JVI Accepts, published online ahead of print on 9 April 2014 J. Virol. doi:10.1128/JVI.00100-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Development of a high yield live attenuated H7N9 influenza vaccine that provides

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protection against homologous and heterologous H7 wild-type viruses in ferrets

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Running title: Live attenuated H7N9 influenza vaccines in ferrets

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Celia Santos2, Kanta Subbarao2, Hong Jin1, Yumiko Matsuoka2

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Zhongying Chen1#, Mariana Baz 2, Janine Lu1, Myeisha Paskel2,

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MedImmune LLC, 319 North Bernardo Ave., Mountain View, CA, USA Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, MD 20892, USA

Keywords: Influenza, H7N9, vaccines, ferrets.

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Corresponding author: Zhongying Chen, PhD MedImmune LLC Mountain View, CA Phone: 650-603-2481 Fax: 650-603-3481 Email: [email protected] Abstract word #: 196 Text word#: 4317

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ABSTRACT

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Live attenuated H7N9 influenza vaccine viruses that possess the hemagglutinin

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(HA) and neuraminidase (NA) gene segments from the newly emerged wild-type (wt)

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A/Anhui/1/2013 (H7N9) and six internal protein gene segments from the cold-adapted

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influenza virus A/Ann Arbor/6/60 (AA ca) were generated by reverse genetics. The

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reassortant virus containing the original wt A/Anhui/1/2013 HA and NA sequences

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replicated poorly in eggs. Multiple variants with amino acid substitutions in the HA head

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domain that improved viral growth were identified by viral passage in eggs and MDCK

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cells. The selected vaccine virus containing two amino acid changes (N133D/G198E) in

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the HA improved viral titer by more than 10-fold (reached a titer of 108.6 Fluorescent

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Focus Units/mL) without affecting viral antigenicity. Introduction of these amino acid

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changes into an H7N9 PR8 reassortant also significantly improved viral titers and HA

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protein yield in eggs. The H7N9 ca vaccine virus was immunogenic in ferrets. A single

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dose of vaccine conferred complete protection of ferrets from homologous wt

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A/Anhui/1/2013 (H7N9) and near complete protection from heterologous wt

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A/Netherlands/219/2013 (H7N7) challenge infection. Therefore, this H7N9 LAIV

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candidate has been selected for vaccine manufacture and clinical evaluation to protect

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humans from wt H7N9 virus infection.

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IMPORTANCE In response to the recent avian H7N9 influenza virus infection in humans, we developed a live attenuated H7N9 influenza vaccine (LAIV) with two amino acid

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substitutions in the viral HA protein that improved vaccine yield by 10-fold in chicken

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embryonated eggs, the substrate for vaccine manufacture. The two amino acids also

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improved the antigen yield for inactivated H7N9 vaccines, demonstrating that this finding

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could great facilitate the efficiency of H7N9 vaccine manufacture. The candidate H7N9

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LAIV was immunogenic and protected ferrets against homologous and heterologous

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wild-type H7 virus challenge, making it suitable for use in protecting humans from H7

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infection.

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INTRODUCTION

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Avian influenza A viruses pose a threat of influenza pandemics because most

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people are serologically naive toward most hemagglutinin (HA) and neuraminidase (NA)

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subtypes. Avian influenza H7 subtype viruses have caused occasional human infection

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since 1959 (1-4). In 2003, an outbreak of a highly pathogenic avian influenza (HPAI)

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H7N7 virus in poultry farms in the Netherlands resulted in 89 cases of human infection

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including one fatal case and 3 cases of possible human-to-human transmission (5). In

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2004, an outbreak of HPAI H7N3 virus infection in 57 poultry workers with

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conjunctivitis or influenza-like symptoms was reported in Canada (6, 7). In 2012, HPAI

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H7N3 infection in two poultry workers was reported during H7N3 outbreaks in Mexican

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poultry (8). From February 2013, a novel avian-origin H7N9 subtype influenza virus

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emerged in China causing severe lower respiratory tract disease in humans (9). A total of

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135 human cases including 45 deaths occurred in the first wave from February to May

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2013 (including 2 cases in July). From October 2013 a second wave of human infection

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has been occurring that caused 240 cases including 70 deaths as of February 28, 2014

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(http://www.who.int/influenza/human_animal_interface/influenza_h7n9/140225_H7N9R

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A_for_web_20140306FM.pdf ). Most cases occurred among middle-aged and older

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adults who had direct exposure to poultry (10, 11). Although cross-reactive antibodies

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against influenza viruses may exist, the pre-existing antibodies against the novel H7N9

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virus were not detectable in any age group (12). The H7N9 virus possesses several

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genetic features contributing to its ability to infect humans (9, 13, 14). Structural and

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receptor binding analyses have demonstrated that the H7N9 viruses bind to both avian-

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like α2,3-linked sialic acid (SA) receptors and mammalian-like α2,6-linked SA

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receptors. The Q226L change, which has been associated with reduced binding to α2,3-

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SA and increased binding to α2,6- SA (15, 16), and other residues in the H7N9 HA

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contribute to this receptor binding specificity (12, 17-19). Although sustained human-to-

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human transmission has not been reported, the H7N9 virus can be transmitted via aerosol

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in ferrets, raising concerns about its pandemic potential (20-22).

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Several strategies have been used to develop vaccines against avian influenza

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viruses (23). Live attenuated influenza vaccines (LAIVs) bearing the HA and NA of the

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viruses of interest and remaining genes from the cold-adapted A/Ann Arbor/6/60 ca virus

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(AA ca) have several potential advantages as pandemic vaccines. LAIVs are based on

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licensed technology, can be produced at high yield, and elicit antibodies (systemic and

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mucosal) and cell-mediated immune responses (24, 25). We have generated an H7N7

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(A/Netherlands/219/2003, NL03) and H7N3 (A/chicken/British Columbia/CN-6/2004,

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BC04) LAIV viruses that induced cross-reactive antibody responses in animals (mice,

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ferrets and monkeys) that conferred protection against challenge with either homologous

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or heterologous H7 viruses (26, 27). In addition, the H7N3 LAIV evaluated in a Phase I

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clinical trial was shown to be immunogenic in humans (28). We recently showed that

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ferret antisera against these H7 ca viruses had cross-reactivity to the H7N9 virus (29).

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The cross-reactivity between divergent H7 viruses was also reported for the inactivated

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virus, recombinant protein or virus-like particle (VLP) vaccines studied in mice (30-32)

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or humans (33). Another Eurasian lineage H7N3 LAIV reassortant with an alternative

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internal gene backbone was reported to induce cross-reactive antibodies to H7N9 (34),

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indicating an H7 LAIV might be protective against a divergent H7 strain.

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In this study, we describe the generation of a live attenuated H7N9 vaccine

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candidate (H7N9 ca) containing the HA and NA gene segments of the recently emerged

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H7N9 A/Anhui/1/2013 wt virus and six internal protein gene segments of the AA ca virus

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by reverse genetics and the identification of critical residues in the HA that improved

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vaccine virus yield in eggs. The final selected LAIV candidate demonstrated high yield in

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eggs, good immunogenicity and protection against challenge infection with wt

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homologous and heterologous H7 viruses in ferrets.

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MATERIALS AND METHODS

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Viruses. The HPAI A/Netherlands/219/2003 (NL03, H7N7) and the A/Anhui/1/2013

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(AH13, H7N9) wt influenza viruses used for the evaluation of the efficacy of the vaccine

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candidate were kindly provided by Dr. Nancy Cox, Influenza Division, Centers for

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Disease Control and Prevention (CDC), Atlanta, GA and David Swayne at South East

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Poultry Research Laboratories (USDA). Virus stocks were propagated in the allantoic

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cavity of 9- to 11- day-old specific-pathogen-free embryonated hen’s eggs (Charles River

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Laboratories, North Franklin, CT) at 35°C. The allantoic fluid from eggs was harvested

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24 h post-inoculation and tested for hemagglutinating activity, and stored at -80°C. The

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50% tissue culture infectious dose (TCID50) for each virus was determined by titration of

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serially diluted virus in Madin-Darby canine kidney (MDCK) cells and calculated by the

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Reed and Muench method (35).

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Generation of H7N9 reassortant viruses by reverse genetics. Viral RNA isolated from

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egg amplified A/Anhui/1/2013 wt was received from the CDC. The HA and NA gene

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segments of A/Anhui/1/2013 were amplified from viral RNA by reverse transcription-

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polymerase chain reaction (RT-PCR) using the primers that are universal to the HA and

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NA gene end sequences and cloned into the plasmid vector pAD3000 (36). Site-directed

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mutagenesis was performed to introduce specific changes into the HA genes using the

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QuikChange® Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA)

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and the HA sequence was confirmed. The 6:2 reassortant vaccine viruses were generated

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by co-transfecting eight cDNA plasmids encoding the HA and NA of the H7N9 virus and

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the 6 internal protein gene segments of the AA ca master donor strain into co-cultured

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293T and MDCK cells. At 3 to 5 days post-transfection, the transfected cell supernatants

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were inoculated into 10- to 11-day-old embryonated hen’s eggs and incubated at 33°C for

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64±4 hours. Virus titers were determined by the fluorescence focus assay using an anti-

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NP monoclonal antibody and expressed as log10FFU (fluorescent focus units)/mL or by

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plaque assay in MDCK cells as previously described (37). The HA and NA sequences of

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the rescued viruses were verified by sequencing cDNAs amplified from viral RNA by

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RT-PCR.

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The 6:2 PR8 reassortant viruses, containing the HA and NA protein gene

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segments from the H7N9 virus and the 6 internal protein gene segments from the PR8

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strain, were generated by plasmid rescue as described above.

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Ferret studies. The ferret studies were conducted in AAALAC certified facilities under

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protocols approved by Institutional Animal Care and Use Committee (IACUC) at

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MedImmune for the vaccine virus replication and immunogenicity studies and Southern

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Research Institute for the H7 wt challenge studies.

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To evaluate the immunogenicity of the H7N9 ca variants, groups of 3 individually

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housed 8-12 week old male or female ferrets from Simonson (Gilroy, CA) were

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inoculated intranasally (i.n.) with 107 FFU of virus in 0.2 mL. Ferrets were bled 14 days

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post-immunization (p.i.) and sera were assessed for antibody titers by a hemagglutination

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inhibition (HAI) assay (37).

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To assess the replication of the H7N9 ca vaccine candidate, ferrets were

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inoculated with the vaccine virus as described above. Three days after inoculation, virus

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titers in the nasal turbinates (NT) and lungs were determined by egg infectivity and

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expressed as 50% egg infectious dose (EID50) per gram of tissue.

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To evaluate the protective efficacy of the H7N9 vaccine candidate, groups of 15-

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to 16-week-old female ferrets (N=4) that were seronegative for antibodies to circulating

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H3N2 and H1N1 human influenza viruses were immunized i.n. with 1 (Day 28) or 2

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doses (Day 0 and day 28) of 107 FFU of H7N9 ca or PBS (mock immunized) in 0.2 mL

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and serum samples were collected on days 0, 14, 28, 42 p.i.. The animals were transferred

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to an animal biological safety level 3 (ABSL3) facility (Southern Research Institute,

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Frederick, MD) for challenge infection with wt H7 viruses. On day 55 or day 56 p.i, sera

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were collected and the ferrets were challenged i.n. with 107 TCID50 of the homologous

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A/Anhui/1/2013 (H7N9) or heterologous A/Netherlands/219/2003 (H7N7) wt viruses,

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respectively. The serum antibody response against homologous and heterologous wt H7

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viruses was determined in a microneutralization assay (26). The animals were euthanized

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5 days post-challenge and NTs and lungs (right middle lobe and the caudal portion of the

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left cranial lobe) were harvested. Tissue homogenates of NTs and lungs were titrated in

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MDCK cells and the virus titers were expressed as TCID50 per gram of tissue.

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Assessment of viral protein yield. The H7N9 PR8 reassortant viruses were propagated

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in 25 embryonated hen’s eggs as described above. Virus in allantoic fluid was purified by

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sucrose gradient centrifugation. The virus band was collected, pelleted by

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ultracentrifugation and resuspended in 1 mL of the NTE buffer (10mM TrisCl, 100mM

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NaCl, 10mM EDTA, pH7.5). Total protein was quantitated with a BCA assay kit from

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Pierce (Rockford, IL). An equal volume (25 μl) of each purified virus was

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electrophoresed on a 4-20% SDS-PAGE followed by Coomassie Blue staining.

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RESULTS

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Generation of A/Anhui/1/2013 reassortant vaccine variants The HA and NA of vRNA isolated from egg grown wt A/Anhui/1/2013 virus was

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sequenced. The HA gene contained egg adaptation sequence changes at positions 133,

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135 and 158 (H3 numbering throughout the paper) compared to the HA sequence of wt

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A/Anhui/1/2013 from a human specimen (GISAID accession # EPI439507) (Table 1).

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The sequence of the NA gene was identical to that from the same specimen (GISAID

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accession # EPI439509). The RT-PCR amplified HA and NA gene segments were cloned

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and each clone was sequenced. From 20 HA clones analyzed, 6 variants were isolated:

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V1 (5%) contained the same sequence as the original wt sequence, V2 (45%) had a

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N158D change, V3 (25%) had a N133D/N158D double mutation, V4 (5%) had a N133D

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change, V5 (15%) and V6 (5%) contained single mutations of A135T and N199D,

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respectively. Reassortant vaccine viruses with the HA plasmid of each HA variant

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together with the NA plasmid of A/Anhui/1/2013 and the 6 internal protein gene

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plasmids from A/Ann Arbor/6/60 (AA ca) were obtained. The variants had different

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levels of replication in eggs, with titers ranging from 107.2 to 108.3 FFU/mL (Table 1). V1

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with the wt HA sequence grew poorly in eggs with a titer of only 107.2 FFU/mL and

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formed tiny plaques in MDCK cells (Figure 1). V2 (N158D) and V3 (N133D/N158D)

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with the indicated egg adaptation changes in the HA had the highest titers in eggs (108.2

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and 108.3 FFU/mL respectively), indicating that the N158D change greatly improved the

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vaccine virus growth in eggs. V4 and V6 with a single mutation of N133D or N199D in

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the HA had titers higher than V1 but lower than 108 FFU/mL. The V5 variant (A135T),

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which acquired a potential glycosylation site at N133, reached a titer of 108.1 FFU/mL.

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V1, V4 and V6 had lower titers than V2, V3 and V5, and formed small or mixed

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plaques in MDCK cells (Figure 1). After another round of egg passage, the titer of V4

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improved by acquiring an additional G198E mutation in the HA. Further egg passages of

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V1 and V6 produced mixed plaque sizes. Viruses with larger plaque size were isolated,

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amplified in eggs and the HA sequences were determined. The following amino acid

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changes in the HA were identified: N224D in V6, A160T, R220S or K193N in V1. Each

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of these identified mutations was introduced into the HA of V4, V6 or V1 and additional

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vaccine variants (V7-V11) were rescued. All the variants exhibited higher titers (>108

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FFU/mL) than the parental viruses, and V7 (N133D/G198E) had the highest titer of 108.6

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FFU/mL (Table 1).

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HA N133D/G198E changes significantly improved the yield of H7N9 PR8

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reassortant in eggs

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A PR8 reassortant (A/Shanghai/2/2013, RG32A) was generated by CDC for

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manufacture of inactivated H7N9 vaccines. This reassortant contained the 6 internal

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protein gene segments from A/Puerto Rico/8/34 (PR8) and the HA and NA gene

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segments from A/Shanghai/2/2013 (H7N9). The HA amino acid sequence of

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A/Shanghai/2/2013 is identical to A/Anhui/1/2013. To evaluate whether the HA protein

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yield of the PR8-H7N9 reassortant could be improved by introduction of the

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N133D/G198E (V7) amino acid substitutions in the HA, 6:2 reassortant influenza viruses

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comprising the 6 internal protein gene segments from PR8, the NA gene segment from 12

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A/Anhui/1/2013, and the HA gene segment from A/Anhui/1/2013-V1 or V7 were

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generated by plasmid rescue and resulting viruses were amplified in eggs. The PR8-V7

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virus had a titer of 108.9, which was significantly higher than PR8-V1 (108.1) and the CDC

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reassortant RG32A (107.9). Corresponding to the virus titers in eggs, the PR8-V7 virus

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had higher viral protein yield than the PR8-V1 and RG32A reassortants (Figure 2). These

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results demonstrate that the two amino acid substitutions in V7 (N133D/G198E) greatly

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improved the viral growth and HA protein yield of a PR8 reassortant and the PR8-V7

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variant could be a candidate for manufacture of inactivated H7N9 vaccines.

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Immunogenicity and antigenicity of the vaccine variants

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The sequence changes identified in the HA variants are located in the head region

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of the HA trimer structure which may impact the immunogenicity and antigenicity of the

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vaccine viruses (Figure 3). To examine the immunogenicity and antigenicity of the

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variants, ferrets were inoculated intranasally with 107 FFU of each variant in 0.2 mL.

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Post-immunization serum samples were collected on day 14 and antibody titers were

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evaluated by HAI assay against the homologous virus and the reference virus V1 using

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chicken erythrocytes (Table 2). V1 was selected as a reference virus for antigenicity

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assessment of each virus because V1 contained the wt HA sequence and was confirmed

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to be antigenically identical to BPL-inactivated wt A/Anhui/1/2013 in HAI (data not

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shown). The V1, V2, V6, V7 and V11 variants all elicited good antibody titers (GMT

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HAI titers ≥ 64) against the homologous virus. The V3, V5, V8, V9 and V10 variants

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induced lower HAI antibody titers (GMT HAI titers 19-40). V2, V3, V10 and V11

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variants cross-reacted with V1 with titers that were ≥ 4-fold than the titers to the

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homologous viruses, indicating that the N158D, R220S or K193N changes affected viral

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antigenicity. V5, V6, V7, V8 and V9-post-infection sera cross-reacted well with V1,

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indicating that the changes of A135T, N199D, N133D, G198E, N224D or A160T in HA

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did not significantly change viral antigenicity. Based on its high growth in eggs, authentic

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antigenicity and good immunogenicity, the A/Anhui/1/2013 ca-V7 containing the

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N133D/G198E substitutions in HA was selected as the final H7N9 ca vaccine seed for

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manufacture.

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The A/Anhui/1/2013 ca vaccine is attenuated and offers protection against

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homologous and heterologous wt virus challenge infection in ferrets

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The A/Anhui/1/2013 ca-V7 (AH13 ca) vaccine virus replicated in the nasal

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turbinates (NT) of ferrets on day 3 post-vaccination with an average titer of 104.9

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EID50/mL. A low level of viral titers (less than 2.0 TCID50/mL) was detected in nasal

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washes within 3 days of vaccination; no titer was detected after 3 days (data not shown).

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The vaccine virus was not detected in ferret lungs (Fig. 4A). Lung tissues from

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vaccinated ferrets showed no abnormal histopathology findings (data not shown). These

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data demonstrated that the AH13 ca vaccine virus had the desired attenuation phenotype.

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One dose of the AH13 ca vaccine elicited a robust neutralizing antibody response

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with a GMT of 220 (ranging from 63 to 640) and 63 (ranging from 10 to 202) against the

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homologous A/Anhui/1/2013 wt and heterologous A/Netherlands/219/2003 (NL03) wt viruses,

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respectively, on day 28 p.i. (Table 3). In the group of ferrets that received 2 doses of the

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vaccine virus, the first dose elicited homologous neutralizing antibody with a GMT of 63

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(ranging from 10 to 113) on day 28 p.i.. The second dose of vaccine further boosted the

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neutralizing antibody response to a GMT of 294 (ranging from 202 to 453). The

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difference in the neutralizing antibody titers following one dose of vaccine in the two

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groups (GMT 220 versus 63), may be due to the fact that ferrets are outbred or that at the

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time of immunization the ferrets that received only one dose of vaccine were a month

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older than the ferrets that received 2 doses of vaccine. The GMT of cross-neutralizing

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antibodies against the heterologous NL03 wt virus following one and two doses of the

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AH13 ca virus were 16 and 87, respectively (Table 3). Overall the anti-AH13 ca ferret

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antisera cross-reacted to NL03 with titers that were 2-4 fold lower than the homologous

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neutralizing antibody titers. An HAI assay showed that the anti-AH13 ca antisera

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similarly cross-reacted to NL03, an NL03 mutant (T135A) without the glycosylation site

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at HA position 133, as well as a North American H7N3 strain A/British Columbia/CN-

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6/2004 (BC04), with 2-4 fold titer reduction compared to homologous HAI titers (data

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not shown), indicating that the unique glycosylation site in NL03 did not affect viral

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antigenicity and the AH13 ca virus induced broadly cross-reactive antibodies to H7

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viruses. Consistently, anti-NL03 and anti-BC04 ferret antisera cross-reacted with

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A/Anhui/1/2013 with approximately 3-fold reduction in neutralization titers (29),

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demonstrating the cross-reactivity of divergent H7 viruses.

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The protective efficacy of 1 or 2 doses of the AH13 ca vaccine in preventing the

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replication of the homologous and heterologous wt challenge viruses in the respiratory

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tract of ferrets was evaluated on day 5 following challenge, when the challenge viruses

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were predicted to be present at a high titer in the lungs of mock-immunized ferrets (21).

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The H7 wt challenge viruses A/Anhui/1/2013 (H7N9) and A/Netherlands/219/2003

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(H7N7) replicated well in the respiratory tract of mock-vaccinated ferrets with mean

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titers of 106.4 and 107.1 TCID50/g, respectively in the NT, and mean titers of 105.8 and 104.8

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TCID50/g, respectively in the lungs. Immunization with one or two doses of the AH13 ca

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vaccine fully or nearly fully protected ferrets from replication of the homologous and

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heterologous wt, respectively, in both NTs and lungs. In the homologous H7N9 wt

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challenge group, none of the immunized ferrets had detectable titer in both NTs and

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lungs. In the heterologous H7N7 wt challenge group, only three ferrets had a titer of 102.0

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TCID50/g in the NT and one ferret had a titer of 102.0 TCID50/g in the lung (Figure 4B).

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DISCUSSION

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The novel avian-origin H7N9 virus has been associated with significant morbidity

316

and mortality in humans. This virus possesses several genetic features, including binding

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to human-like 2,6-SA receptors, a deletion in the NA protein and the E627K mutation in

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the PB2 protein, that raise concerns about its pandemic potential (9, 12, 14). In contrast to

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the H7N7 and H7N3 ca vaccine viruses that grew to high titer in eggs (26, 27), the novel

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H7N9 ca virus did not grow well in eggs. In order to respond to the potential need to

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immunize people against the H7N9 virus, we identified the HA variants that can be used

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to produce a high yield vaccine for manufacture. The candidate H7N9 ca vaccine is

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highly immunogenic and cross-reacts well to divergent H7 viruses. A single dose

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provides complete protection against wt H7N9 and H7N7 challenge infection in ferrets.

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The HA residues identified in H1N1pdm and seasonal influenza viruses that

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improve vaccine virus growth in eggs are generally at or near the receptor binding site

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(RBS) (38-40). The changes identified in the H7N9 HA at residues 133, 135, 158, 160,

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193, 198, 199, 220, 224 following egg and MDCK cell passage are also located in

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proximity of the RBS (Figure 2). Residues 133 and 135 are located in the 130 loop on the

330

side of the RBS, residues 193 and 198 are located in the 190 helix, and the 211 and 215

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residues are located in the 220 loop (18, 41). We speculate that these changes create an

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optimized receptor binding structure for host specific viral replication. It was noticed that

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the N133D, N158D, G198E, N199D and N224D changes resulted in the presence of

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negatively charged acidic residues aspartate (D) or glutamate (E) on the surface of the

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HA. The K193N and R220S changes reduce positive charge on the HA trimer surface.

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Similar changes were also identified in the HA of the H1N1pdm viruses, K122E, A189D,

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N128D, D130E and K212E (H3#), that improved vaccine virus growth in eggs and

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MDCK cells. Further studies indicated that the acidic residue substitutions in H1N1pdm

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did not affect viral entry and replication but greatly improved viral spread in the host

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cells (40). These negatively charged residues possibly decrease HA and sialic acid

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interaction and thus facilitate the release of progeny viruses from infected cells for

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efficient multi-cycle replication.

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Glycosylation of the HA protein affects receptor binding, fusion and antigenicity

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and has been shown to play important roles in virus replication, host restriction, virulence

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and transmission (42, 43). The A135T and A160T changes create potential N-linked

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glycosylation sites at the N133 and N158 residues that improve virus growth in eggs. We

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speculate the glycosylation may improve virus receptor binding and infectivity in eggs

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based on similar findings reported for the HPAI A/Netherlands/219/2003 (H7N7) in

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which glycosylation at N133 increased its binding affinity to avian-type α2-3-linked

350

sialosides (44). It was also reported that glycosylation at HA position 188 (H3# 197) near

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the receptor binding site increased the virulence of an avian H7N7 strain in chicken (45).

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On the contrary, the loss of a glycosylation site at HA position 133 of human H3N2

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viruses is associated with better viral growth in MDCK cells (46). For the H5N1 viruses,

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the loss of the glycosylation site at HA residue 158 improved viral transmission and

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vaccine immunogenicity in ferrets (47-49). Thus, the effect of HA glycosylation is strain

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and host-specific.

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Amino acid changes at antigenic sites on the surface of the HA molecule could

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alter viral antigenicity. Thus, each vaccine variant was evaluated for antigenicity in the

18

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HAI assay. The N158D (V2, V3) and the K193N (V11) changes at antigenic site B, and

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the R220S (V10) change at antigenic site D affected viral antigenicity of the H7N9 virus.

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The 158 residue has previously been shown to alter the viral antigenicity in seasonal

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H3N2 and H1N1pdm viruses (37, 50).

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One dose of the H7N9 ca vaccine virus induced neutralizing antibody in ferrets

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that cross-reacted with the H7N7 virus in ferrets. A second dose of the H7N9 ca vaccine

365

greatly boosted serum antibody titers. The immunogenicity of the H7N9 ca vaccine virus

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was as great or greater than the immunogenicity of the H7N3 or H7N7 ca vaccines that

367

were previously generated and evaluated in our laboratory (26, 27). The poorer

368

immunogenicity of the A/Netherland/219/2003 (H7N7) vaccine virus could be attributed

369

to its 2,3-SA receptor binding specificity. The introduction of Q226L/G228S that

370

increased the 2,6-SA receptor binding improved immunogenicity of the H7N7 ca vaccine

371

virus in ferrets (29). The novel H7N9 HA contains L226, which may contribute to its

372

higher replication in the upper respiratory tract and greater immunogenicity in ferrets.

373

Pandemic vaccines are evaluated for safety and immunogenicity in clinical trials

374

but efficacy data can only come from studies in experimentally infected animals. In this

375

study, we demonstrated that a single dose of the H7N9 ca vaccine conferred complete

376

protection, in both NT and lungs, against homologous wt virus challenge infection and

377

near complete protection against the heterologous wt virus. This finding correlated with

378

the robust neutralizing antibody response induced after one dose of the vaccine. In our

379

previous studies, one dose of the H7N3 or H7N7 ca vaccines conferred protection from

380

pulmonary replication of the homologous and heterologous wt virus challenge but not

381

replication in the upper respiratory tract (26, 27).

19

382

In summary, we generated a high-yield H7N9 vaccine candidate that is highly

383

immunogenic and efficacious in a ferret challenge study. The candidate vaccine with

384

amino acid changes at HA residues 133 and 198 maintained the attenuation phenotype

385

conferred by the six internal protein gene segments of AA ca. Based on this promising

386

preclinical data, this vaccine is currently being evaluated in phase I clinical studies.

387

20

ACKNOWLEDGEMENTS

388 389 390

This study was funded in part by federal funds from Biomedical Advanced

391

Research and Development Authority (BARDA) of the U.S. Department of Health and

392

Human Services (HHS) under the contract No HHSO100201200012I and by the

393

Intramural Research Program of NIAID, NIH. The work is conducted under a

394

Cooperative Research and Development Agreement (CRADA) between MedImmune and

395

NIAID/NIH. We thank Drs. Michael Shaw and Nancy Cox at CDC for providing the

396

H7N9 virus and viral RNA, the staff of the animal care facilities at MedImmune and SRI

397

for their assistance with ferret studies, MedImmune’s strain variant team for their

398

support, Dr. Christopher Cotter for technical assistance, Dr. JoAnn Suzich for critical

399

review of the manuscript.

400

21

401

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26

597 598

Table 1. HA sequences and virus titers of A/Anhui/1/2013 (H7N9) ca variants Amino acid at the HA position H3# (H7#) 6:2 variant

133 (123)

135 (125)

wt

N

A

N

vRNA

N/D

A/T

D/N

158 160 193 198 199 220 (149) (151) (184) (189) (190) (211)

A

K

G

N

R

224 (215)

Titer in eggs (log10FFU/mL)

N

n/a n/a

V1

7.2

V2 V3

D

V4

D

V5

D

8.2

D

8.3 7.7

T*

8.1

V6 V7

D D

E

V8 V9

599 600 601 602 603 604

8.6 D

D

T*

8.2 8.4

V10 V11

7.8

S N

8.3 8.4

The wt virus sequence was downloaded from GISAID database. The vRNA was isolated from egg grown A/Anhui/1/2013. The titers represent the average of at least two independent experiments. a. The amino acid changes from the wt virus are shown. *A135T and A160T changes introduce potential N-glycosylation sites.

27

605 606

Table 2. Immunogenicity and antigenicity of A/Anhui/1/2013 ca HA variants Serum HAI titers (GMT) of ferrets immunized with A/Anhui/1/2013 ca

HA variants: Virus

V1

V2

V3

V5

V6

V7

V8

V9

V10

V11

V1 (wt)

81

16

10

16

32

32

10

32

5

32

Homologous Virus

n/a

128

40

19

64

64

20

40

20

256

607 608

Ferrets (n=3) were inoculated i.n. with 107FFU of the indicated A/Anhui/1/2013 ca HA

609

variants (V1-V11). Serum was collected 14 days p.i. and the serum antibody titers were

610

determined by HAI assay against the homologous virus and the V1 reference virus. The

611

values represent the geometric mean titers (GMT) from three ferrets. n/a: not applicable.

28

612 613 614 615

Table 3. Serum neutralizing antibody responses in ferrets following one or two doses of AH13 ca vaccine

Test antigen

616 617 618 619 620 621 622

Serum neutralizing antibody titers (GMT) in ferrets immunized with the AH13 ca vaccine 2 dosesa

1 dose D14

D28

D14

D28

D42

D56

AH13 (H7N9) wt

30

220

22

63

281

294

NL03 (H7N7) wt

11

63

11

16

99

87

Ferrets (n=8) were inoculated i.n. with 1 or 2 doses of 107 FFU of the AH13 ca vaccine. Serum was collected at the indicated days after the first immunization and assessed by the microneutralization assay. Antibodies were not detected in preimmunization sera and in sera from mock-immunized ferrets. a Ferrets received 2 doses of vaccine administered 28 days apart.

29

623 624

FIGURE LEGEND

625

Figure 1. Plaque morphology of A/Anhui/1/2013 ca variants

626

Viruses were rescued by reverse genetics and propagated once in embryonated hen’s

627

eggs. Plaque assay was performed in MDCK cells and incubated at 33°C for 4 days and

628

stained with crystal violet.

629

(A) A/Anhui/1/2013 ca variants (V1-V6) isolated from vRNA

630

(B) A/Anhui/1/2013 ca variants (V7-V11) with introduced HA sequence changes

631 632

Figure 2. HA protein yield of PR8 reassortants. PR8-A/Anhui/1/2013-V1, PR8-

633

A/Anhui/1/2013-V7 and PR8-A/shanghai/2/2013 (RG32A) were propagated in eggs and

634

the virus titers were indicated on the bottom of each lane. Viral harvest from 25 eggs was

635

purified by sucrose gradient. Purified virus was resuspended in 1mL of NTE and total

636

viral protein was measured and expressed as mg/100eggs. An equal volume of each

637

purified virus was loaded onto a SDS-PAGE for electrophoresis and stained with

638

Coomassie Blue.

639 640

Figure 3. The location of the identified HA residues that improve the growth of H7N9

641

viruses on the HA 3D structure (PDB# 4KOL, only one monomer shown). RBS: receptor

642

binding site.

643 644

Figure 4. A. Attenuation study. Ferrets were inoculated with AH13 ca intranasally with a

645

dose of 107 FFU. After 3 days, the nasal turbinate (NT) and lung tissues were collected

646

and viral titers were expressed as EID50 per gram of tissue. The dashed lines indicate the

647

limit of detection. B and C. Wild-type virus challenge study. Ferrets were inoculated

648

with AH13 ca (H7N9 ca) intranasally with one or two doses of 107 FFU. One month after

649

the final dose of vaccine was administered, ferrets were challenged with wt

650

A/Anhui/1/2013 (H7N9) (B) or wt A/Netherlands/219/2003 (H7N7) (C) viruses. After 5

651

days, the NT and lung tissues were collected and viral titers were expressed as TCID50

652

per gram of tissue. Vaccinated groups had a statistically significant reduction in virus

30

653

titers of the homologous H7N9 (P ≤0.0003) and heterologous H7N7 (P ≤0.05) compared

654

to the titers in the mock-immunized group.

31