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.

Engineering of novel hemagglutinin biosensors for rapid detection and drug screening of Influenza A H7N9 virus.

New pathogenic influenza virus strains are constantly emerging, posing a serious risk to both human health and economic growth. To effectively control the spread of this virus, there is an urgent need for early, rapid, sensitive, simple, and cost-effective detection technologies, as well as new and effective antiviral drugs. In this study, we have successfully achieved a significant milestone by successfully fusing the H7N9 influenza virus hemagglutinin (HA) protein with the nano-luciferase component, resulting in the development of a novel set of biosensors. This remarkable achievement marks the first instance of utilizing this biosensor technology for influenza antibody detection. Our biosensor technology also has the potential to facilitate the development of antiviral drugs targeting specific epitopes of the HA protein, providing a promising avenue for the treatment of H7N9 influenza virus infections. Furthermore, our biosensors have broad applications beyond H7N9 influenza virus detection, as they can be expanded for the detection of other pathogens and drug screening applications in the future. By providing a novel and effective solution to the detection and treatment of influenza viruses, our biosensors have the potential to revolutionize the field of infectious disease control.

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.

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.

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.

Exploring the determinants of influenza A/H7N9 control intervention efficacy in China: Disentangling the effect of the '1110' policy and poultry vaccination.

The influenza A virus of the H7N9 subtype (FLUAV H7N9) emerged in Eastern China provinces in 2013 causing illness in both poultry and humans. Most reported FLUAV H7N9 human cases were related to those associated with the live poultry market chain. From 2013 to 2017, there were five epidemic waves of human infections, and from the end of 2016, the number of human cases increased sharply. To control FLUAV H7N9 in the market chain, the so-called '1110' policy at live poultry markets and a national vaccination programme were implemented. The relative efficacy of these two measures on the number of poultry and human infections has not been quantified and compared. To explore their efficacy, a cross-sectional study was conducted in six provinces of China, and the vaccination and surveillance data of H7N9 were analysed. Our survey data showed that poultry vendors were not widely aware of and did not accept the '1110' policy. For subjective and objective factors, some measures of the '1110' policy were not implemented in live bird markets (LBMs). However, the national vaccination programme achieved good immune effects and sharply decreased poultry FLUAV H7N9 infections. The detection rates of FLUAV H7N9 in LBMs and farms gradually decreased since the vaccination programme was implemented. Our analysis also indicated that human infections were closely related to poultry virus carriage rates; therefore, controlling FLUAV H7N9 circulation in poultry was an effective measure to control FLUAV H7N9 infections in humans. Although LBMs play a significant role in human infections, the management measures may not be implemented efficiently; hence, we need to conduct more investigations before developing related policies.

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.

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.

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.

A Phase 1 Randomized Placebo-Controlled Study to Assess the Safety, Immunogenicity and Genetic Stability of a New Potential Pandemic H7N9 Live Attenuated Influenza Vaccine in Healthy Adults.

This study describes a double-blind randomized placebo-controlled phase I clinical trial in healthy adults of a new potential pandemic H7N9 live attenuated influenza vaccine (LAIV) based on the human influenza virus of Yangtze River Delta hemagglutinin lineage (ClinicalTrials.gov Identifier: NCT03739229). Two doses of H7N9 LAIV or placebo were administered intranasally to 30 and 10 subjects, respectively. The vaccine was well-tolerated and not associated with increased rates of adverse events or with any serious adverse events. Vaccine virus was detected in nasal swabs during the 6 days after vaccination or revaccination. A lower frequency of shedding was observed after the second vaccination. Twenty-five clinical viral isolates obtained after the first and second doses of vaccine retained the temperature-sensitive and cold-adapted phenotypic characteristics of LAIV. There was no confirmed transmission of the vaccine strain from vaccinees to placebo recipients. After the two H7N9 LAIV doses, an immune response was observed in 96.6% of subjects in at least one of the assays conducted.

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.

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.

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.

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.

Human antigen presenting cells stimulated with Salmonella delivered influenza antigens induce cytokine production and proliferation of human CD4+ T cells in vitro.

This study aimed to investigate whether the human antigen presenting cells (APCs) can process and present Salmonella expressing H7N9 hemagglutinin (Sal-HA), neuraminidase (Sal-NA) or M2 ectodomain (Sal-M2e) to T cells and subsequently activate CD4+ T cell responses in vitro. In this study, APCs generated from human peripheral blood mononuclear cells (PBMCs) were first treated with mitomycin-C, followed by stimulation with Sal-HA, Sal-M2e, Sal-NA or Salmonella alone for 24 h. Subsequently, stimulated APCs were coincubated with untreated PBMCs (1:10) of the same individual for 24 or 72 h and then analysed for cytokine induction and T cell proliferations by qRT-PCR assay and flow cytometry, respectively. Our results demonstrated that APCs stimulated with Sal-HA, Sal-M2e or Sal-NA induced significantly (p < .05) higher CD3+CD4+ T cell proliferations compared to the APCs treated with Salmonella alone. Our data further revealved that APCs treated with Sal-HA induced significantly (p < .05) higher CD3+CD4+ T cell responses compared to the APCs treated with either Sal-M2e or Sal-NA, which both induced almost comparable levels. The T cell proliferation responses were further measured by lymphocyte proliferation assay and the results showed that Sal-HA and Sal-M2e stimulated APCs induced significantly (p < .05) higher proliferations in T cells compared to the APCs stimulated with either Sal-NA or Salmonella alone. With respect to cytokine inductions, APCs treated with either Sal-HA or Sal-M2e induced significantly (p < .05) higher mRNA transcription levels of proinflammatory (IL-1ß, IL-6, IL-12 and IL-23), Th1 (IFN-γ), Th17 (IL-17 and IL-21) and Th2 (IL-10 and TGF-ß) cytokines in T cells compared to Sal-NA or Salmonella alone treated APCs. In conclusion, we show that Salmonella system can efficiently deliver vaccine antigens to APCs and is, thus, capable to elicit heterologous antigen-specific adaptive immunity.

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.

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.

Measurement of anti-influenza neuraminidase antibody induced by H7N9 influenza vaccine / 中华微生物学和免疫学杂志

Objective To develop an enzyme-linked lectin assay ( ELLA ) for measuring neuraminidase inhibition (NI) antibody titers in subjects vaccinated with H7N9 influenza vaccine. Methods Neuraminidase substrate, the dilution and incubation time of enzyme-labeled antibody, the concentration of influenza antigen for coating and pH value of the dilution buffer were optimized. Based on that, ELLA was established and used to detect anti-influenza neuraminidase antibody titers in serum samples of 34 subjects before and after vaccination with H7N9 influenza vaccine. Results The optimal neuraminidase substrate was fetuin at a coating concentration of 7. 5 μg/ml. The optimal dilution of enzyme-labeled antibody was 1 : 500. The virus strain of influenza H7N9 vaccine was used as antigen at a concentrations of 4. 5lgCCID50/ml in solution with a pH of 6. 5. Influenza-specific NI titers detected after immunization with vaccine were significantly higher than those before vaccination (P<0. 001). In the 34 subjects receiving H7N9 vaccine, the seroconversion rate of NI antibody was 47％ (≥40 in NI titer ), which was lower than that of HI antibody (P<0. 05). Conclusions An ELLA with natural substrate for measurement of anti-in-fluenza NI antibody was developed. It is simple and practical and might be used in the establishment of im-mune evaluation system for influenza vaccines and NI antibody.