ATCC)) were maintained in DMEM supplemented with acquired the amino acid commonly found at this position. The 10% fetal bovine serum. MDCK cells (obtained from ATCC) were grown in MEM biological significance of the PB2-I504V mutation, which is containing 5% new born calf serum. Vero cells (obtained from ATCC) were grown located at the surface of PB2 (ref. 57), is yet not known. in MEM containing 10% fetal bovine serum. Vaccine virus NYMC X-181 (derived The significance of the PB1-M40L and PB1-G180W mutations from A/California/07/2009 (pandemic H1N1)) and NYMC X-223A (derived from A/Texas/50/2012 (seasonal H3N2)) were kindly provided by the National Institute is also currently not known. At position 40, methionine is highly for Biological Standards and Control (NIBSC; Potters Bar, UK). All other viruses conserved among human and avian PB1 proteins. By contrast, at were generated by use of reverse genetics. position 180 of PB1, 499% of human and avian PB1 proteins encode glutamic acid, whereas glycine (as encoded by PR8 Construction of plasmids. The detoxified HA and NA for VN04 were generated viruses) is found very rarely (Supplementary Table 13). Interest- previously . The wild-type sequences for the HA and NA genes of A/Hubei/1/ ingly, an analysis of the PB1 proteins of recent seasonal influenza 2010 (H5N1), A/Egypt/N03072/2010 (H5N1), A/Indonesia/05/2005 (H5N1) and vaccines found a PB1-G180E mutation in 11 of 21 vaccine A/Anhui/1/2013 (H7N9) were obtained from GISAID.org (Supplementary Table 14). The H5N1 gene segments (NA and detoxified HA) were engineered by viruses . Tryptophan (as found in our study) may have been site-directed mutagenesis of related sequences or were chemically synthesized selected from our mutant virus library because its length is similar (Genescript, USA), then amplified by PCR and inserted into the RNA polymerase I to that of glutamic acid, although these amino acids differ in vector pHH21 (ref. 60). The Anhui13 HA and NA sequences were chemically ‘bulkiness’, hydrophobicity and charge. synthesized and cloned into pHH21 as above. The HA and NA genes of X-181 and X-223A were amplified by PCR with reverse transcription (RT–PCR) from the The PA-R401 and NP-I116 residues are highly conserved respective vaccine virus and cloned into the pHH21 vector. among human and avian influenza A virus proteins, while the To rapidly construct chimeric HA and NA segments, universal chimeric HA lysine and leucine residues found at these positions of the PR8- and NA vectors were constructed. Briefly, the pHH21-PR8-HA and pHH21-PR8- HY backbone are very rare (Supplementary Table 13). No NA plasmids were modified to contain two BsmBI restriction enzyme sites that were then used to replace the HA and NA ecto-domain sequences, respectively. biological functions have been identified for these residues. This cloning strategy does not introduce unwanted amino-acid changes into the Likewise, no functions have been identified for the amino acids at HA and NA sequences. positions 30 and 118 of NS1. The NS1-A30P mutation detected in PR8-HY has not been observed in nature (Supplementary Construction of plasmid libraries. Random mutations were introduced into the Table 13). The NS1-K118 residue encoded by the PR8-HY six internal genes of UW-PR8 virus by error-prone PCR using GeneMorph II backbone is expressed by roughly one-third of avian NS1 Random Mutagenesis Kit. Briefly, PCR reaction conditions and target DNA tem- proteins, while most avian and almost all human NS1 proteins plate amounts were optimized to generate 1–4 amino acids substitutions per possess arginine at this position (Supplementary Table 13). protein. The randomly mutated cDNAs were then inserted into RNA polymerase 12 NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9148 ARTICLE I vector to generate randomly mutated plasmid libraries. The diversity of the SDS–PAGE electrophoresis. One microlitre of virus concentrate was mixed with plasmid libraries was confirmed by sequence analysis: we found that 75–92% of the water to a total volume of 10ml. Loading dye (2.5ml) with 2% (v/v) b-mercap- sequenced clones retained the start codon, lacked premature stop codons, and toethanol as the reducing agent was added to each sample. Samples were heated to possessed, on average, 1.95–3.9 amino acid changes per PCR product. 95 C for 5 min before loading onto the NuPage 4–12% Bris-Tris precast gel (Life technology). Gels were run at 150 V for 120 min using 1  MES buffer (Bio-Rad) and then stained with SYPRO-Ruby (Sigma). Quantification of protein amounts was carried out by using ImageJ software (National Institutes of Health). To cal- Virus rescue and virus library generation. All viruses and virus libraries used in this study were generated by means of reverse genetics, using eight pHH21-based culate the HA content, we divided the HA amount (calculated by summing the amounts of HA1 and HA2) by the sum of the amounts of HA1, HA2, NP and M1, RNA polymerase I plasmids for viral RNA synthesis and four protein-expressing plasmids to synthesize the viral replication complex as described by Neumann and multiplied this value by the amount of total viral protein in the samples analysed by use of gel electrophoresis. et al. Virus stocks were generated by infecting MDCK cells, Vero cells or embryonated chicken eggs with 100ml of virus-containing supernatant derived from plasmid-transfected 293T cells. The titres of all virus stocks were determined Virulence studies in mice. To determine the MLD , three mice/group (a sample by means of plaque assays in MDCK cells. To generate virus libraries, cells were size adequate to detect large effects between groups) of 6-week-old female BALB/c transfected with a mutant plasmid library instead of the wild-type construct. At mice (Jackson Laboratory, Bar Harbor, ME, USA) were anaesthetized with iso- 48 h post transfection, supernatants derived from plasmid-transfected 293T cells 1 6 flurane and inoculated intranasally with 10-fold dilutions of virus (from 10 to 10 were passaged in MDCK and Vero cells by infecting them at a MOI of 0.01. All PFU) in a volume of 50ml. For these experiments, mice were randomized and experiments involving wild type or reassortant A/Anhui/1/2013 (H7N9) virus were investigators were not blinded. Body weight changes and survival were recorded carried out in biosafety level 3 containment. Experiments with H5N1 viruses that daily until day 14, and the MLD was calculated by the method of Reed and were exempt from Select Agent status by APHIS and approved by the University of Muench. To assess virus replication in mice, 10 PFU of recombinant viruses were Wisconsin-Madison Institutional Biosafety Committee for work at BSL-2 were used to infect six additional mice. At days 3 and 5 post infection, three mice in each used at that containment level. group were euthanized and their lungs were collected and homogenized. Virus titres were determined by plaque assays in MDCK cells. Evaluation of viral growth kinetics. To analyse the growth characteristics of viruses, Vero or MDCK cells were infected in triplicate (a sample size adequate to Genetic stability testing. To evaluate the genetic stability of the high-yield detect large effects between groups) with recombinant viruses at a MOI of 0.001 backbone, the recombinant viruses were consecutively passaged 10 times in Vero (MDCK cell infection) or 0.005 (Vero cell infection). One hour after incubation at cells at an MOI of 0.1. In parallel, these viruses were also consecutively passaged 10 37 C, the cells were washed once with phosphate-buffered saline (PBS), and fresh times in embryonated chicken eggs at an inoculation dosage of 2  10 PFU per MEM/BSA medium with 0.5mgml tosyl phenylalanyl chloromethyl ketone egg. Viruses sampled after the 10th passage in the supernatants of Vero cells and (TPCK) trypsin was added. Supernatants were collected at the indicated time the allantoic fluid of embryonated chicken eggs were sequenced by means of Sanger points and the virus titres in the supernatants were determined by plaque assays in sequencing. MDCK cells. For Vero cell infections, fresh TPCK trypsin was added to the supernatants every day (0.5mgml ). To analyse viral growth kinetics in Virus genome sequencing. To determine the mutations present in the 36 high- embryonated chicken eggs, 2  10 PFU of virus was inoculated into 10-day-old yield candidate viruses isolated from the MDCK passages, we performed whole embryonated chicken eggs. The allantoic fluids of four eggs each were harvested genome sequencing on the Ion Torrent PGM System (Life Technologies). Briefly, at the indicated time points. Virus titres were determined by plaque assays in 5ml of total RNA extracted from the supernatant of virus-infected cells was used to MDCK cells. amplify all eight viral segments in a multi-segment RT–PCR reaction . Barcoded, The haemagglutination (HA) titres of supernatants derived from infected 200-base insert libraries were then produced using the Ion Xpress Plus Fragment MDCK and Vero cells or allantoic fluid derived from inoculated eggs were Library Kit (Life Technologies), with an eight-cycle limited PCR to increase the determined by using an HA assay. Briefly, 50ml of virus sample was serially diluted numbers of fragments with adaptors. Final library pools were constructed 2-fold in 96-well U-bottom microtitre plates (Thermo Scientific) that contained containing equimolar amounts of each barcoded component, and the pools were 50ml of PBS per well, and then 50ml of 0.5% turkey red blood cells was added to run on Ion Torrent 314 chips. each well. After a 45-min incubation at room temperature, the highest agglutinating well was read as the virus HA titre. Statistical analysis. The data were analysed by using the R software (www.r- project.org), version 3.1. For comparisons of multiple groups with measurements Virus concentration and purification. Two 4-Layers Easy-Fill Cell Factories collected independently at different time points (that is, viral growth curves in (Thermo Scientific) of Vero cells were infected with recombinant viruses, and fresh embryonated chicken eggs), we used two-way analysis of variance followed by –1 TPCK trypsin was added every day (0.5mgml ). Alternatively, viruses were grown Tukey’s post-hoc test. For comparisons of measurements from multiple groups in 10-day-old embryonated chicken eggs. Infected cell culture supernatants were collected at a single time point, we used one-way analysis of variance also followed collected at 72 h post infection, whereas allantoic fluids were collected 48 h after egg by Tukey’s post-hoc test. For comparisons of multiple groups with dependent inoculation. Cell culture supernatants or allantoic fluids were clarified by cen- measurements (that is, viral growth curves in cell culture for which aliquots were trifugation (3,500 r.p.m., 15 min, 4 C). Viruses were then pelleted by centrifugation collected from the same culture at different time points), we fitted a linear mixed- (18,500 r.p.m., 90 min at 4 C in a Beckman Type19 rotor), resuspended in 5 ml of effects model to the data using the R package NLME, and the time, the virus PBS and loaded onto 30 ml, 20–50% continuous sucrose gradients that were cen- strains, and the interaction between these two factors were considered. Next we trifuged at 25,000 r.p.m. for 90 min at 4 C in a Beckman SW32 rotor. The virus built a contrast matrix to compare the strains in a pairwise fashion at the same time band was collected, diluted in PBS, pelleted by centrifugation (25,000 r.p.m., points (for example, group_1 versus group_2 at 24 h post infection, group_1 versus 90 min, 4 C in a Beckman SW32 rotor), and the final virus pellet was resuspended group_3 at 24 h post infection, group_2 versus group_3 at 24 h post infection), in 400ml of PBS (including 0.1% b-propiolactone (BPL)) overnight at 4 Cto using the R package PHIA. Because the comparisons were performed individually, inactivate the virus particles. Then, samples were incubated at 37 C for 45 min to the final P-values were adjusted using Holm’s method to account for multiple inactivate the BPL, aliquoted, and stored at  80 C. comparisons. Finally, for comparisons involving only two groups with measure- A/Anhui/1/2013 (H7N9) recombinant viruses were amplified in BSL-3 ments at single time points, we used two-tailed, unpaired t-tests; if multiple containment and inactivated by treatment with 0.1% BPL overnight. Virus comparisons were performed, the P-values were adjusted using Holm’s method. inactivation was confirmed by negative HA assays after two consecutive passages In all cases, except for the data shown in Fig. 7, raw data were converted to the in embryonated chicken eggs (the method and validation of virus inactivation logarithmical scale before the analysis and the results were considered statistically were approved by the Select Agent Program of the University of Wisconsin- significant if we obtained P-values (or adjusted P-values)o0.05, and the variance Madison). between groups was assessed using Levene’s test (it was similar for the groups being compared, with P-value40.05). Total protein assay. Total protein content of virus concentrates was determined by using the Pierce BCA protein assay kit (Thermo Scientific) according to the Ethics and biosafety. Our experiments in mice followed the University of Wis- manufacturer’s instructions. consin-Madison’s Animal Care and Use Protocol. All experiments were approved by the Animal Care and Use Committee of the University of Wisconsin-Madison (protocol number V00806), which acknowledged and accepted both the legal and Deglycosylation of viral proteins using PNGase F. Virus proteins were degly- ethical responsibility for the animals, as specified in the Fundamental Guidelines cosylated by using PNGase F (New England Biolabs). 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J.P. and G.N. wrote the contribute to high virulence of influenza A virus in mice. J. Virol. 83, manuscript. 6673–6680 (2009). 57. Pflug, A., Guilligay, D., Reich, S. & Cusack, S. Structure of influenza A polymerase bound to the viral RNA promoter. Nature 516, 355–360 (2014). 58. Plant, E. P., Liu, T. M., Xie, H. & Ye, Z. Mutations to A/Puerto Rico/8/34 PB1 Additional information gene improves seasonal reassortant influenza A virus growth kinetics. Vaccine Accession codes. The sequences of the six internal viral RNA segments of the high-yield 31, 207–212 (2012). PR8 backbone have been deposited in the GenBank under accession codes KT314334 to 59. Imai, M. et al. Experimental adaptation of an influenza H5 HA confers KT314339. respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Supplementary information accompanies this paper at http://www.nature.com/ Nature 486, 420–428 (2012). naturecommunications 60. Neumann, G. et al. Generation of influenza A viruses entirely from cloned cDNAs. Proc. Natl Acad. Sci. USA 96, 9345–9350 (1999). Competing financial interests: J.P., C.A.N., E.G., C.A.M., A.F., M.I, E.A.M, and T.J.S.L. 61. Zhou, B. et al. Single-reaction genomic amplification accelerates sequencing have no competing interests. G.N. and Y.K. are Co-founders of FluGen. Y.K. is also a paid and vaccine production for classical and Swine origin human influenza a consultant of Crucell. viruses. J. Virol. 83, 10309–10313 (2009). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Acknowledgements We thank Susan Watson for scientific editing. We thank the National Institute for How to cite this article: Ping, J. et al. Development of high-yield influenza A virus Biological Standards and Control (NIBSC, Potters Bar, UK) for the vaccine viruses vaccine viruses. Nat. Commun. 6:8148 doi: 10.1038/ncomms9148 (2015). NYMC X-181 and NYMC X-223A. This work was supported by the NIAID-funded Center for Research on Influenza Pathogenesis (CRIP, HHSN266200700010C), by the This work is licensed under a Creative Commons Attribution 4.0 Wisconsin Alumni Research Foundation, by the Japan Initiative for Global Research International License. The images or other third party material in this Network on Infectious Diseases from the Ministry of Education, Culture, Sports, Science, article are included in the article’s Creative Commons license, unless indicated otherwise and Technology, Japan, by ERATO, Japan, Strategic Basic Research Programs of Japan in the credit line; if the material is not included under the Creative Commons license, Science and Technology Agency, by the Advanced Research & Development Programs for Medical Innovation from Japan Agency for Medical Research and development, users will need to obtain permission from the license holder to reproduce the material. AMED, and by the Bill & Melinda Gates Foundation (OPPGH5383). To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ NATURE COMMUNICATIONS | 6:8148 | DOI: 10.1038/ncomms9148 | www.nature.com/naturecommunications 15 & 2015 Macmillan Publishers Limited. All rights reserved.

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