© The American Society of Gene & Cell Therapy
original article
H7N9 Live Attenuated Influenza Vaccine Is Highly Immunogenic, Prevents Virus Replication, and Protects Against Severe Bronchopneumonia in Ferrets Jørgen de Jonge1, Irina Isakova-Sivak2, Harry van Dijken1, Sanne Spijkers1,5, Justin Mouthaan1,6, Rineke de Jong3, Tatiana Smolonogina2, Paul Roholl4 and Larisa Rudenko2 1 Centre for Infectious Disease Control, National Institute of Public Health and the Environment (RIVM), Bilthoven, the Netherlands; 2Department of Virology, Institute of Experimental Medicine, Saint Petersburg, Russia; 3Department of Virology, Central Veterinary Institute of Wageningen UR, Lelystad, the Netherlands; 4Microscope Consultancy, Weesp, the Netherlands; 5Current address: BioNovion, Oss, the Netherlands; 6Current address: Genmab, Utrecht, the Netherlands
Avian influenza viruses continue to cross the species barrier, and if such viruses become transmissible among humans, it would pose a great threat to public health. Since its emergence in China in 2013, H7N9 has caused considerable morbidity and mortality. In the absence of a universal influenza vaccine, preparedness includes development of subtype-specific vaccines. In this study, we developed and evaluated in ferrets an intranasal live attenuated influenza vaccine (LAIV) against H7N9 based on the A/Leningrad/134/17/57 (H2N2) cold-adapted master donor virus. We demonstrate that the LAIV is attenuated and safe in ferrets and induces high hemagglutination- and neuraminidase-inhibiting and virusneutralizing titers. The antibodies against hemagglutinin were also cross-reactive with divergent H7 strains. To assess efficacy, we used an intratracheal challenge ferret model in which an acute severe viral pneumonia is induced that closely resembles viral pneumonia observed in severe human cases. A single- and two-dose strategy provided complete protection against severe pneumonia and prevented virus replication. The protective effect of the two-dose strategy appeared better than the single dose only on the microscopic level in the lungs. We observed, however, an increased lymphocytic infiltration after challenge in single-vaccinated animals and hypothesize that this a side effect of the model. Received 2 October 2015; accepted 22 December 2015; advance online publication 1 March 2016. doi:10.1038/mt.2016.23
INTRODUCTION
Avian influenza viruses continue to cross the species barrier, and although infections detected in humans are incidental, they often have a severe outcome. In February 2013, a novel influenza virus of the subtype H7N9 emerged in China and has since continued to infect humans in a seasonal-like fashion. As of May 2015, 657 cases were confirmed to have been infected with H7N9 of which
261 have perished.1 Infections were mostly observed in people who visited live bird markets. Since the virus is mainly detected in poultry at these markets, it is assumed that H7N9 is transmitted from poultry to humans.2 The disease starts with typical influenza-like illness including fever and cough. Severe cases present viral pneumonia, acute respiratory distress syndrome (ARDS), and multiorgan failure. However, these cases usually have underlying chronic conditions.3 Despite the fact that H7N9 has not yet been capable of establishing a sustained transmission among humans, the virus is considered to have potential to become pandemic in humans for several reasons.2,4 Firstly, as the virus is low pathogenic in birds,5 it slips under the radar unless active monitoring is performed. Secondly, since the first detection, the virus continues to emerge, expands geographically, and has diverged into different clades,6 increasing the chance of acquiring the ability to transmit between humans. Furthermore, H7N9 viruses bind both avian-type (α2,3-linked sialic acid) and, to a lesser extent, human-type (α2,6linked sialic acid) receptors.7,8 Regardless of the limited respiratory droplet transmission between ferrets, this dual-receptor specificity can be a critical feature for the acquisition of sustained human-to-human transmission in case more adaptive changes occur in the receptor-binding site.9,10 Moreover, several isolates have been shown to be resistant to antivirals, which impedes preventive measures and treatment.11,12 Obviously, the human population is naive to H7N9,4 which allows the virus to spread easily without an immunological barrier once it becomes transmissible between humans. To anticipate possible future pandemics, the World Health Organization (WHO) has initiated several programs, the Global Action Plan (GAP) for influenza vaccines is one among them.13,14 This program includes the development of vaccines against potentially pandemic influenza viruses. Since for influenza, a universal vaccine is not yet available, development of subtype-specific vaccines up to clinical phase 1/2 is a manner to be able to rapidly act in the event of a pandemic. Careful consideration of which influenza viruses have a potency to initiate a pandemic is however
Correspondence: Jørgen de Jonge, Centre for Infectious Disease Control, National Institute of Public Health and the Environment (RIVM), 3720 BA, Bilthoven, the Netherlands. E-mail: [email protected] Molecular Therapy vol. 24 no. 5, 991–1002 may 2016
991
H7N9 LAIV Is Immunogenic and Effective in Ferrets
required and still provides no guarantee. Based on the arguments above, H7N9 was recently included. Within the GAP program, we developed and evaluated a live attenuated influenza vaccine (LAIV) against H7N9 based on the A/Leningrad/134/17/57 (H2N2) cold-adapted master donor virus. Prior to the H7N9 emergence, a number of H7 candidate vaccine viruses had been developed and tested in clinical trials, however, unfortunately none of them contained N9 neuraminidase (NA).15–17 Antibodies against NA block the sialidase activity of neuraminidase and, as a consequence, impair viral budding and limit the infection.18 Existing H7 vaccines contain N1, N3, or N7 NA genes; however, since their amino acid homology with N9 is less than 50%, there will be most likely no cross-reactive antibodies to N9. Although some studies show that previously developed vaccines against divergent H7 provide cross protection against H7N9,19–21 this would only be a temporal solution in the absence of specific H7N9 vaccines. Vaccines that induce cross-reactive antibodies against H7 only and no antibodies against neuraminidase are likely less effective and therefore urge development of vaccines specific for the current H7N9 outbreak. Inactivated influenza vaccines (IIVs) provide strain-specific humoral immunity that does not protect against antigenic variations of the influenza virus. Moreover, IIVs do not induce mucosal immunity and thus fail to protect at the gate. Inactivated vaccines against H7N9 prove to be weak immunogenic, and adjuvation is required for sero-conversion,22 which makes the vaccine more complex. LAIVs are believed to be immunologically superior vaccines due to their potential to induce all arms of the adaptive immune responses, including induction of serum antibodies, mucosal immunity, and cytotoxic T lymphocytes targeted to conserved virus epitopes.23–25 Other advantages of LAIVs over traditional IIVs are a much cheaper and quicker manufacturing process and easier administration by intranasal spray.14 We developed an intranasal H7N9 live attenuated vaccine and evaluated safety, immunogenicity, and efficacy in an intratracheally induced severe pneumonia ferret model. We show that the vaccine is safe and raises high hemagglutination-inhibiting (HI) and neuraminidase-inhibiting (NI) titers and virus-neutralizing (VN) titers, which provide sterilizing immunity in challenged ferrets. Importantly, the vaccine protects against weight loss, fever, clinical disease, (severe) pneumonia, and death.
RESULTS Generation and in vitro characterization of the H7N9 LAIV candidate
To prepare for the event that H7N9 becomes transmissible between humans and a pandemic is on the verge, an H7N9 LAIV based on the A/Anhui/1/2013 isolate representative for currently circulating H7N9 influenza in China was generated. The LAIV was constructed by classical reassortment in developing chicken embryos using the cold-adapted and temperature-sensitive master donor virus (MDV) A/Leningrad/134/17/57 (H2N2) as a backbone.26 The resulting A/17/Anhui/2013/61 (H7N9 LAIV) vaccine strain inherited HA and NA genes from wild-type (WT) H7N9 A/Anhui/1/2013 parental virus and six internal genes from the MDV as confirmed by partial sequencing.27 Full-length genome sequencing confirmed nucleotide sequences of six internal genes 992
© The American Society of Gene & Cell Therapy
identical to the internal genes of the MDV. However, the HA gene of A/17/Anhui/2013/61 reassortant virus revealed two nucleotide changes of A to G at positions 421 and 499 (ORF numbering) compared with WT H7N9 (GISAID: EPI439507). These changes resulted in changes at the amino acid level of Asn-123-Asp and Asn-149-Asp by H7 numbering (Figure 1). In addition, the NA gene of the reassortant also contained a nucleotide change of C to T, which resulted in Thr-10-Ile change compared with NA of WT virus (GISAID: EPI439509). To reveal whether these mutations were spontaneous or result from egg adaptation, we sequenced WT A/Anhui/1/2013 passaged three, four, and five times in eggs and three times in eggs followed by one passage on Madin–Darby canine kidney (MDCK) cells (Figure 1). Interestingly, mutation at position 421 (Asn-123-Asp) was appearing gradually during virus passaging in eggs, and after four sequential passages, the original nucleotide was detected at a very low level (approximately 5%) and almost disappeared by fifth egg passage. Noteworthy, one passage in MDCK cells could significantly restore the original population of WT virus, and the heterogeneity was seen at approximately 45% level. Similarly, the second mutation at position 499 could be partially restored after single passage in MDCK cells, whereas all egg-grown variants shared Asn-149-Asp mutation without quasi species. The Thr-10-Ile change in the NA protein was also appearing gradually during egg passaging and could be partially restored by a single passage in a mammalian host (Figure 1). These data demonstrate a strong connection between egg passaging of WT H7N9 virus with the appearance of Asn-123-Asp and Asn149-Asp mutations in HA and Thr-10-Ile mutation in NA, i.e., these mutations are egg adapted. Passaging in mammalian cell culture can restore these mutations to the original residues. Therefore, during the generation of LAIV reassortant virus using the egg-based system, it is impossible to maintain the original mammalian cell–adapted component of WT virus HA and NA, and the presence of e gg-adapted component is inevitable.
H7N9 LAIV has a cold-adapted and temperaturesensitive phenotype LAIVs are characterized by their temperature-sensitive (ts) and cold-adapted (ca) phenotype. The first phenotype is characterized by the inability to grow at temperatures that represent fever in humans and the latter by the ability to grow at low (26 °C) temperatures. In chicken embryos, H7N9 LAIV (A/17/Anhui/2013/61) exhibited high reproductive activity at an optimum incubation temperature of 33 °C (Supplementary Table S1). Similar to the MDV (A/Leningrad/134/17/57 (H2N2)), the H7N9 LAIV acquired the ts and ca phenotypes. It reproduced well at a reduced temperature of 26 °C and practically lost the ability to reproduce at temperature elevated to 38–39 °C. The WT parent virus A/Anhui/1/2013 (H7N9), in contrast, was distinguished by the ability to reproduce to high titers at 38–39 °C and low reproduction at 26 °C. H7N9 LAIV is attenuated in ferrets Next, attenuation of the H7N9 LAIV was evaluated in a ferret safety test performed according to the WHO biosafety guidelines on production and quality control of H7N9 vaccines.28 When ferrets were intranasally infected with WT H7N9 (A/Anhui/1/2013, www.moleculartherapy.org vol. 24 no. 5 may 2016
© The American Society of Gene & Cell Therapy
H7N9 LAIV Is Immunogenic and Effective in Ferrets
Figure 1 Mutations as a result of egg adaptation. Sequencing diagrams of HA (nt 421 and 499) and NA (nt 29) of WT A/Anhui/1/2013 (H7N9) influenza after multiple passaging on eggs and a single passage on MDCKs.
a
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Figure 2 Virus replication in the respiratory tract and brain. (a) Virus titers in the nasal turbinates, trachea, lung, olfactory bulb (OB), and brain of ferrets sacrificed 4 days after intranasal infection with either WT H7N9 (red triangles), H2N2 master donor virus (MDV; blue circles), or H7N9 live attenuated influenza vaccine (LAIV) (black squares). No virus replication was detected in the intestines or the spleen (not shown). (b) Nose and (c) throat swabs were performed on all animals (six per group) 1 day prior to challenge and 2 and 4 days after infection. After termination of three animals per group 4 days post infection (d.p.i.), sampling was continued in the remaining three animals on days 6, 8, and 10. The virus titers in the homogenized tissue samples or transport buffer of the swabs were determined by end-point titration on Madin–Darby canine kidney cells using a fivefold serial dilution. WT H7N9 titrations were incubated at 37 °C whereas H2N2 MD and H7N9 LAIV titrations were performed at 32 °C. Presented are the individual 10 log transformed titers connected with a line through the averages (b and c only). Dotted horizontal lines indicate limit of detection. Dotted vertical lines indicate day of termination of first three ferrets.
Molecular Therapy vol. 24 no. 5 may 2016
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H7N9 LAIV Is Immunogenic and Effective in Ferrets
107 TCID50), they did not develop any clear clinical signs of influenza illness. They did develop a mild fever and showed limited weight loss over a period of 14 days (Supplementary Table S2). In contrast, H7N9 LAIV and MDV did not induce fever or weight loss. High viral titers were detected 4 days after infection with WT H7N9 in the upper (nasal turbinates) and lower (trachea and lung) respiratory tract and exterior of the respiratory system in the olfactory bulb (OB) and brain (Figure 2a), but not systemically (spleen and intestine—data not shown). In the H7N9 LAIV and MDV infected ferrets, viral titers were only detected in the upper respiratory tract (nasal turbinates). WT H7N9 replicated at high levels in the nose and throat and continued until 6 and 8 days after infection, respectively, whereas only low levels in the nose and high levels in the throat were detected in the H7N9 LAIV infected ferrets until 4 days after infection (Figure 2b,c). Infection with the MDV resulted in low levels of virus replication in the nose and substantial virus replication in the throat, however, this continued until day 10. Gross pathology performed on the lungs of ferrets sacrificed 4 and 14 days after infection showed only minor lesions in the lungs of WT H7N9-infected ferrets, which were approximately equal in MDV infected ferrets and less in the H7N9 LAIV infected ferrets (data not shown). Thus, the absence of clinical signs of disease, limitation of virus replication to only the upper respiratory tract, a reduced time span of viral replication, and reduced gross pathology confirmed the attenuated phenotype of the H7N9 LAIV.
Single and booster vaccination with H7N9 LAIV induce high HI, NI, and VN antibody titers To investigate the immunogenicity of H7N9 LAIV, ferrets were intranasally vaccinated according to a single (1×LAIV) and booster (2×LAIV) strategy. As observed in the safety study (Figure 2), 2 days after first vaccination, high virus titers were detected in the throat of H7N9 LAIV-vaccinated ferrets. However, after booster vaccination (21 days after primary vaccination), no virus was detected, indicating that the booster had been neutralized by the antibodies induced at primary vaccination. H7N9 LAIV induced high HI, NI, and VN antibody titers as detected in serum collected on days 13, 20, and 23 after single vaccination and on day 17 after booster vaccination (Figure 3). Seven days after first vaccination, no relevant titers could be detected, but the responses peak at 14 (HI) and 21 days after first vaccination (NI and VN). Despite booster vaccination, HI and VN titers declined but NI titers increased; however, none of the effects were significant. We next evaluated the cross-reactive potency of the vaccine-induced H7 antibodies against A/Netherlands/33/03 (H7N7) and A/mallard/Netherlands/12/00 (H7N3) strains in sera obtained 23 days after single vaccination and 17 days after booster vaccination (all obtained the day before challenge). The H7N7 strain was isolated from a human case during the 2003 outbreak in the Netherlands, and the H7N3 is a low-pathogenic avian influenza strain also used in a vaccine strain.15 The H7N7 and H7N3 strains share 96.3% and 97.1% homology with A/Anhui/1/2013 (H7N9), respectively. The H7N9 LAIV vaccine induces cross-reactive antibodies against both H7 variants (Figure 3d), although higher responses were observed for the H7N3 strains, which also reflects the higher homology with H7N9.
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© The American Society of Gene & Cell Therapy
Thus, H7N9 is highly immunogenic and induces functional antibodies against hemagglutinin and neuraminidase surface proteins that result in high VN antibody titers. Moreover, the H7-directed antibodies cross react with divergent H7.
H7N9 LAIV protects against severe disease and death Since intranasal infection only induced mild disease in ferrets (Supplementary Table S2),9,29 which does not represent the severe disease observed in H7N9-infected humans, the intratracheal challenge model was chosen to establish the protective efficacy of H7N9 LAIV. In a dose-escalation infection study, a dose of 107 TCID50 WT H7N9 influenza consistently induced severe pneumonia (data not shown), similar to as previously reported.30 Since at this dose, the disease progressed rapidly, termination was performed 3 days post challenge (d.p.c.) to be able to compare end-point measurements. When placebo-vaccinated ferrets were challenged accordingly, they became less active (score 1) that progressed to inactive behavior (score 2) after 2–3 days. They initially presented faster breathing (score 1) which developed to heavy stomach breathing (score 2) by 2–3 d.p.c. (Supplementary Table S3). Finally, three out of six animals succumbed to the infection prior to termination of the study. Ferrets vaccinated with H7N9 LAIV, on the other hand, only developed mild disease and showed none to some inactivity (score 1) and few animals displayed faster breathing (score 1), and none of the animals died. Less than 1 d.p.c., placebo-vaccinated ferrets developed a substantial fever, which lasted for approximately 1 day and after a small dip returned for another day (Figure 4a.1). During the 3 d.p.c., the average temperature was ~1.2 °C higher than normal, and the highest recorded fever was 3 °C above baseline (Supplementary Table S4). In H7N9 LAIV-vaccinated ferrets, only a very mild induction of fever was registered after challenge (Figure 4a.2, a.3). Vaccination significantly reduced the average temperature increase with 0.7–0.8 °C and the highest recorded fever with ~1.5 °C (Supplementary Table S4). P lacebo-vaccinated ferrets lost between 5% and 15% of their body weight by 3 d.p.c. (Figure 4b), the average weight was 8% lower than baseline and the average maximum recorded weight loss was 10% (Supplementary Table S4). These effects were significantly (P
original article
H7N9 Live Attenuated Influenza Vaccine Is Highly Immunogenic, Prevents Virus Replication, and Protects Against Severe Bronchopneumonia in Ferrets Jørgen de Jonge1, Irina Isakova-Sivak2, Harry van Dijken1, Sanne Spijkers1,5, Justin Mouthaan1,6, Rineke de Jong3, Tatiana Smolonogina2, Paul Roholl4 and Larisa Rudenko2 1 Centre for Infectious Disease Control, National Institute of Public Health and the Environment (RIVM), Bilthoven, the Netherlands; 2Department of Virology, Institute of Experimental Medicine, Saint Petersburg, Russia; 3Department of Virology, Central Veterinary Institute of Wageningen UR, Lelystad, the Netherlands; 4Microscope Consultancy, Weesp, the Netherlands; 5Current address: BioNovion, Oss, the Netherlands; 6Current address: Genmab, Utrecht, the Netherlands
Avian influenza viruses continue to cross the species barrier, and if such viruses become transmissible among humans, it would pose a great threat to public health. Since its emergence in China in 2013, H7N9 has caused considerable morbidity and mortality. In the absence of a universal influenza vaccine, preparedness includes development of subtype-specific vaccines. In this study, we developed and evaluated in ferrets an intranasal live attenuated influenza vaccine (LAIV) against H7N9 based on the A/Leningrad/134/17/57 (H2N2) cold-adapted master donor virus. We demonstrate that the LAIV is attenuated and safe in ferrets and induces high hemagglutination- and neuraminidase-inhibiting and virusneutralizing titers. The antibodies against hemagglutinin were also cross-reactive with divergent H7 strains. To assess efficacy, we used an intratracheal challenge ferret model in which an acute severe viral pneumonia is induced that closely resembles viral pneumonia observed in severe human cases. A single- and two-dose strategy provided complete protection against severe pneumonia and prevented virus replication. The protective effect of the two-dose strategy appeared better than the single dose only on the microscopic level in the lungs. We observed, however, an increased lymphocytic infiltration after challenge in single-vaccinated animals and hypothesize that this a side effect of the model. Received 2 October 2015; accepted 22 December 2015; advance online publication 1 March 2016. doi:10.1038/mt.2016.23
INTRODUCTION
Avian influenza viruses continue to cross the species barrier, and although infections detected in humans are incidental, they often have a severe outcome. In February 2013, a novel influenza virus of the subtype H7N9 emerged in China and has since continued to infect humans in a seasonal-like fashion. As of May 2015, 657 cases were confirmed to have been infected with H7N9 of which
261 have perished.1 Infections were mostly observed in people who visited live bird markets. Since the virus is mainly detected in poultry at these markets, it is assumed that H7N9 is transmitted from poultry to humans.2 The disease starts with typical influenza-like illness including fever and cough. Severe cases present viral pneumonia, acute respiratory distress syndrome (ARDS), and multiorgan failure. However, these cases usually have underlying chronic conditions.3 Despite the fact that H7N9 has not yet been capable of establishing a sustained transmission among humans, the virus is considered to have potential to become pandemic in humans for several reasons.2,4 Firstly, as the virus is low pathogenic in birds,5 it slips under the radar unless active monitoring is performed. Secondly, since the first detection, the virus continues to emerge, expands geographically, and has diverged into different clades,6 increasing the chance of acquiring the ability to transmit between humans. Furthermore, H7N9 viruses bind both avian-type (α2,3-linked sialic acid) and, to a lesser extent, human-type (α2,6linked sialic acid) receptors.7,8 Regardless of the limited respiratory droplet transmission between ferrets, this dual-receptor specificity can be a critical feature for the acquisition of sustained human-to-human transmission in case more adaptive changes occur in the receptor-binding site.9,10 Moreover, several isolates have been shown to be resistant to antivirals, which impedes preventive measures and treatment.11,12 Obviously, the human population is naive to H7N9,4 which allows the virus to spread easily without an immunological barrier once it becomes transmissible between humans. To anticipate possible future pandemics, the World Health Organization (WHO) has initiated several programs, the Global Action Plan (GAP) for influenza vaccines is one among them.13,14 This program includes the development of vaccines against potentially pandemic influenza viruses. Since for influenza, a universal vaccine is not yet available, development of subtype-specific vaccines up to clinical phase 1/2 is a manner to be able to rapidly act in the event of a pandemic. Careful consideration of which influenza viruses have a potency to initiate a pandemic is however
Correspondence: Jørgen de Jonge, Centre for Infectious Disease Control, National Institute of Public Health and the Environment (RIVM), 3720 BA, Bilthoven, the Netherlands. E-mail: [email protected] Molecular Therapy vol. 24 no. 5, 991–1002 may 2016
991
H7N9 LAIV Is Immunogenic and Effective in Ferrets
required and still provides no guarantee. Based on the arguments above, H7N9 was recently included. Within the GAP program, we developed and evaluated a live attenuated influenza vaccine (LAIV) against H7N9 based on the A/Leningrad/134/17/57 (H2N2) cold-adapted master donor virus. Prior to the H7N9 emergence, a number of H7 candidate vaccine viruses had been developed and tested in clinical trials, however, unfortunately none of them contained N9 neuraminidase (NA).15–17 Antibodies against NA block the sialidase activity of neuraminidase and, as a consequence, impair viral budding and limit the infection.18 Existing H7 vaccines contain N1, N3, or N7 NA genes; however, since their amino acid homology with N9 is less than 50%, there will be most likely no cross-reactive antibodies to N9. Although some studies show that previously developed vaccines against divergent H7 provide cross protection against H7N9,19–21 this would only be a temporal solution in the absence of specific H7N9 vaccines. Vaccines that induce cross-reactive antibodies against H7 only and no antibodies against neuraminidase are likely less effective and therefore urge development of vaccines specific for the current H7N9 outbreak. Inactivated influenza vaccines (IIVs) provide strain-specific humoral immunity that does not protect against antigenic variations of the influenza virus. Moreover, IIVs do not induce mucosal immunity and thus fail to protect at the gate. Inactivated vaccines against H7N9 prove to be weak immunogenic, and adjuvation is required for sero-conversion,22 which makes the vaccine more complex. LAIVs are believed to be immunologically superior vaccines due to their potential to induce all arms of the adaptive immune responses, including induction of serum antibodies, mucosal immunity, and cytotoxic T lymphocytes targeted to conserved virus epitopes.23–25 Other advantages of LAIVs over traditional IIVs are a much cheaper and quicker manufacturing process and easier administration by intranasal spray.14 We developed an intranasal H7N9 live attenuated vaccine and evaluated safety, immunogenicity, and efficacy in an intratracheally induced severe pneumonia ferret model. We show that the vaccine is safe and raises high hemagglutination-inhibiting (HI) and neuraminidase-inhibiting (NI) titers and virus-neutralizing (VN) titers, which provide sterilizing immunity in challenged ferrets. Importantly, the vaccine protects against weight loss, fever, clinical disease, (severe) pneumonia, and death.
RESULTS Generation and in vitro characterization of the H7N9 LAIV candidate
To prepare for the event that H7N9 becomes transmissible between humans and a pandemic is on the verge, an H7N9 LAIV based on the A/Anhui/1/2013 isolate representative for currently circulating H7N9 influenza in China was generated. The LAIV was constructed by classical reassortment in developing chicken embryos using the cold-adapted and temperature-sensitive master donor virus (MDV) A/Leningrad/134/17/57 (H2N2) as a backbone.26 The resulting A/17/Anhui/2013/61 (H7N9 LAIV) vaccine strain inherited HA and NA genes from wild-type (WT) H7N9 A/Anhui/1/2013 parental virus and six internal genes from the MDV as confirmed by partial sequencing.27 Full-length genome sequencing confirmed nucleotide sequences of six internal genes 992
© The American Society of Gene & Cell Therapy
identical to the internal genes of the MDV. However, the HA gene of A/17/Anhui/2013/61 reassortant virus revealed two nucleotide changes of A to G at positions 421 and 499 (ORF numbering) compared with WT H7N9 (GISAID: EPI439507). These changes resulted in changes at the amino acid level of Asn-123-Asp and Asn-149-Asp by H7 numbering (Figure 1). In addition, the NA gene of the reassortant also contained a nucleotide change of C to T, which resulted in Thr-10-Ile change compared with NA of WT virus (GISAID: EPI439509). To reveal whether these mutations were spontaneous or result from egg adaptation, we sequenced WT A/Anhui/1/2013 passaged three, four, and five times in eggs and three times in eggs followed by one passage on Madin–Darby canine kidney (MDCK) cells (Figure 1). Interestingly, mutation at position 421 (Asn-123-Asp) was appearing gradually during virus passaging in eggs, and after four sequential passages, the original nucleotide was detected at a very low level (approximately 5%) and almost disappeared by fifth egg passage. Noteworthy, one passage in MDCK cells could significantly restore the original population of WT virus, and the heterogeneity was seen at approximately 45% level. Similarly, the second mutation at position 499 could be partially restored after single passage in MDCK cells, whereas all egg-grown variants shared Asn-149-Asp mutation without quasi species. The Thr-10-Ile change in the NA protein was also appearing gradually during egg passaging and could be partially restored by a single passage in a mammalian host (Figure 1). These data demonstrate a strong connection between egg passaging of WT H7N9 virus with the appearance of Asn-123-Asp and Asn149-Asp mutations in HA and Thr-10-Ile mutation in NA, i.e., these mutations are egg adapted. Passaging in mammalian cell culture can restore these mutations to the original residues. Therefore, during the generation of LAIV reassortant virus using the egg-based system, it is impossible to maintain the original mammalian cell–adapted component of WT virus HA and NA, and the presence of e gg-adapted component is inevitable.
H7N9 LAIV has a cold-adapted and temperaturesensitive phenotype LAIVs are characterized by their temperature-sensitive (ts) and cold-adapted (ca) phenotype. The first phenotype is characterized by the inability to grow at temperatures that represent fever in humans and the latter by the ability to grow at low (26 °C) temperatures. In chicken embryos, H7N9 LAIV (A/17/Anhui/2013/61) exhibited high reproductive activity at an optimum incubation temperature of 33 °C (Supplementary Table S1). Similar to the MDV (A/Leningrad/134/17/57 (H2N2)), the H7N9 LAIV acquired the ts and ca phenotypes. It reproduced well at a reduced temperature of 26 °C and practically lost the ability to reproduce at temperature elevated to 38–39 °C. The WT parent virus A/Anhui/1/2013 (H7N9), in contrast, was distinguished by the ability to reproduce to high titers at 38–39 °C and low reproduction at 26 °C. H7N9 LAIV is attenuated in ferrets Next, attenuation of the H7N9 LAIV was evaluated in a ferret safety test performed according to the WHO biosafety guidelines on production and quality control of H7N9 vaccines.28 When ferrets were intranasally infected with WT H7N9 (A/Anhui/1/2013, www.moleculartherapy.org vol. 24 no. 5 may 2016
© The American Society of Gene & Cell Therapy
H7N9 LAIV Is Immunogenic and Effective in Ferrets
Figure 1 Mutations as a result of egg adaptation. Sequencing diagrams of HA (nt 421 and 499) and NA (nt 29) of WT A/Anhui/1/2013 (H7N9) influenza after multiple passaging on eggs and a single passage on MDCKs.
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Figure 2 Virus replication in the respiratory tract and brain. (a) Virus titers in the nasal turbinates, trachea, lung, olfactory bulb (OB), and brain of ferrets sacrificed 4 days after intranasal infection with either WT H7N9 (red triangles), H2N2 master donor virus (MDV; blue circles), or H7N9 live attenuated influenza vaccine (LAIV) (black squares). No virus replication was detected in the intestines or the spleen (not shown). (b) Nose and (c) throat swabs were performed on all animals (six per group) 1 day prior to challenge and 2 and 4 days after infection. After termination of three animals per group 4 days post infection (d.p.i.), sampling was continued in the remaining three animals on days 6, 8, and 10. The virus titers in the homogenized tissue samples or transport buffer of the swabs were determined by end-point titration on Madin–Darby canine kidney cells using a fivefold serial dilution. WT H7N9 titrations were incubated at 37 °C whereas H2N2 MD and H7N9 LAIV titrations were performed at 32 °C. Presented are the individual 10 log transformed titers connected with a line through the averages (b and c only). Dotted horizontal lines indicate limit of detection. Dotted vertical lines indicate day of termination of first three ferrets.
Molecular Therapy vol. 24 no. 5 may 2016
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H7N9 LAIV Is Immunogenic and Effective in Ferrets
107 TCID50), they did not develop any clear clinical signs of influenza illness. They did develop a mild fever and showed limited weight loss over a period of 14 days (Supplementary Table S2). In contrast, H7N9 LAIV and MDV did not induce fever or weight loss. High viral titers were detected 4 days after infection with WT H7N9 in the upper (nasal turbinates) and lower (trachea and lung) respiratory tract and exterior of the respiratory system in the olfactory bulb (OB) and brain (Figure 2a), but not systemically (spleen and intestine—data not shown). In the H7N9 LAIV and MDV infected ferrets, viral titers were only detected in the upper respiratory tract (nasal turbinates). WT H7N9 replicated at high levels in the nose and throat and continued until 6 and 8 days after infection, respectively, whereas only low levels in the nose and high levels in the throat were detected in the H7N9 LAIV infected ferrets until 4 days after infection (Figure 2b,c). Infection with the MDV resulted in low levels of virus replication in the nose and substantial virus replication in the throat, however, this continued until day 10. Gross pathology performed on the lungs of ferrets sacrificed 4 and 14 days after infection showed only minor lesions in the lungs of WT H7N9-infected ferrets, which were approximately equal in MDV infected ferrets and less in the H7N9 LAIV infected ferrets (data not shown). Thus, the absence of clinical signs of disease, limitation of virus replication to only the upper respiratory tract, a reduced time span of viral replication, and reduced gross pathology confirmed the attenuated phenotype of the H7N9 LAIV.
Single and booster vaccination with H7N9 LAIV induce high HI, NI, and VN antibody titers To investigate the immunogenicity of H7N9 LAIV, ferrets were intranasally vaccinated according to a single (1×LAIV) and booster (2×LAIV) strategy. As observed in the safety study (Figure 2), 2 days after first vaccination, high virus titers were detected in the throat of H7N9 LAIV-vaccinated ferrets. However, after booster vaccination (21 days after primary vaccination), no virus was detected, indicating that the booster had been neutralized by the antibodies induced at primary vaccination. H7N9 LAIV induced high HI, NI, and VN antibody titers as detected in serum collected on days 13, 20, and 23 after single vaccination and on day 17 after booster vaccination (Figure 3). Seven days after first vaccination, no relevant titers could be detected, but the responses peak at 14 (HI) and 21 days after first vaccination (NI and VN). Despite booster vaccination, HI and VN titers declined but NI titers increased; however, none of the effects were significant. We next evaluated the cross-reactive potency of the vaccine-induced H7 antibodies against A/Netherlands/33/03 (H7N7) and A/mallard/Netherlands/12/00 (H7N3) strains in sera obtained 23 days after single vaccination and 17 days after booster vaccination (all obtained the day before challenge). The H7N7 strain was isolated from a human case during the 2003 outbreak in the Netherlands, and the H7N3 is a low-pathogenic avian influenza strain also used in a vaccine strain.15 The H7N7 and H7N3 strains share 96.3% and 97.1% homology with A/Anhui/1/2013 (H7N9), respectively. The H7N9 LAIV vaccine induces cross-reactive antibodies against both H7 variants (Figure 3d), although higher responses were observed for the H7N3 strains, which also reflects the higher homology with H7N9.
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Thus, H7N9 is highly immunogenic and induces functional antibodies against hemagglutinin and neuraminidase surface proteins that result in high VN antibody titers. Moreover, the H7-directed antibodies cross react with divergent H7.
H7N9 LAIV protects against severe disease and death Since intranasal infection only induced mild disease in ferrets (Supplementary Table S2),9,29 which does not represent the severe disease observed in H7N9-infected humans, the intratracheal challenge model was chosen to establish the protective efficacy of H7N9 LAIV. In a dose-escalation infection study, a dose of 107 TCID50 WT H7N9 influenza consistently induced severe pneumonia (data not shown), similar to as previously reported.30 Since at this dose, the disease progressed rapidly, termination was performed 3 days post challenge (d.p.c.) to be able to compare end-point measurements. When placebo-vaccinated ferrets were challenged accordingly, they became less active (score 1) that progressed to inactive behavior (score 2) after 2–3 days. They initially presented faster breathing (score 1) which developed to heavy stomach breathing (score 2) by 2–3 d.p.c. (Supplementary Table S3). Finally, three out of six animals succumbed to the infection prior to termination of the study. Ferrets vaccinated with H7N9 LAIV, on the other hand, only developed mild disease and showed none to some inactivity (score 1) and few animals displayed faster breathing (score 1), and none of the animals died. Less than 1 d.p.c., placebo-vaccinated ferrets developed a substantial fever, which lasted for approximately 1 day and after a small dip returned for another day (Figure 4a.1). During the 3 d.p.c., the average temperature was ~1.2 °C higher than normal, and the highest recorded fever was 3 °C above baseline (Supplementary Table S4). In H7N9 LAIV-vaccinated ferrets, only a very mild induction of fever was registered after challenge (Figure 4a.2, a.3). Vaccination significantly reduced the average temperature increase with 0.7–0.8 °C and the highest recorded fever with ~1.5 °C (Supplementary Table S4). P lacebo-vaccinated ferrets lost between 5% and 15% of their body weight by 3 d.p.c. (Figure 4b), the average weight was 8% lower than baseline and the average maximum recorded weight loss was 10% (Supplementary Table S4). These effects were significantly (P
