Specific Monoclonal Antibodies Targeting Unique HA Epitopes Block H7N9 Influenza A Viral Replication.

The H7N9 subtype influenza A viruses pose a serious threat to public health, and there is still a lack of vaccines or drugs for humans against H7N9 influenza viruses. In this study, we screened two monoclonal antibodies (MAbs), 4H1E8 and 7H9A6, that specifically recognize the hemagglutinin (HA) protein of H7N9 influenza virus and display highly neutralizing activity against H7N9 virus. The epitopes recognized by two MAbs are nearly all conserved within all known H7 subtypes. Characteristic identification showed that two MAbs have high avidity for the HA protein but no hemagglutinin inhibition activity or antibody-dependent cellular cytotoxicity. Mechanistically, the 4H1E8 and 7H9A6 antibodies inhibit the pH-dependent conformational change of HA and block the HA-mediated membrane fusion. More importantly, 4H1E8 and 7H9A6 exhibit promising prophylactic and therapeutic effects against lethal challenge with H7N9 virus. Moreover, 4H1E8- and 7H9A6-treated mice displayed inhibition of pulmonary viral replication and reduced lung lesions after viral challenge. Together, these findings indicate that antibodies 4H1E8 and 7H9A6 recognize unique epitopes in the HA protein and possess the neutralizing activity and protective efficacy against the H7N9 influenza A viruses. IMPORTANCE In 2013, H7N9 influenza viruses appeared in China and other countries resulting in more than 1,500 individual infections or death. There are still limited studies on vaccines or drugs for humans against H7N9 influenza viruses. Alternative approaches against H7N9 virus infection need to be developed. Here, we identified two monoclonal antibodies (4H1E8 and 7H9A6) that possess neutralizing activity by blocking the pH-dependent HA-mediated membrane fusion. Additionally, the two monoclonal antibodies protect mice against the H7N9 virus challenge prophylactically or therapeutically. Therefore, our study demonstrates that 4H1E8 and 7H9A6 could be used for the prevention and treatment of the H7N9 influenza virus, and the conserved epitopes we identified may contribute to the development of a broad H7N9 vaccine and provide insights into unique antiviral approaches.

A Replication-Defective Influenza Virus Harboring H5 and H7 Hemagglutinins Provides Protection against H5N1 and H7N9 Infection in Mice.

The recent highly pathogenic avian influenza (HPAI) H5N1 and H7N9 viruses have caused hundreds of human infections with high mortality rates. Although H5N1 and H7N9 viruses have been limited mainly to avian species, there is high potential for these viruses to acquire human-to-human transmission and initiate a pandemic. A highly safe and effective vaccine is needed to protect against a potential H5N1 or H7N9 influenza pandemic. Here, we report the generation and evaluation of two reassortant influenza viruses, PR8-H5-H7NA and PR8-H7-H5NA These viruses contain six internal segments from A/Puerto Rico/8/1934 (PR8), the HA segment from either A/Alberta/01/2014 (H5N1) [AB14 (H5N1)] or A/British Columbia/01/2015 (H7N9) [BC15 (H7N9)], and a chimeric NA segment with either the BC15 (H7N9) HA gene or the AB14 (H5N1) HA gene flanked by the NA packaging signals of PR8. These viruses expressed both H5 and H7 HAs in infected cells, replicated to high titers when exogenous NA was added to the culture medium in vitro, and were replication defective and nonvirulent when administered intranasally in mice. Moreover, intranasal vaccination with PR8-H5-H7NA elicited robust immune responses to both H5 and H7 viruses, conferring complete protection against both AB14 (H5N1) and BC15 (H7N9) challenges in mice. Conversely, vaccination with PR8-H7-H5NA only elicited robust immune responses toward the H7 virus, which conferred complete protection against BC15 (H7N9) but not against AB14 (H5N1) in mice. Therefore, PR8-H5-H7NA has strong potential to serve as a vaccine candidate against both H5 and H7 subtypes of influenza viruses.IMPORTANCE Avian influenza H5N1 and H7N9 viruses infected humans with high mortality rates. A highly safe and effective vaccine is needed to protect against a potential pandemic. We generated and evaluated two reassortant influenza viruses, PR8-H5-H7NA and PR8-H7-H5NA, as vaccine candidates. Each virus contains one type of HA in segment 4 and the other subtype of HA in segment 6, thereby expressing both H5 and H7 subtypes of the HA molecule. The replication of viruses is dependent on the addition of exogenous NA in cell culture and is replication defective in vivo Vaccination of PR8-H5-H7NA virus confers protection to both H5N1 and H7N9 virus challenge; conversely, vaccination of PR8-H7-H5NA provides protection only to H7N9 virus challenge. Our data revealed that when engineering such a virus, the H5 or H7 HA in segment 6 affects the immunogenicity. PR8-H5-H7NA has strong potential to serve as a vaccine candidate against both H5 and H7 subtypes of influenza viruses.

In Silico Studies Reveal Peramivir and Zanamivir as an Optimal Drug Treatment Even If H7N9 Avian Type Influenza Virus Acquires Further Resistance.

An epidemic of avian type H7N9 influenza virus, which took place in China in 2013, was enhanced by a naturally occurring R294K mutation resistant against Oseltamivir at the catalytic site of the neuraminidase. To cope with such drug-resistant neuraminidase mutations, we applied the molecular docking technique to evaluate the fitness of the available drugs such as Oseltamivir, Zanamivir, Peramivir, Laninamivir, L-Arginine and Benserazide hydrochloride concerning the N9 enzyme with single (R294K, R119K, R372K), double (R119_294K, R119_372K, R294_372K) and triple (R119_294_372K) mutations in the pocket. We found that the drugs Peramivir and Zanamivir score best amongst the studied compounds, demonstrating their high binding potential towards the pockets with the considered mutations. Despite the fact that mutations changed the shape of the pocket and reduced the binding strength for all drugs, Peramivir was the only drug that formed interactions with the key residues at positions 119, 294 and 372 in the pocket of the triple N9 mutant, while Zanamivir demonstrated the lowest RMSD value (0.7 Å) with respect to the reference structure.

Emergence and Adaptation of a Novel Highly Pathogenic H7N9 Influenza Virus in Birds and Humans from a 2013 Human-Infecting Low-Pathogenic Ancestor.

Since its emergence in 2013, the H7N9 low-pathogenic avian influenza virus (LPAIV) has been circulating in domestic poultry in China, causing five waves of human infections. A novel H7N9 highly pathogenic avian influenza virus (HPAIV) variant possessing multiple basic amino acids at the cleavage site of the hemagglutinin (HA) protein was first reported in two cases of human infection in January 2017. More seriously, those novel H7N9 HPAIV variants have been transmitted and caused outbreaks on poultry farms in eight provinces in China. Herein, we demonstrate the presence of three different amino acid motifs at the cleavage sites of these HPAIV variants which were isolated from chickens and humans and likely evolved from the preexisting LPAIVs. Animal experiments showed that these novel H7N9 HPAIV variants are both highly pathogenic in chickens and lethal to mice. Notably, human-origin viruses were more pathogenic in mice than avian viruses, and the mutations in the PB2 gene associated with adaptation to mammals (E627K, A588V, and D701N) were identified by next-generation sequencing (NGS) and Sanger sequencing of the isolates from infected mice. No polymorphisms in the key amino acid substitutions of PB2 and HA in isolates from infected chicken lungs were detected by NGS. In sum, these results highlight the high degree of pathogenicity and the valid transmissibility of this new H7N9 variant in chickens and the quick adaptation of this new H7N9 variant to mammals, so the risk should be evaluated and more attention should be paid to this variant.IMPORTANCE Due to the recent increased numbers of zoonotic infections in poultry and persistent human infections in China, influenza A(H7N9) virus has remained a public health threat. Most of the influenza A(H7N9) viruses reported previously have been of low pathogenicity. Now, these novel H7N9 HPAIV variants have caused human infections in three provinces and outbreaks on poultry farms in eight provinces in China. We analyzed the molecular features and compared the relative characteristics of one H7N9 LPAIV and two H7N9 HPAIVs isolated from chickens and two human-origin H7N9 HPAIVs in chicken and mouse models. We found that all HPAIVs both are highly pathogenic and have valid transmissibility in chickens. Strikingly, the human-origin viruses were more highly pathogenic than the avian-origin viruses in mice, and dynamic mutations were confirmed by NGS and Sanger sequencing. Our findings offer important insight into the origin, adaptation, pathogenicity, and transmissibility of these viruses to both poultry and mammals.

Multiple amino acid substitutions involved in the adaption of three avian-origin H7N9 influenza viruses in mice.

BACKGROUND: Avian influenza A H7N9 virus has caused five outbreak waves of human infections in China since 2013 and posed a dual challenge to public health and poultry industry. The number of reported H7N9 virus human cases confirmed by laboratory has surpassed that of H5N1 virus. However, the mechanism for how H7N9 influenza virus overcomes host range barrier has not been clearly understood. METHODS: To generate mouse-adapted H7N9 influenza viruses, we passaged three avian-origin H7N9 viruses in mice by lung-to-lung passages independently. Then, the characteristics between the parental and mouse-adapted H7N9 viruses was compared in the following aspects, including virulence in mice, tropism of different tissues, replication in MDCK cells and molecular mutations. RESULTS: After ten passages in mice, MLD50 of the H7N9 viruses reduced >750-3,160,000 folds, and virus titers in MDCK cells increased 10-200 folds at 48 hours post-inoculation. Moreover, the mouse-adapted H7N9 viruses showed more expanded tissue tropism and more serious lung pathological lesions in mice. Further analysis of the amino acids changes revealed 10 amino acid substitutions located in PB2 (E627K), PB1 (W215R and D638G), PA (T97I), HA (H3 numbering: R220G, L226S, G279R and G493R) and NA (P3Q and R134I) proteins. Moreover, PB2 E627K substitution was shared by the three mouse-adapted viruses (two viruses belong to YRD lineage and one virus belongs to PRD lineage), and PA T97A substitution was shared by two mouse-adapted viruses (belong to YRD lineage). CONCLUSIONS: Our result indicated that the virulence in mice and virus titer in MDCK cells of H7N9 viruses significantly increased after adapted in mouse model. PB2 E627K and PA T97A substitutions are vital in mouse adaption and should be monitored during epidemiological study of H7N9 virus.

NS1 of H7N9 Influenza A Virus Induces NO-Mediated Cellular Senescence in Neuro2a Cells.

BACKGROUND/AIMS: The novel avian H7N9 influenza A virus has been detected in brain tissues and associated with central nervous system (CNS) symptoms in infected human and mice. Roles of its virulence factor, NS1 protein in influenza virus infected neuron has yet to be explored. METHODS: Nitric oxide (NO) release and inducible nitric oxide synthase (iNOS) expression in H7N9/NS1-expressed Neuro2a cells were detected by Griess test and western blotting. Cell proliferation rate of H7N9/NS1-expressing cells was recorded by Cell Counting Kit-8. Effects of H7N9/NS1 on cellular senescence were investigated by senescence-associated ß-galactosidase (SA-ß-gal) staining, immunofluorescent staining of phosphorylated heterochromatin protein 1γ (pHP1γ) and qPCR analysis of IL-6 and IL-8. RESULTS: H7N9/NS1 in Neuro2a cells and primary cultured mouse cortical neurons increased the expression of iNOS and boosted NO release. Neuro2a cells constitutively expressing NS1 displayed a reduced proliferative ability, enhanced SA-ß-gal staining, increased level of IL-6 and IL-8 and a typical punctuate structure of pHP1γ in nuclei. In addition, p38 MAPK was elevated in NS1-expressing Neuro2a cells. Reduced iNOS expression and subdued cellular senescence effect was found in p38 MAPK inhibitor-treated NS1-expressing Neuro2a cells. CONCLUSION: Our results suggest that H7N9/NS1 protein increases the iNOS expression and NO release in Neuro2a cells, which can induce cell growth arrest and cellular senescence.

Novel avian-origin human influenza A(H7N9) can be transmitted between ferrets via respiratory droplets.

The outbreak of human infections caused by novel avian-origin influenza A(H7N9) in China since March 2013 underscores the need to better understand the pathogenicity and transmissibility of these viruses in mammals. In a ferret model, the pathogenicity of influenza A(H7N9) was found to be less than that of an influenza A(H5N1) strain but comparable to that of 2009 pandemic influenza A(H1N1), based on the clinical signs, mortality, virus dissemination, and results of histopathologic analyses. Influenza A(H7N9) could replicate in the upper and lower respiratory tract, the heart, the liver, and the olfactory bulb. It is worth noting that influenza A(H7N9) exhibited a low level of transmission between ferrets via respiratory droplets. There were 4 mutations in the virus isolated from the contact ferret: D678Y in the gene encoding PB2, R157K in the gene encoding hemagglutinin (H3 numbering), I109T in the gene encoding nucleoprotein, and T10I in the gene encoding neuraminidase. These data emphasized that avian-origin influenza A(H7N9) can be transmitted between mammals, highlighting its potential for human-to-human transmissibility.

The R292K mutation that confers resistance to neuraminidase inhibitors leads to competitive fitness loss of A/Shanghai/1/2013 (H7N9) influenza virus in ferrets.

BACKGROUND: Neuraminidase (NA) inhibitors are the only licensed therapeutic option for human zoonotic H7N9 infections. An NA-R292K mutation that confers broad-spectrum resistance to NA inhibitors has been documented in H7N9 patients after treatment. METHODS: We evaluated the transmission potential of a human influenza A H7N9 isolate with a NA-R292K mutation in the ferret model followed by genotyping assay to monitor its competitive fitness in vivo. RESULTS: Plaque-purified A/Shanghai/1/2013 wild-type and NA-R292K viruses transmitted at comparable efficiency to direct or respiratory droplet contact ferrets. In ferrets inoculated with the plaque-purified A/Shanghai/1/2013 NA-R292K virus with dominant K292 (94%), the resistant K292 genotype was outgrown by the wild-type R292 genotype during the course of infection. Transmission of the resistant K292 genotype was detected in 3/4 direct contact and 3/4 respiratory droplet contact ferrets at early time points but was gradually replaced by the wild-type genotype. In the respiratory tissues of inoculated or infected ferrets, the wild-type R292 genotype dominated in the nasal turbinate, whereas the resistant K292 genotype was more frequently detected in the lungs. CONCLUSIONS: The NA inhibitor-resistant H7N9 virus with the NA-R292K mutation may transmit among ferrets but showed compromised fitness in vivo while in competition with the wild-type virus.

R229I substitution from oseltamivir induction in HA1 region significantly increased the fitness of a H7N9 virus bearing NA 292K.

The proportion of human isolates with reduced neuraminidase inhibitors (NAIs) susceptibility in highly pathogenic avian influenza (HPAI) H7N9 virus was high. These drug-resistant strains showed good replication capacity without serious loss of fitness. In the presence of oseltamivir, R229I substitution were found in HA1 region of the HPAI H7N9 virus before NA R292K appeared. HPAI H7N9 or H7N9/PR8 recombinant viruses were developed to study whether HA R229I could increase the fitness of the H7N9 virus bearing NA 292K. Replication efficiency was assessed in MDCK or A549 cells. Neuraminidase enzyme activity and receptor-binding ability were analyzed. Pathogenicity in C57 mice was evaluated. Antigenicity analysis was conducted through a two-way HI test, in which the antiserum was obtained from immunized ferrets. Transcriptomic analysis of MDCK infected with HPAI H7N9 24hpi was done. It turned out that HA R229I substitution from oseltamivir induction in HA1 region increased (1) replication ability in MDCK(P < 0.05) and A549(P < 0.05), (2) neuraminidase enzyme activity, (3) binding ability to both α2,3 and α2,6 receptor, (4) pathogenicity to mice(more weight loss; shorter mean survival day; viral titer in respiratory tract, P < 0.05; Pathological changes in pneumonia), (5) transcriptome response of MDCK, of the H7N9 virus bearing NA 292K. Besides, HA R229I substitution changed the antigenicity of H7N9/PR8 virus (>4-fold difference of HI titre). It indicated that through the fine-tuning of HA-NA balance, R229I increased the fitness and changed the antigenicity of H7N9 virus bearing NA 292K. Public health attention to this mechanism needs to be drawn.

The transmembrane replacement H7N9-VLP vaccine displays high levels of protection in mice.

The incidence of infections caused by the H7N9 subtype of the influenza virus has expanded rapidly in China in recent decades, generating massive economic loss and posing a significant threat to public health. In the absence of specialized antiviral treatments or long-term effective preventative vaccinations, it is critical to constantly enhance vaccines and create effective antiviral drugs to prevent the recurrence of pandemics. In the present study, a transmembrane-substituted (TM) virus-like particle (VLP)-based vaccine was created by replacing the transmembrane region of hemagglutinin (HA) protein with the transmembrane region of the H3 HA protein and then used to immunize BALB/c mice. Sera and T cells were collected from the immunized mice to evaluate the passive immune effects. Our results showed that naïve mice achieved 80-100% protection against homologous and heterologous H7N9 influenza strains after receiving passive serum immunization; the protective effect of the TM VLPs was more evident than that of the wild-type HA VLPs. In contrast, mice immunized with passive T cells achieved only 20 to 80% protection against homologous or heterologous strains. Our findings significantly contribute to understanding the control of the H7N9 virus and the development of a vaccine.

A flow-through chromatography purification process for Vero cell-derived influenza virus (H7N9).

Immunization is the most effective way to respond to an influenza epidemic. To produce Vero cell-derived influenza vaccines, a more efficient, stable and economical purification process is required. In this study, we purified the H7N9 influenza virus grown in Vero cells that were cultured in a serum-free medium by using a combination of anion exchange chromatography (AEC) and ligand-activated core chromatography (LCC), which avoids the virus capture step. After purification, 99.95 % host cell DNA (hcDNA) (final concentration: 28.69 pg/dose) and 98.87 % host cell protein (HCP) (final concentration: 28.28 ng/dose) were removed. The albumin content was 11.36 ng/dose. All these remnants met the current Chinese Pharmacopoeia and WHO requirements. The final virus recovery rate was 58.74 %, with the concentration of hemagglutinin recorded at 132.12 µg/mL. The flow-through chromatography purification process represents an alternative to the existing processes for cell-derived influenza viruses and might be suitable for the purification of other viruses as well.

H7N9 influenza virus-like particle based on BEVS protects chickens from lethal challenge with highly pathogenic H7N9 avian influenza virus.

H7N9 avian influenza virus poses a dual threat to both poultry industry and public health. Therefore, it is highly urgent to develop an effective vaccine to reduce its pandemic potential. Virus-like particles (VLP) represent an effective approach for pandemic vaccine development. In this study, a recombinant baculovirus co-expressing the HA, NA and M1 genes of the H7N9 virus was constructed for generation of H7N9 VLP. Single immunization of chickens with 15 µg of the VLP or the commercial whole virus inactivated vaccine stimulates high hemagglutination inhibition, virus neutralizing and HA-specific IgY antibodies. Moreover, the antiserum had a good cross-reactivity with H7N9 field strains isolated in different years. Within 14 days after a lethal challenge with highly pathogenic (HP) H7N9 virus, no clinical symptoms and death were observed in the vaccinated chickens, and no virus was recovered from the organs. Compared to the non-vaccinated chickens, H7N9 VLP significantly reduced the proportion of animals shedding virus. Only 30 % of the VLP-vaccinated birds shed virus, whereas virus shedding was detected in 50 % of the chickens immunized with the commercial vaccine. Moreover, both vaccines dramatically alleviated pulmonary lesions caused by HP H7N9 virus, with a greater degree observed for the VLP. Altogether, our results indicated that the H7N9 VLP vaccine candidate confers a complete clinical protection against a lethal challenge with HP H7N9 virus, significantly inhibits virus shedding and abolishes viral replication in chickens. The VLP generated in this study represents a promising alternative strategy for the development of novel H7N9 avian influenza vaccines for chickens.

The virulence modulator PA-X protein has minor effect on the pathogenicity of the highly pathogenic H7N9 avian influenza virus in mice.

PA-X is a novel discovered accessory protein encoded by the PA mRNA of the influenza A virus. Accumulated studies have demonstrated the crucial role of this protein in regulating the virulence of various subtypes of influenza virus, including H1N1, H5N1, H9N2, H1N2, H3N8 and H3N2 virus. However, the role of PA-X protein in regulating the virulence of the highly pathogenic avian H7N9 virus was unknown. In this study, we firstly generated two recombinant H7N9 viruses which have lower PA-X expression level than the parental H7N9 virus. We then systematically compared their difference in virus replication, polymerase activity, virulence and virus-induced host immune responses in mice. The results showed that the PA-X deficient viruses significantly increased viral replication in madin darby canine kidney cells and slightly increased viral replication in mouse lung. In addition, loss of PA-X expression significantly increased viral polymerase activity and alleviated the host-shutoff activity mediated by the parental PA protein. However, in contrast with the usual function of PA-X in regulating the virulence in different subtype influenza virus, no obvious effect on viral virulence in mice was observed by H7N9 PA-X protein. Furthermore, among the 12 kinds of cytokines and 2 kinds of complement derived components that we tested, the PA-X deficiency viruses only induced significantly higher expression levels of MX1 than the parental virus. Altogether, these results showed that PA-X has little effect on viral virulence and viral induced innate immune response of the H7N9 subtype virus. Our study adds further information for the growing understanding of the complexity of PA-X in regulating viral virulence and host innate immune response of different influenza virus.

Computational predicting the human infectivity of H7N9 influenza viruses isolated from avian hosts.

The genome composition of a given avian influenza virus is the primary determinant of its potential for cross-species transmission from birds to humans. Here, we introduce a viral genome-based computational tool that can be used to evaluate the human infectivity of avian isolates of influenza A H7N9 viruses, which can enable prediction of the potential risk of these isolates infecting humans. This tool, which is based on a novel class weight-biased logistic regression (CWBLR) algorithm, uses the sequences of the eight genome segments of an H7N9 strain as the input and gives the probability of this strain infecting humans (reflecting its human infectivity). We examined the replication efficiency and the pathogenicity of several H7N9 avian isolates that were predicted to have very low or high human infectivity by the CWBLR model in cell culture and in mice, and found that the strains with high predicted human infectivity replicated more efficiently in mammalian cells and were more infective in mice than those that were predicted to have low human infectivity. These results demonstrate that our CWBLR model can serve as a powerful tool for predicting the human infectivity and cross-species transmission risks of H7N9 avian strains.

Protective efficacy of anti-neuraminidase monoclonal antibodies against H7N9 influenza virus infection.

The H7N9 influenza virus has been circulating in China for more than six years. The neuraminidase (NA) has gained great concern for the development of antiviral drugs, therapeutic antibodies, and new vaccines. In this study, we screened seven mouse monoclonal antibodies (mAbs) and compared their protective effects against H7N9 influenza virus. The epitope mapping from escape mutants showed that all the seven mAbs could bind to the head region of the N9 NA close to the enzyme activity sites, and four key sites of N9 NA were reported for the first time. The mAbs D3 and 7H2 could simultaneously inhibit the cleavage of the sialic acid of fetuin protein with large molecular weight and NA-XTD with small molecule weight in the NA inhibition experiment, prevent the formation of virus plaque at a low concentration, and effectively protect the mice from the challenge of the lethal dose of H7N9 virus.

Development and characterization of standard reagents for cell-based prepandemic influenza vaccine products.

Outbreaks of infection by novel avian influenza virus strains in humans cause public health issues worldwide, and the development of vaccines against such novel strains is the most effective method for the prevention of these virus outbreaks. All types of vaccines must be tested for potency before use; thus, quantitative potency assays are needed for influenza vaccines. The single radial immunodiffusion (SRID) assay is considered the gold standard for quantification of influenza virus antigens, and the SRID reference reagents are essential for the determination of vaccine potency. However, it remains debatable whether reference reagents derived from egg-based vaccine platforms can be used to precisely quantify non-egg-derived vaccines; thus, influenza vaccine production using cell-based platforms has attracted increasing attention. To evaluate the utility of reference reagents derived from a cell-based influenza vaccine platform, we prepared cell-based reference reagents from MDCK cell-grown viruses and compared them with egg-derived reference reagents. A primary liquid standard (PLS) was purified from cell-derived candidate influenza vaccine viruses, and hemagglutinin (HA) antigen content was determined by a densitometric method. The produced PLS could be stored at 4°C for more than 10 months. We also established a simple HA protein purification method for goat antiserum preparation, and the performance of the resulting antiserum was compared to that of standard reagents obtained using different production platforms. The results of this study indicate that these reference reagents can be used for both cell-based and egg-based production platforms and that the differences between these two types of platforms are negligible.

Evaluation of the immune response of a H7N9 candidate vaccine virus derived from the fifth wave A/Guangdong/17SF003/2016.

Highly pathogenic influenza H7N9 viruses that emerged in the fifth wave of H7N9 outbreak pose a risk to human health. The World Health Organization has updated the candidate vaccine viruses for H7N9 viruses recently. In this study, we evaluated the immune response to an updated H7N9 candidate vaccine virus, which derived from the highly pathogenic A/Guangdong/17SF003/2016 (GD/16) in mice and rhesus macaques. GD/16 vaccination elicited robust neutralizing, virus-specific immunoglobulin G antibodies and effective protection, but poor hemagglutination inhibition antibody titers. Furthermore, mouse and rhesus macaque serum raised against the previous H7N9 CVV A/Anhui/1/2013 (AH/13) were tested for its cross-reactivity to GD/16 virus. We found that although AH/13-immune serum has poor hemagglutination inhibition reactivity against GD/16 virus, AH/13 elicit efficient cross-neutralizing antibodies and in vivo protection against GD/16. Further studies showed that the hemagglutinin of GD/16 has strong receptor binding avidity, which might be associated with the decreased hemagglutination inhibition assay sensitivity. This study underscores the point that receptor binding avidity should be taken into account when performing quantitative interpretation of hemagglutination inhibition data. A combination of multiple serological assays is required for accurate vaccine evaluation and antigenic analysis of influenza viruses.

The optimized fusion protein HA1-2-FliCΔD2D3 promotes mixed Th1/Th2 immune responses to influenza H7N9 with low induction of systemic proinflammatory cytokines in mice.

H7N9 influenza virus has an unusually high fatality rate of approximately 40%, and a safe and effective vaccine against this subtype is urgently needed. Flagellin, a Toll-like receptor (TLR) 5 agonist, has been deemed as a potent adjuvant candidate. However, its high antigenicity and potential for causing inflammatory injury might restrict its clinical application. Previously, we demonstrated that a fusion protein, HA1-2-FliC, comprising the hemagglutinin globular head protein (HA1-2) of H7N9 influenza virus and the full-length Salmonella typhimurium flagellin protein (FliC), had high efficiency against H7N9 in mouse and chicken models. Here, we constructed an improved fusion protein, HA1-2-FliCΔD2D3, with HA1-2 fused to the FliCΔD2D3 (lacking the hypervariable-region domains D2 and D3 of FliC). HA1-2-FliCΔD2D3 exhibited efficient immunoreactivity and TLR5 agonist efficacy, and promoted innate immune-response activation in mouse macrophages, peripheral blood mononuclear cells, and splenocytes, based on cytokine- and chemokine-expression profiles. Mice immunized with HA1-2-FliCΔD2D3 showed significantly lower systemic inflammatory responses (compared with HA1-2-FliC) and highly reduced flagellin-specific antibody production, without affecting HA1-2-specific antibody production and cellular immune responses. Enhanced IFN-γ/IL-4 generation suggested that HA1-2-FliCΔD2D3 maintained balanced Th1/Th2 immune responses. Furthermore, virus challenge was performed in a chicken model. The results showed that chickens receiving FliCΔD2D3 adjuvant vaccine induced high levels of serum neutralizing antibodies, and exhibited a significant reduction of viral loads in throat and cloaca compared to chickens receiving only HA1-2. In conclusion, we constructed the H7N9 influenza subunit vaccine candidate HA1-2-FliCΔD2D3, with reduced immunogenicity against FliC and lower adverse events. The improved adjuvant FliCΔD2D3 can potentially help in developing safe and effective universal protein-based influenza vaccines for humans.

Structural Basis of Protection against H7N9 Influenza Virus by Human Anti-N9 Neuraminidase Antibodies.

Influenza virus neuraminidase (NA) is a major target for small-molecule antiviral drugs. Antibodies targeting the NA surface antigen could also inhibit virus entry and egress to provide host protection. However, our understanding of the nature and range of target epitopes is limited because of a lack of human antibody structures with influenza neuraminidase. Here, we describe crystal and cryogenic electron microscopy (cryo-EM) structures of NAs from human-infecting avian H7N9 viruses in complex with five human anti-N9 antibodies, systematically defining several antigenic sites and antibody epitope footprints. These antibodies either fully or partially block the NA active site or bind to epitopes distant from the active site while still showing neuraminidase inhibition. The inhibition of antibodies to NAs was further analyzed by glycan array and solution-based NA activity assays. Together, these structural studies provide insights into protection by anti-NA antibodies and templates for the development of NA-based influenza virus vaccines and therapeutics.

Vaccination of poultry successfully eliminated human infection with H7N9 virus in China.

The H7N9 viruses that emerged in China in 2013 were nonpathogenic in chickens but mutated to a highly pathogenic form in early 2017 and caused severe disease outbreaks in chickens. The H7N9 influenza viruses have caused five waves of human infection, with almost half of the total number of human cases (766 of 1,567) being reported in the fifth wave, raising concerns that even more human infections could occur in the sixth wave. In September 2017, an H5/H7 bivalent inactivated vaccine for chickens was introduced, and the H7N9 virus isolation rate in poultry dropped by 93.3% after vaccination. More importantly, only three H7N9 human cases were reported between October 1, 2017 and September 30, 2018, indicating that vaccination of poultry successfully eliminated human infection with H7N9 virus. These facts emphasize that active control of animal disease is extremely important for zoonosis control and human health protection.