Inhibitory effects of aprotinin on influenza A and B viruses in vitro and in vivo.

Influenza viruses cause significant morbidity and mortality worldwide. Long-term or frequent use of approved anti-influenza agents has resulted in drug-resistant strains, thereby necessitating the discovery of new drugs. In this study, we found aprotinin, a serine protease inhibitor, as an anti-influenza candidate through screening of compound libraries. Aprotinin has been previously reported to show inhibitory effects on a few influenza A virus (IAV) subtypes (e.g., seasonal H1N1 and H3N2). However, because there were no reports of its inhibitory effects on the other types of influenza viruses, we investigated the inhibitory effects of aprotinin in vitro on a wide range of influenza viruses, including avian and oseltamivir-resistant influenza virus strains. Our cell-based assay showed that aprotinin had inhibitory effects on seasonal human IAVs (H1N1 and H3N2 subtypes), avian IAVs (H5N2, H6N5, and H9N2 subtypes), an oseltamivir-resistant IAV, and a currently circulating influenza B virus. We have also confirmed its activity in mice infected with a lethal dose of influenza virus, showing a significant increase in survival rate. Our findings suggest that aprotinin has the capacity to inhibit a wide range of influenza virus subtypes and should be considered for development as a therapeutic agent against influenza.

Development of American-Lineage Influenza H5N2 Reassortant Vaccine Viruses for Pandemic Preparedness.

Novel low-pathogenic avian influenza (LPAI) H5N2 viruses hit poultry farms in Taiwan in 2003, and evolved into highly pathogenic avian influenza (HPAI) viruses in 2010. These viruses are reassortant viruses containing HA and NA genes from American-lineage H5N2 and six internal genes from local H6N1 viruses. According to a serological survey, the Taiwan H5N2 viruses can cause asymptomatic infections in poultry workers. Therefore, a development of influenza H5N2 vaccines is desirable for pandemic preparation. In this study, we employed reverse genetics to generate a vaccine virus having HA and NA genes from A/Chicken/CY/A2628/2012 (E7, LPAI) and six internal genes from a Vero cell-adapted high-growth H5N1 vaccine virus (Vero-15). The reassortant H5N2 vaccine virus, E7-V15, presented high-growth efficiency in Vero cells (512 HAU, 107.6 TCID50/mL), and passed all tests for qualification of candidate vaccine viruses. In ferret immunization, two doses of inactivated whole virus antigens (3 µg of HA protein) adjuvanted with alum could induce robust antibody response (HI titre 113.14). In conclusion, we have established reverse genetics to generate a qualified reassortant H5N2 vaccine virus for further development.

A two dose immunization with an inactivated reassortant H5N2 virus protects chickens against lethal challenge with homologous 2.3.2.1 clade and heterologous 2.2 clade highly pathogenic avian influenza H5N1 viruses.

The present study was aimed at generating a reassortant vaccine candidate virus with clade 2.3.2.1 Hemagglutinin (HA) and its evaluation in a challenge study for protection against homologous (2.3.2.1 clade) and heterologous (2.2 clade) highly pathogenic avian influenza (HPAI) H5N1 viruses. Plasmid-based reverse genetics technique was used to rescue a 5 + 3 reassortant H5N2 strain containing the modified HA of H5N1 (clade 2.3.2.1), the Neuraminidase (NA) of H9N2, the Matrix (M) of H5N1 and the internal genes of A/WSN/33 H1N1. In addition, another 6 + 2 reassortant virus containing modified HA from H5N1 (clade 2.3.2.1), the NA from H9N2 and the internal genes of A/WSN/33 H1N1 was also rescued. The 5 + 3 reassortant H5N2 virus could grow to a higher titer in both MDCK cells and chicken eggs compared to the 6 + 2 reassortant H5N2 virus. The vaccine containing the inactivated 5 + 3 reassortant H5N2 virus was used in a two-dose immunization regime which protected specific pathogen free (SPF) chickens against two repeated challenges with homologous 2.3.2.1 clade and heterologous 2.2 clade HPAI H5N1 viruses. The 5 + 3 reassortant H5N2 virus based on clade 2.3.2.1 generated in this study can be effective in protecting chickens in the case of an outbreak caused by antigenically different clade 2.2 HPAI H5N1 viruses and opens the way to explore its applicability as potential vaccine candidate especially in the Asian countries reporting these clades frequently. The study also indicates that sequential immunization can broaden protection level against antigenically diverse strains of H5N1 viruses.

Influenza viruses production: Evaluation of a novel avian cell line DuckCelt®-T17.

The influenza vaccine manufacturing industry is looking for production cell lines that are easily scalable, highly permissive to multiple viruses, and more effective in term of viral productivity. One critical characteristic of such cell lines is their ability to grow in suspension, in serum free conditions and at high cell densities. Influenza virus causing severe epidemics both in human and animals is an important threat to world healthcare. The repetitive apparition of influenza pandemic outbreaks in the last 20years explains that manufacturing sector is still looking for more effective production processes to replace/supplement embryonated egg-based process. Cell-based production strategy, with a focus on avian cell lines, is one of the promising solutions. Three avian cell lines, namely duck EB66®cells (Valneva), duck AGE.CR® cells (Probiogen) and quail QOR/2E11 cells (Baxter), are now competing with traditional mammalian cell platforms (Vero and MDCK cells) used for influenza vaccine productions and are currently at advance stage of commercial development for the manufacture of influenza vaccines. The DuckCelt®-T17 cell line presented in this work is a novel avian cell line developed by Transgene. This cell line was generated from primary embryo duck cells with the constitutive expression of the duck telomerase reverse transcriptase (dTERT). The DuckCelt®-T17 cells were able to grow in batch suspension cultures and serum-free conditions up to 6.5×106cell/ml and were easily scaled from 10ml up to 3l bioreactor. In the present study, DuckCelt®-T17 cell line was tested for its abilities to produce various human, avian and porcine influenza strains. Most of the viral strains were produced at significant infectious titers (>5.8 log TCID50/ml) with optimization of the infection conditions. Human strains H1N1 and H3N2, as well as all the avian strains tested (H5N2, H7N1, H3N8, H11N9, H12N5) were the most efficiently produced with highest titre reached of 9.05 log TCID50/ml for A/Panama/2007/99 influenza H3N2. Porcine strains were also greatly rescued with titres from 4 to 7 log TCID50/ml depending of the subtypes. Interestingly, viral kinetics showed maximal titers reached at 24h post-infection for most of the strains, allowing early harvest time (Time Of Harvest: TOH). The B strains present specific production kinetics with a delay of 24h before reaching the maximal viral particle release. Process optimization on H1N1 2009 human pandemic strain allowed identifying best operating conditions for production (MOI, trypsin concentration, cell density at infection) allowing improving the production level by 2 log. Our results suggest that the DuckCelt®-T17 cell line is a very promising platform for industrial production of influenza viruses and particularly for avian viral strains.

Vaccine Efficacy of Inactivated, Chimeric Hemagglutinin H9/H5N2 Avian Influenza Virus and Its Suitability for the Marker Vaccine Strategy.

In order to produce a dually effective vaccine against H9 and H5 avian influenza viruses that aligns with the DIVA (differentiating infected from vaccinated animals) strategy, we generated a chimeric H9/H5N2 recombinant vaccine that expressed the whole HA1 region of A/CK/Korea/04163/04 (H9N2) and the HA2 region of recent highly pathogenic avian influenza (HPAI) A/MD/Korea/W452/14 (H5N8) viruses. The chimeric H9/H5N2 virus showed in vitro and in vivo growth properties and virulence that were similar to those of the low-pathogenic avian influenza (LPAI) H9 virus. An inactivated vaccine based on this chimeric virus induced serum neutralizing (SN) antibodies against both H9 and H5 viruses but induced cross-reactive hemagglutination inhibition (HI) antibody only against H9 viruses. Thus, this suggests its compatibility for use in the DIVA strategy against H5 strains. Furthermore, the chimeric H9/H5N2 recombinant vaccine protected immunized chickens against lethal challenge by HPAI H5N8 viruses and significantly attenuated virus shedding after infection by both H9N2 and HPAI H5N8 viruses. In mice, serological analyses confirmed that HA1- and HA2 stalk-specific antibody responses were induced by vaccination and that the DIVA principle could be employed through the use of an HI assay against H5 viruses. Furthermore, each HA1- and HA2 stalk-specific antibody response was sufficient to inhibit viral replication and protect the chimeric virus-immunized mice from lethal challenge with both mouse-adapted H9N2 and wild-type HPAI H5N1 viruses, although differences in vaccine efficacy against a homologous H9 virus (HA1 head domain immune-mediated protection) and a heterosubtypic H5 virus (HA2 stalk domain immune-mediated protection) were observed. Taken together, these results demonstrate that the novel chimeric H9/H5N2 recombinant virus is a low-pathogenic virus, and this chimeric vaccine is suitable for a DIVA vaccine with broad-spectrum neutralizing antibody against H5 avian influenza viruses.IMPORTANCE Current influenza virus killed vaccines predominantly induce antihemagglutinin (anti-HA) antibodies that are commonly strain specific in that the antibodies have potent neutralizing activity against homologous strains but do not cross-react with HAs of other influenza virus subtypes. In contrast, the HA2 stalk domain is relatively well conserved among subtypes, and recently, broadly neutralizing antibodies against this domain have been isolated. Therefore, in light of the need for a vaccine strain that applies the DIVA strategy utilizing an HI assay and induces broad cross-protection against H5N1 and H9N2 viruses, we generated a novel chimeric H9/H5N1 virus that expresses the entire HA1 portion from the H9N2 virus and the HA2 region of the heterosubtypic H5N8 virus. The chimeric H9/H5N2 recombinant vaccine protected immunized hosts against lethal challenge with H9N2 and HPAI H5N1 viruses with significantly attenuated virus shedding in immunized hosts. Therefore, this chimeric vaccine is suitable as a DIVA vaccine against H5 avian influenza viruses.

Adaptive mutations in PB2 gene contribute to the high virulence of a natural reassortant H5N2 avian influenza virus in mice.

The highly pathogenic A/chicken/Hebei/1102/2010 (HB10) H5N2 virus is a natural reassortant derived from circulating H5N1 and endemic H9N2 avian influenza viruses (AIV). To evaluate the potential of its interspecies transmission, we previously serially passaged the non-virulent HB10 virus in the mouse lung and obtained a high virulence variant (HB10-MA). Genomic sequencing revealed five mutations (HA-S227N, PB2-Q591K, PB2-D701N, PA-I554V and NP-R351K) that distinguished HB10-MA virus from its parental HB10 virus. In this study, we further investigated the molecular basis for the enhanced virulence of HB10-MA in mice. By generating a series of reassortants between the two viruses and evaluating their virulence in mice, we found that both PB2 and PA genes contribute to the high virulence of HB10-MA in mice, whereas PB2 gene carrying the 591K and/or 701N had a dominant function. In addition, the two amino acids showed a cumulative effect on the virulence, virus replication, and polymerase activity of HB10 or HB10-MA. Therefore, our results collectively emphasized the crucial role of PB2 gene, particularly the paired mutations of Q591K and D701N in the host adaptation of the novel reassortant H5N2 AIV in mammals, which may provide helpful insights into the pathogenic potential of emerging AIV in human beings.

Serial passage in ducks of a low-pathogenic avian influenza virus isolated from a chicken reveals a high mutation rate in the hemagglutinin that is likely due to selection in the host.

A comparative study of the ability of three low-pathogenic avian influenza virus (LPAIV) isolates to be transmitted from duck to duck was performed. Pekin ducks were inoculated with two LPAIV isolates from chickens (A/Ck/PA/13609/93 [H5N2], H5N2-Ck; A/Ck/TX/167280-4/02 [H5N3], H5N3-Ck) and one isolate from a wild bird (A/Mute Swan/ MI/451072/06 [H5N1], H5N1-WB). During the establishment of the passage model, only two viruses (H5N1, H5N2) were able to be transmitted from duck to duck. Transmission of these isolates was dependent on the inoculation dose and route of infection. Analysis of swab samples taken from ducks revealed that the wild-bird isolate, H5N1-WB, was primarily shed via the cloacal route. The chicken isolate, H5N2-Ck, was isolated from cloacal as well as oro-pharyngeal swabs. Analysis of the amino acid sequences of the viral surface glycoproteins showed that the hemagglutinin (HA) of the H5N2-Ck isolate was under a stronger evolutionary pressure than the HA of the H5N1-WB isolate, as indicated by the presence of a larger number of amino acid changes observed during passage. The neuraminidase (NA) of both viruses showed either no (in the case of H5N1-WB) or very few amino acid changes.

[The use of apoptosis inducers in the therapy of experimental influenza infection and preventing of chronic post-influenza lung damage].

Influenza is a respiratory infection widely spread around the world. Influenza complications are various in nature and in most cases involve the excessive proliferation of cells in respiratory tract as a factor of pathogenesis. In the present work the efficacy of the use of apoptosis inducer 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtalenecarboxylic acid (AHPN) for prophylaxis of chronic damage on the stage of post- influenza pneumonia has been studied. Mice were infected with influenza virus A/mallard/Pennsylvania/10218/84(H5N2) with further study of the level of influenza virus reproduction in the lungs, specific mortality of animals and morphology of the foci of post-influenza pneumonia on the 15th day post inoculation. AHPN was shown to decrease the infectious activity of the virus in the lungs by 1.2-1.5 log10 EID50/0.2 mL depending on the dose as compared to the control group, in a weak decrease in mortality of animals (protection index was 12.5-37.5%). The application of AHPN restricted both the proliferative and infiltrative component in chronic post-influenza lesions. It demonstrated the most pronounced effect on the lung morphology when applied on days 4 to 7 post inoculation, i. e. in the period of maximal activation of inflammatory tissue infiltration and regeneration of bronchiolar epithelium. In conclusion, the use of apoptosis inducers can partially prevent the development of chronic post-influenza lesions with proliferative component.

Evaluation of the U.S. Department of Agriculture's egg pasteurization processes on the inactivation of high-pathogenicity avian influenza virus and velogenic Newcastle disease virus in processed egg products.

Globally, 230,662 metric tons of liquid egg products are marketed each year. The presence of highly pathogenic avian influenza (HPAI) or Newcastle disease in an exporting country can legitimately inhibit trade in eggs and processed egg products; development and validation of pasteurization parameters are essential for safe trade to continue. The HPAI virus (HPAIV) A/chicken/Pennsylvania/1370/1983 (H5N2) and velogenic Newcastle disease virus (vNDV) AMPV-1/chicken/California/S01212676/2002 were inoculated into five egg products and heat treated at various times and temperatures to determine thermal inactivation rates to effect a 5-log viral reduction. For HPAIV and vNDV, the pasteurization processes for fortified, sugared, plain, and salted egg yolk, and homogenized whole egg (HPAIV only) products resulted in >5-log reductions in virus at the lower temperature-longer times of U.S. Department of Agriculture (USDA)-approved Salmonella pasteurization processes. In addition, a >5-log reduction of HPAIV was also demonstrated for the five products at the higher temperatures-shorter times of USDA-approved pasteurization processes, whereas the vNDV virus was adequately inactivated in only fortified and plain egg yolk products. For the salted and sugared egg yolk products, an additional 0.65 and 1.6 min of treatment, respectively, at 63.3 °C was necessary to inactivate 5 log of vNDV. Egg substitute with fat does not have standard USDA pasteurization criteria, but the D59-value was 0.75 min, adequate to inactivate 5 log of vNDV in <4 min.

Activation of type I and III interferon signalling pathways occurs in lung epithelial cells infected with low pathogenic avian influenza viruses.

The host response to the low pathogenic avian influenza (LPAI) H5N2, H5N3 and H9N2 viruses were examined in A549, MDCK, and CEF cells using a systems-based approach. The H5N2 and H5N3 viruses replicated efficiently in A549 and MDCK cells, while the H9N2 virus replicated least efficiently in these cell types. However, all LPAI viruses exhibited similar and higher replication efficiencies in CEF cells. A comparison of the host responses of these viruses and the H1N1/WSN virus and low passage pH1N1 clinical isolates was performed in A549 cells. The H9N2 and H5N2 virus subtypes exhibited a robust induction of Type I and Type III interferon (IFN) expression, sustained STAT1 activation from between 3 and 6 hpi, which correlated with large increases in IFN-stimulated gene (ISG) expression by 10 hpi. In contrast, cells infected with the pH1N1 or H1N1/WSN virus showed only small increases in Type III IFN signalling, low levels of ISG expression, and down-regulated expression of the IFN type I receptor. JNK activation and increased expression of the pro-apoptotic XAF1 protein was observed in A549 cells infected with all viruses except the H1N1/WSN virus, while MAPK p38 activation was only observed in cells infected with the pH1N1 and the H5 virus subtypes. No IFN expression and low ISG expression levels were generally observed in CEF cells infected with either AIV, while increased IFN and ISG expression was observed in response to the H1N1/WSN infection. These data suggest differences in the replication characteristics and antivirus signalling responses both among the different LPAI viruses, and between these viruses and the H1N1 viruses examined. These virus-specific differences in host cell signalling highlight the importance of examining the host response to avian influenza viruses that have not been extensively adapted to mammalian tissue culture.

[Immunogenicity of influenza virus H5N2 vaccine strain samples produced by roller cultivation in media with plant derived components].

AIM: Study in CBA line mice of immunogenicity of cold adapted reassortant influenza virus H5N2 vaccine strain samples produced in rollers in MDCK and Vero cell cultures by using plant derived components. MATERIALS AND METHODS: Antibody levels in blood sera and nasal swabs, lungs and small intestine of experimental vaccine strain sample immunized mice were evaluated by using HI reaction in accordance with WHO recommendations. RESULT: Reassortant vaccine strain A/17/duck/Potsdam/86/92 (H5N2) produced in MDCK and Vero cells by using plant derived components (rice and soy flour hydrolyzate and plant protease based nutrient medium) after intranasal immunization of mice induced local and humoral antibodies, and the latter not only against homologous virus, but also against highly pathogenic avian influenza virus strains A/ Chicken/Suzdalka/Nov-11/2005 and A/Chicken/Kurgan/05/2005. CONCLUSION: Immunogenicity studies of reassortant influenza virus A/17/duck/Potsdam/86/92 (H5N2) vaccine strain samples cultivated in MDCK and Vero cells by using media with plant derived components in mice show high levels of humoral and secretory immunity.

[Use of plant-origin components in roller cultivation of vaccine reassortant influenza virus strain H5N2].

AIM: To study the optimal conditions for roller cultivation of cold-adapted reassortant vaccine strain of influenza virus A/17/Duck/ Potsdam/86/92 (H5N2) in MDCK and Vero cell cultures grown on nutrient medium based on soy and rice flour hydrolysates obtained using trypsin and bromeline. MATERIALS AND METHODS: Vaccine strain was cultivated on MDCK and Vero cells in rollers in the presence of plant proteases. Obtained culture samples of vaccine strains were lyophilized and their infectivity was assessed. RESULTS: Cultivation of vaccine strain on MDCK and Vero cells grown in experimental media containing reduced quantity (2 and 3% respectively) of fetal calf serum ("Gibco", USA) resulted in high titers of the virus in the presence of plant proteases (4 mcg/ml of papain and 20 mcg/ml bromeline). CONCLUSION: Use of plant enzymes and nutrient media based on enzymic plant hydrolysates, including those obtained with bromeline, for cultivation of vaccine strain on MDCK and Vero cell cultures in rollers could make the manufacturing process of live influenza vaccines safer and more cost effective.

Thermal inactivation of H5N2 high-pathogenicity avian influenza virus in dried egg white with 7.5% moisture.

High-pathogenicity avian influenza viruses (HPAIV) cause severe systemic disease with high mortality in chickens. Isolation of HPAIV from the internal contents of chicken eggs has been reported, and this is cause for concern because HPAIV can be spread by movement of poultry products during marketing and trade activity. This study presents thermal inactivation data for the HPAIV strain A/chicken/PA/1370/83 (H5N2) (PA/83) in dried egg white with a moisture content (7.5%) similar to that found in commercially available spray-dried egg white products. The 95% upper confidence limits for D-values calculated from linear regression of the survival curves at 54.4, 60.0, 65.5, and 71.1 degrees C were 475.4, 192.2, 141.0, and 50.1 min, respectively. The line equation y = [0.05494 x degrees C] + 5.5693 (root mean square error = 0.0711) was obtained by linear regression of experimental D-values versus temperature. Conservative predictions based on the thermal inactivation data suggest that standard industry pasteurization protocols would be very effective for HPAIV inactivation in dried egg white. For example, these calculations predict that a 7-log reduction would take only 2.6 days at 54.4 degrees C.

Immunization with influenza A NP-expressing vaccinia virus recombinant protects mice against experimental infection with human and avian influenza viruses.

Two-fold immunization of Balb/c mice with a vaccinia virus recombinant expressing the NP protein of influenza A/PR8/34 (H1N1) virus under the control of a strong synthetic promoter induced specific antibodies and protected animals against low-dose challenge by mouse-adapted heterosubtypic variants of human A/Aichi2/68 (H3N2) and avian A/Mallard/Pennsylvania/10218/84 (H5N2) influenza virus strains. The surviving immunized animals had lower anti-hemagglutinin antibody titers compared to non-immunized mice. There was no difference in viral titers in lungs of immunized and non-immunized animals that succumbed to the infection. In order to try to increase immune system presentation of NP-protein-derived peptides, and thereby increase their immunogenicity, we constructed another vaccinia-based NP-expressing recombinant containing a rapid proteolysis signal covalently bound to the NP protein. This sequence, derived from the mouse ornithine decarboxylase gene has been shown to increase degradation of various proteins. However, we found that when used as part of a recombinant NP, this signal neither increased its proteolytic degradation, nor was it more efficient in the induction of a protective response against influenza infection.