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Journal of Virology logoLink to Journal of Virology
. 2014 Jun;88(12):7016–7023. doi: 10.1128/JVI.00100-14

Development of a High-Yield Live Attenuated H7N9 Influenza Virus Vaccine That Provides Protection against Homologous and Heterologous H7 Wild-Type Viruses in Ferrets

Zhongying Chen a,✉, Mariana Baz b, Janine Lu a, Myeisha Paskel b, Celia Santos b, Kanta Subbarao b, Hong Jin a, Yumiko Matsuoka b
Editor: A García-Sastre
PMCID: PMC4054345  PMID: 24719414

ABSTRACT

Live attenuated H7N9 influenza vaccine viruses that possess the hemagglutinin (HA) and neuraminidase (NA) gene segments from the newly emerged wild-type (wt) A/Anhui/1/2013 (H7N9) and six internal protein gene segments from the cold-adapted influenza virus A/Ann Arbor/6/60 (AA ca) were generated by reverse genetics. The reassortant virus containing the original wt A/Anhui/1/2013 HA and NA sequences replicated poorly in eggs. Multiple variants with amino acid substitutions in the HA head domain that improved viral growth were identified by viral passage in eggs and MDCK cells. The selected vaccine virus containing two amino acid changes (N133D/G198E) in the HA improved viral titer by more than 10-fold (reached a titer of 108.6 fluorescent focus units/ml) without affecting viral antigenicity. Introduction of these amino acid changes into an H7N9 PR8 reassortant virus also significantly improved viral titers and HA protein yield in eggs. The H7N9 ca vaccine virus was immunogenic in ferrets. A single dose of vaccine conferred complete protection of ferrets from homologous wt A/Anhui/1/2013 (H7N9) and nearly complete protection from heterologous wt A/Netherlands/219/2003 (H7N7) challenge infection. Therefore, this H7N9 live attenuated influenza vaccine (LAIV) candidate has been selected for vaccine manufacture and clinical evaluation to protect humans from wt H7N9 virus infection.

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 substitutions in the viral HA protein that improved vaccine yield by 10-fold in chicken embryonated eggs, the substrate for vaccine manufacture. The two amino acids also improved the antigen yield for inactivated H7N9 vaccines, demonstrating that this finding could great facilitate the efficiency of H7N9 vaccine manufacture. The candidate H7N9 LAIV was immunogenic and protected ferrets against homologous and heterologous wild-type H7 virus challenge, making it suitable for use in protecting humans from H7 infection.

INTRODUCTION

Avian influenza A viruses pose a threat of influenza pandemics because most people are serologically naive toward most hemagglutinin (HA) and neuraminidase (NA) subtypes. Avian influenza H7 subtype viruses have caused occasional human infection since 1959 (1–4). In 2003, an outbreak of a highly pathogenic avian influenza (HPAI) H7N7 virus in poultry farms in the Netherlands resulted in 89 cases of human infection, including one fatal case and three cases of possible human-to-human transmission (5). In 2004, an outbreak of HPAI H7N3 virus infection in 57 poultry workers with conjunctivitis or influenza-like symptoms was reported in Canada (6, 7). In 2012, HPAI H7N3 infection in two poultry workers was reported during H7N3 outbreaks in Mexican poultry (8). From February 2013, a novel avian-origin H7N9 subtype influenza virus emerged in China causing severe lower respiratory tract disease in humans (9). A total of 135 human cases, including 45 deaths, occurred in the first wave from February to May 2013 (including 2 cases in July). Since October 2013 a second wave of human infection has been occurring that has caused 240 cases, including 70 deaths as of 28 February 28 2014 (10). Most cases occurred among middle-aged and older adults who had direct exposure to poultry (11, 12). Although cross-reactive antibodies against influenza viruses may exist, the preexisting antibodies against the novel H7N9 virus were not detectable in any age group (13). The H7N9 virus possesses several genetic features contributing to its ability to infect humans (9, 14, 15). Structural and receptor binding analyses have demonstrated that the H7N9 viruses bind to both avian-like α2,3-linked sialic acid (SA) receptors and mammalian-like α2,6-linked SA receptors. The Q226L change, which has been associated with reduced binding to α2,3-SA and increased binding to α2,6-SA (16, 17), and other residues in the H7N9 HA contribute to this receptor binding specificity (13, 18–20). Although sustained human-to-human transmission has not been reported, the H7N9 virus can be transmitted via aerosol in ferrets, raising concerns about its pandemic potential (21–23).

Several strategies have been used to develop vaccines against avian influenza viruses (24). Live attenuated influenza vaccines (LAIVs) bearing the HA and NA of the viruses of interest and remaining genes from the cold-adapted A/Ann Arbor/6/60 virus (AA ca) have several potential advantages as pandemic vaccines. LAIVs are based on licensed technology, can be produced at high yield, and elicit antibodies (systemic and mucosal) and cell-mediated immune responses (25, 26). We have generated H7N7 (A/Netherlands/219/2003 [NL03]) and H7N3 (A/chicken/British Columbia/CN-6/2004 [BC04]) LAIV viruses that induced cross-reactive antibody responses in animals (mice, ferrets, and monkeys) that conferred protection against challenge with either homologous or heterologous H7 viruses (27, 28). In addition, the H7N3 LAIV evaluated in a phase I clinical trial was shown to be immunogenic in humans (29). We recently showed that ferret antiserum against these H7 ca viruses had cross-reactivity to the H7N9 virus (30). The cross-reactivity between divergent H7 viruses was also reported for the inactivated virus, recombinant protein, or virus-like particle (VLP) vaccines studied in mice (31–33) or humans (34). Another Eurasian-lineage H7N3 LAIV reassortant with an alternative internal gene backbone was reported to induce cross-reactive antibodies to H7N9 (35), indicating that an H7 LAIV might be protective against a divergent H7 strain.

In this study, we describe the generation by reverse genetics of a live attenuated H7N9 vaccine candidate (H7N9 ca) containing the HA and NA gene segments of the recently emerged H7N9 A/Anhui/1/2013 wt virus and six internal protein gene segments of the AA ca virus and the identification of critical residues in the HA that improved vaccine virus yield in eggs. The final selected LAIV candidate demonstrated high yield in eggs, good immunogenicity, and protection against challenge infection with wt homologous and heterologous H7 viruses in ferrets.

MATERIALS AND METHODS

Viruses.

The HPAI A/Netherlands/219/2003 (NL03; H7N7) and the A/Anhui/1/2013 (AH13; H7N9) wt influenza viruses used for the evaluation of the efficacy of the vaccine candidate were kindly provided by Nancy Cox, Influenza Division, Centers for Disease Control and Prevention (CDC), Atlanta, GA, and David Swayne at Southeast Poultry Research Laboratories (U.S. Department of Agriculture). Virus stocks were propagated in the allantoic cavity of 9- to 11-day-old specific-pathogen-free embryonated hen eggs (Charles River Laboratories, North Franklin, CT) at 35°C. The allantoic fluid from eggs was harvested at 24 h postinoculation and tested for hemagglutinating activity and stored at −80°C. The 50% tissue culture infectious dose (TCID50) for each virus was determined by titration of serially diluted virus in Madin-Darby canine kidney (MDCK) cells and calculated by the Reed and Muench method (36).

Generation of H7N9 reassortant viruses by reverse genetics.

Viral RNA (vRNA) isolated from egg-amplified A/Anhui/1/2013 wt was received from the CDC. The HA and NA gene segments of A/Anhui/1/2013 were amplified from viral RNA by reverse transcription-PCR (RT-PCR) using the primers that are universal to the HA and NA gene end sequences and cloned into the plasmid vector pAD3000 (37). Site-directed mutagenesis was performed to introduce specific changes into the HA genes using a QuikChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA), and the HA sequence was confirmed. The 6:2 reassortant vaccine viruses were generated by cotransfecting eight cDNA plasmids carrying the HA and NA of the H7N9 virus and the six internal protein gene segments of the AA ca master donor strain into cocultured 293T and MDCK cells. At 3 to 5 days posttransfection, the transfected cell supernatants were inoculated into 10- to 11-day-old embryonated hen eggs and incubated at 33°C for 64 ± 4 h. Virus titers were determined either by a fluorescence focus assay using an anti-NP monoclonal antibody and expressed as log10 fluorescent focus units (FFU)/ml or by plaque assay in MDCK cells as previously described (38). The HA and NA sequences of the rescued viruses were verified by sequencing cDNAs amplified from viral RNA by RT-PCR.

The 6:2 PR8 reassortant viruses, containing the HA and NA protein gene segments from the H7N9 virus and the six internal protein gene segments from the PR8 strain, were generated by plasmid rescue as described above.

Ferret studies.

The ferret studies were conducted in AAALAC-certified facilities under protocols approved by Institutional Animal Care and Use Committee (IACUC) at MedImmune for the vaccine virus replication and immunogenicity studies and Southern Research Institute for the H7 wt challenge studies.

To evaluate the immunogenicity of the H7N9 ca variants, groups of three individually housed 8- to 12-week-old male or female ferrets from Simonson (Gilroy, CA) were inoculated intranasally (i.n.) with 107 FFU of virus in 0.2 ml. Ferrets were bled at 14 days postimmunization (p.i.), and sera were assessed for antibody titers by a hemagglutination inhibition (HAI) assay (38).

To assess the replication of the H7N9 ca vaccine candidate, ferrets were inoculated with the vaccine virus as described above. Three days after inoculation, virus titers in the nasal turbinates (NTs) and lungs were determined by egg infectivity and expressed as 50% egg infectious dose (EID50) per gram of tissue.

To evaluate the protective efficacy of the H7N9 vaccine candidate, groups of 15- to 16-week-old female ferrets (n = 4) that were seronegative for antibodies to circulating H3N2 and H1N1 human influenza viruses were immunized i.n. with 1 (day 28) or 2 doses (day 0 and day 28) of 107 FFU of H7N9 ca or phosphate-buffered saline ([PBS] mock immunized) in 0.2 ml, and serum samples were collected on days 0, 14, 28, and 42 p.i. The animals were transferred to an animal biological safety level 3 (ABSL3) facility (Southern Research Institute, Frederick, MD) for challenge infection with wt H7 viruses. On day 55 or day 56 p.i., serum samples were collected, and the ferrets were challenged i.n. with 107 TCID50 of the homologous A/Anhui/1/2013 (H7N9) or heterologous A/Netherlands/219/2003 (H7N7) wt virus, respectively. The serum antibody response against homologous and heterologous wt H7 viruses was determined in a microneutralization assay (27). The animals were euthanized 5 days postchallenge, and NTs and lungs (right middle lobe and the caudal portion of the left cranial lobe) were harvested. Tissue homogenates of NTs and lungs were titrated in MDCK cells, and the virus titers were expressed as TCID50 per gram of tissue.

Assessment of viral protein yield.

The H7N9 PR8 reassortant viruses were propagated in 25 embryonated hen eggs as described above. Virus in allantoic fluid was purified by sucrose gradient centrifugation. The virus band was collected, pelleted by ultracentrifugation, and resuspended in 1 ml of the NTE buffer (10 mM Tris-Cl, 100 mM NaCl, 10 mM EDTA, pH 7.5). Total protein was quantitated with a bicinchoninic acid (BCA) assay kit from Pierce (Rockford, IL). An equal volume (25 μl) of each purified virus was electrophoresed on a 4 to 20% SDS-PAGE gel, followed by Coomassie blue staining.

RESULTS

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 sequenced. The HA gene contained egg adaptation sequence changes at positions 133, 135, and 158 (H3 numbering throughout the paper) compared to the HA sequence of wt A/Anhui/1/2013 from a human specimen (Global Initiative on Sharing All Influenza Data [GISAID] DNA accession number 439507) (Table 1). The sequence of the NA gene was identical to that from the same specimen (GISAID DNA accession number 439509). The RT-PCR-amplified HA and NA gene segments were cloned, and each clone was sequenced. From 20 HA clones analyzed, six variants were isolated: V1 (5%) contained the same sequence as the original wt sequence, V2 (45%) had an N158D change, V3 (25%) had an N133D/N158D double mutation, V4 (5%) had an N133D change, and V5 (15%) and V6 (5%) contained single mutations of A135T and N199D, respectively. Reassortant vaccine viruses with the HA plasmid of each HA variant together with the NA plasmid of A/Anhui/1/2013 and the six internal protein gene plasmids from A/Ann Arbor/6/60 (AA ca) were obtained. The variants had different levels of replication in eggs, with titers ranging from 107.2 to 108.3 FFU/ml (Table 1). V1 with the wt HA sequence grew poorly in eggs, with a titer of only 107.2 FFU/ml, and formed tiny plaques in MDCK cells (Fig. 1). V2 (N158D) and V3 (N133D/N158D) with the indicated egg adaptation changes in the HA had the highest titers in eggs (108.2 and 108.3 FFU/ml, respectively), indicating that the N158D change greatly improved growth of the vaccine virus in eggs. V4 and V6 with a single mutation of N133D or N199D in the HA had titers higher than V1 but lower than 108 FFU/ml. The V5 variant (A135T), which acquired a potential glycosylation site at N133, reached a titer of 108.1 FFU/ml.

TABLE 1.

HA sequences and virus titers of A/Anhui/1/2013 (H7N9) ca variants

Virus Amino acid at the HA position in H3# (H7#) virusa
 Titer in eggs (log10 FFU/ml)b
133 (123) 135 (125) 158 (149) 160 (151) 193 (184) 198 (189) 199 (190) 220 (211) 224 (215)
wt N A N A K G N R N NA
vRNA N/D A/T D/N NA
V1 7.2
V2 D 8.2
V3 D D 8.3
V4 D 7.7
V5 T* 8.1
V6 D 7.8
V7 D E 8.6
V8 D D 8.2
V9 T* 8.4
V10 S 8.3
V11 N 8.4
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a

The wt virus sequence of A/Anhui/1/2013 was downloaded from the GISAID database. The vRNA was isolated from egg-grown A/Anhui/1/2013. V1 to V11 are the variants of 6:2 A/Anhui/1/2013 ca viruses. The amino acid changes from the wt virus are shown. A135T and A160T changes (marked with asterisks) introduce potential N-glycosylation sites.

b

The titers represent the average of at least two independent experiments. NA, not applicable.

FIG 1.

FIG 1

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Plaque morphology of A/Anhui/1/2013 ca variants. Viruses were rescued by reverse genetics and propagated once in embryonated hen eggs. A plaque assay was performed in MDCK cells incubated at 33°C for 4 days and stained with crystal violet. (A) A/Anhui/1/2013 ca variants (V1 to V6) isolated from vRNA. (B) A/Anhui/1/2013 ca variants (V7 to V11) with introduced HA sequence changes.

V1, V4, and V6 had lower titers than V2, V3, and V5 and formed small or mixed plaques in MDCK cells (Fig. 1). After another round of egg passage, the titer of V4 improved through the acquisition of an additional G198E mutation in the HA. Further egg passages of V1 and V6 produced mixed plaque sizes. Viruses with larger plaque sizes were isolated and amplified in eggs, and the HA sequences were determined. The following amino acid changes in the HA were identified: N224D in V6 and A160T, R220S, or K193N in V1. Each of these identified mutations was introduced into the HA of V4, V6, or V1 and additional vaccine variants (V7 to V11) were rescued. All the variants exhibited higher titers (>108 FFU/ml) than the parental viruses, and V7 (N133D/G198E) had the highest titer, 108.6 FFU/ml (Table 1).

HA N133D/G198E changes significantly improved the yield of H7N9 PR8 reassortant in eggs.

A PR8 reassortant (A/Shanghai/2/2013 with the RG32A mutation) was generated by the CDC for manufacture of inactivated H7N9 vaccines. This reassortant contained the six internal protein gene segments from A/Puerto Rico/8/34 (PR8) and the HA and NA gene segments from A/Shanghai/2/2013 (H7N9). The HA amino acid sequence of A/Shanghai/2/2013 is identical to that of A/Anhui/1/2013. To evaluate whether the HA protein yield of the PR8 H7N9 reassortant could be improved by introduction of the N133D/G198E (V7) amino acid substitutions in the HA, 6:2 reassortant influenza viruses comprising the six internal protein gene segments from PR8, the NA gene segment from A/Anhui/1/2013, and the HA gene segment from A/Anhui/1/2013 V1 or V7 were generated by plasmid rescue, and the resulting viruses were amplified in eggs. The PR8–V7 virus had a titer of 108.9, which was significantly higher than that of the PR8–V1 (108.1) and the CDC reassortant RG32A (107.9). Corresponding to the virus titers in eggs, the PR8–V7 virus had higher viral protein yield than the PR8–V1 and RG32A reassortants (Fig. 2). These results demonstrate that the two amino acid substitutions in V7 (N133D/G198E) greatly improved the viral growth and HA protein yield of a PR8 reassortant, and the PR8–V7 variant could be a candidate for manufacture of inactivated H7N9 vaccines.

FIG 2.

FIG 2

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HA protein yield of PR8 reassortants. PR8-A/Anhui/1/2013 V1, PR8-A/Anhui/1/2013 V7, and PR8-A/shanghai/2/2013 (RG32A) were propagated in eggs, and the virus titers were indicated on the bottom of each lane. Virus harvested from 25 eggs was purified by sucrose gradient. Purified virus was resuspended in 1 ml of NTE buffer, and total viral protein was measured and expressed as mg/100 eggs. An equal volume of each purified virus was loaded onto an SDS-PAGE gel for electrophoresis and stained with Coomassie blue. MW, molecular weight in thousands.

Immunogenicity and antigenicity of the vaccine variants.

The sequence changes identified in the HA variants are located in the head region of the HA trimer structure, which may impact the immunogenicity and antigenicity of the vaccine viruses (Fig. 3). To examine the immunogenicity and antigenicity of the variants, ferrets were inoculated intranasally with 107 FFU of each variant in 0.2 ml. Postimmunization serum samples were collected on day 14, and antibody titers were evaluated by HAI assay against the homologous virus and the reference virus V1 using chicken erythrocytes (Table 2). V1 was selected as a reference virus for antigenicity assessment of each virus because V1 contained the wt HA sequence and was confirmed to be antigenically identical to beta-propiolactone (BPL)-inactivated wt A/Anhui/1/2013 in an HAI assay (data not shown). The V1, V2, V6, V7, and V11 variants all elicited good antibody titers (HAI geometric mean titers [GMT] of ≥64) against the homologous virus. The V3, V5, V8, V9, and V10 variants induced lower HAI antibody titers (HAI GMT of 19 to 40). V2, V3, V10, and V11 variants cross-reacted with V1 with titers that were ≥4-fold lower than the titers to the homologous viruses, indicating that the N158D, R220S, or K193N change affected viral antigenicity. V5, V6, V7, V8, and V9 postinfection sera cross-reacted well with V1, indicating that the change of A135T, N199D, N133D, G198E, N224D, or A160T in HA did not significantly change viral antigenicity. Based on its high growth in eggs, authentic antigenicity, and good immunogenicity, the A/Anhui/1/2013 ca V7 containing the N133D/G198E substitutions in HA was selected as the final H7N9 ca vaccine seed for manufacture.

FIG 3.

FIG 3

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The location of the identified HA residues that improve the growth of H7N9 viruses on the HA three-dimensional structure (Protein Data Bank [PDB] 4KOL). Only one is monomer shown. RBS, receptor binding site.

TABLE 2.

Immunogenicity and antigenicity of A/Anhui/1/2013 ca HA variants

Virus Serum HAI GMT of ferrets immunized with A/Anhui/1/2013 ca HA variant:a
V1 V2 V3 V5 V6 V7 V8 V9 V10 V11
V1 (wt) 81 16 10 16 32 32 10 32 5 32
Homologous virus NA 128 40 19 64 64 20 40 20 256
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a

Ferrets (n = 3) were inoculated i.n. with 107 FFU of the indicated A/Anhui/1/2013 ca HA variants (V1 to V11). Serum was collected 14 days p.i., and the serum antibody titers were determined by HAI assay against the homologous virus and the V1 reference virus. The values represent the geometric mean titers (GMT) from three ferrets. NA, not applicable.

The A/Anhui/1/2013 ca vaccine is attenuated and offers protection against homologous and heterologous wt virus challenge infection in ferrets.

The A/Anhui/1/2013 ca V7 (AH13 ca) vaccine virus replicated in the nasal turbinates (NTs) of ferrets on day 3 postvaccination with an average titer of 104.9 EID50/ml. A low level of viral titers (less than 2.0 TCID50/ml) was detected in nasal washes within 3 days of vaccination; no titer was detected after 3 days (data not shown). The vaccine virus was not detected in ferret lungs (Fig. 4A). Lung tissues from vaccinated ferrets showed no abnormal histopathology findings (data not shown). These data demonstrated that the AH13 ca vaccine virus had the desired attenuation phenotype.

FIG 4.

FIG 4

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(A) Attenuation study. Ferrets were inoculated with AH13 ca intranasally with a dose of 107 FFU. After 3 days, the nasal turbinates (NTs) and lung tissues were collected, and viral titers were expressed as EID50 per gram of tissue. The dashed lines indicate the limit of detection. (B and C) Wild-type virus challenge study. Ferrets were inoculated with AH13 ca (H7N9 ca) intranasally with one or two doses of 107 FFU. One month after the final dose of vaccine was administered, ferrets were challenged with wt A/Anhui/1/2013 (H7N9) (B) or wt A/Netherlands/219/2003 (H7N7) (C) virus. After 5 days, the NTs and lung tissues were collected, and viral titers were expressed as TCID50 per gram of tissue. Vaccinated groups had a statistically significant reduction in virus titers of the homologous H7N9 (P ≤ 0.0003) and heterologous H7N7 (P ≤ 0.05) virus compared to the titers in the mock-immunized group.

One dose of the AH13 ca vaccine elicited a robust neutralizing antibody response with GMTs of 220 (range, 63 to 640) and 63 (range, 10 to 202) against the homologous A/Anhui/1/2013 wt and heterologous A/Netherlands/219/2003 (NL03) wt viruses, respectively, on day 28 p.i. (Table 3). In the group of ferrets that received two doses of the vaccine virus, the first dose elicited homologous neutralizing antibody with a GMT of 63 (range, 10 to 113) on day 28 p.i. The second dose of vaccine further boosted the neutralizing antibody response to a GMT of 294 (range, 202 to 453). The difference in the neutralizing antibody titers following one dose of vaccine in the two groups (GMT of 220 versus 63) may be due to the fact that ferrets are outbred or that at the time of immunization the ferrets that received only one dose of vaccine were a month older than the ferrets that received two doses of vaccine. The GMT of cross-neutralizing antibodies against the heterologous NL03 wt virus following one and two doses of the AH13 ca virus were 16 and 87, respectively (Table 3). Overall the anti-AH13 ca ferret antiserum cross-reacted to NL03 with titers that were 2- to 4-fold lower than the homologous neutralizing antibody titers. An HAI assay showed that the anti-AH13 ca antiserum similarly cross-reacted to NL03, an NL03 mutant (T135A) without the glycosylation site at HA position 133, and to North American H7N3 strain, A/British Columbia/CN-6/2004 (BC04), with 2- to 4-fold titer reduction compared to homologous HAI titers (data not shown), indicating that the unique glycosylation site in NL03 did not affect viral antigenicity and that the AH13 ca virus induced broadly cross-reactive antibodies to H7 viruses. Consistently, anti-NL03 and anti-BC04 ferret antisera cross-reacted with A/Anhui/1/2013 with approximately a 3-fold reduction in neutralization titers (30), demonstrating the cross-reactivity of divergent H7 viruses.

TABLE 3.

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

Test antigen Serum neutralizing antibody GMT in ferrets immunized with the AH13 ca vaccinea
1 dose
 2 dosesb
Day 14 Day 28 Day 14 Day 28 Day 42 Day 56
AH13 (H7N9) wt 30 220 22 63 281 294
NL03 (H7N7) wt 11 63 11 16 99 87
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a

Ferrets (n = 8) were inoculated i.n. with one or two 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.

b

Ferrets received 2 doses of vaccine administered 28 days apart.

The protective efficacy of one or two doses of the AH13 ca vaccine in preventing the replication of the homologous and heterologous wt challenge viruses in the respiratory tract of ferrets was evaluated on day 5 following challenge when the challenge viruses were predicted to be present at high titers in the lungs of mock-immunized ferrets (22). The H7 wt challenge viruses A/Anhui/1/2013 (H7N9) and A/Netherlands/219/2003 (H7N7) replicated well in the respiratory tract of mock-vaccinated ferrets, with mean titers of 106.4 and 107.1 TCID50/g, respectively, in the NTs and with mean titers of 105.8 and 104.8 TCID50/g, respectively, in the lungs. Immunization with one or two doses of the AH13 ca vaccine fully or nearly fully protected ferrets from replication of the homologous or heterologous wt, respectively, in both NTs and lungs. In the homologous H7N9 wt challenge group, none of the immunized ferrets had a detectable titer in both NTs and lungs. In the heterologous H7N7 wt challenge group, only three ferrets had a titer of 102.0 TCID50/g in the NTs, and one ferret had a titer of 102.0 TCID50/g in the lung (Fig. 4B).

DISCUSSION

The novel avian-origin H7N9 virus has been associated with significant morbidity and mortality in humans. This virus possesses several genetic features, including binding to human-like 2,6-SA receptors, a deletion in the NA protein, and the E627K mutation in the PB2 protein, that raise concerns about its pandemic potential (9, 13, 15). In contrast to the H7N7 and H7N3 ca vaccine viruses that grew to high titers in eggs (27, 28), the novel H7N9 ca virus did not grow well in eggs. In order to respond to the potential need to immunize people against the H7N9 virus, we identified the HA variants that can be used to produce a high-yield vaccine for manufacture. The candidate H7N9 ca vaccine is highly immunogenic and cross-reacts well to divergent H7 viruses. A single dose provides complete protection against wt H7N9 and H7N7 challenge infection in ferrets.

The HA residues identified in H1N1pdm and seasonal influenza viruses that improve vaccine virus growth in eggs are generally at or near the receptor binding site (RBS) (39–41). The changes identified in the H7N9 HA at residues 133, 135, 158, 160, 193, 198, 199, 220, and 224 following egg and MDCK cell passage are also located in proximity to the RBS (Fig. 2). Residues 133 and 135 are located in the 130 loop on the side of the RBS, residues 193 and 198 are located in the 190 helix, and residues 211 and 215 are located in the 220 loop (19, 42). We speculate that these changes create an optimized receptor binding structure for host-specific viral replication. It was noticed that the N133D, N158D, G198E, N199D, and N224D changes resulted in the presence of negatively charged acidic residues aspartate (D) or glutamate (E) on the surface of the HA. The K193N and R220S changes reduce positive charge on the HA trimer surface. Similar changes were also identified in the HA of the H1N1pdm viruses, K122E, A189D, N128D, D130E, and K212E (H3#), that improved vaccine virus growth in eggs and MDCK cells. Further studies indicated that the acidic residue substitutions in H1N1pdm did not affect viral entry and replication but greatly improved viral spread in the host cells (41). These negatively charged residues possibly decrease the HA and sialic acid interaction and thus facilitate the release of progeny viruses from infected cells for efficient multicycle replication.

Glycosylation of the HA protein affects receptor binding, fusion, and antigenicity and has been shown to play important roles in virus replication, host restriction, virulence, and transmission (43, 44). The A135T and A160T changes create potential N-linked glycosylation sites at the N133 and N158 residues that improve virus growth in eggs. We speculate the glycosylation may improve virus receptor binding and infectivity in eggs based on similar findings reported for the HPAI virus A/Netherlands/219/2003 (H7N7) in which glycosylation at N133 increased its binding affinity to avian-type α2,3-linked sialosides (45). It was also reported that glycosylation at HA position 188 (H3# position 197) near the receptor binding site increased the virulence of an avian H7N7 strain in chicken (46). In contrast, the loss of a glycosylation site at HA position 133 of human H3N2 viruses is associated with better viral growth in MDCK cells (47). For the H5N1 viruses, the loss of the glycosylation site at HA residue 158 improved viral transmission and vaccine immunogenicity in ferrets (48–50). Thus, the effect of HA glycosylation is strain and host specific.

Amino acid changes at antigenic sites on the surface of the HA molecule could alter viral antigenicity. Thus, each vaccine variant was evaluated for antigenicity in the HAI assay. The N158D (V2 and V3) and the K193N (V11) changes at antigenic site B and the R220S (V10) change at antigenic site D affected viral antigenicity of the H7N9 virus. The 158 residue has previously been shown to alter viral antigenicity in seasonal H3N2 and H1N1pdm viruses (38, 51).

One dose of the H7N9 ca vaccine virus induced neutralizing antibody in ferrets that cross-reacted with the H7N7 virus in ferrets. A second dose of the H7N9 ca vaccine greatly boosted serum antibody titers. The immunogenicity of the H7N9 ca vaccine virus was as great as or greater than the immunogenicity of the H7N3 or H7N7 ca vaccines that were previously generated and evaluated in our laboratory (27, 28). The poorer immunogenicity of the A/Netherland/219/2003 (H7N7) vaccine virus could be attributed to its 2,3-SA receptor binding specificity. The introduction of Q226L/G228S that increased the 2,6-SA receptor binding improved immunogenicity of the H7N7 ca vaccine virus in ferrets (30). The novel H7N9 HA contains L226, which may contribute to its higher replication in the upper respiratory tract and greater immunogenicity in ferrets.

Vaccines against pandemics are evaluated for safety and immunogenicity in clinical trials, but efficacy data can come only from studies in experimentally infected animals. In this study, we demonstrated that a single dose of the H7N9 ca vaccine conferred complete protection, in both NTs and lungs, against homologous wt virus challenge infection and nearly complete protection against the heterologous wt virus. This finding correlated with the robust neutralizing antibody response induced after one dose of the vaccine. In our previous studies, one dose of the H7N3 or H7N7 ca vaccine conferred protection from pulmonary replication of the homologous and heterologous wt virus challenge but not replication in the upper respiratory tract (27, 28).

In summary, we generated a high-yield H7N9 vaccine candidate that is highly immunogenic and efficacious in a ferret challenge study. The candidate vaccine with amino acid changes at HA residues 133 and 198 maintained the attenuation phenotype conferred by the six internal protein gene segments of AA ca. Based on this promising preclinical data, this vaccine is currently being evaluated in phase I clinical studies.

ACKNOWLEDGMENTS

This study was funded in part by federal funds from Biomedical Advanced Research and Development Authority of the U.S. Department of Health and Human Services under the contract number HHSO100201200012I and by the Intramural Research Program of NIAID, NIH. The work is conducted under a Cooperative Research and Development Agreement between MedImmune and NIAID/NIH.

We thank Michael Shaw and Nancy Cox at CDC for providing the H7N9 virus and viral RNA, the staff of the animal care facilities at MedImmune and SRI for their assistance with ferret studies, MedImmune's strain variant team for their support, Christopher Cotter for technical assistance, and JoAnn Suzich for critical review of the manuscript.

Footnotes

Published ahead of print 9 April 2014

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