SENASICA informa que se aplicaron 80 millones de vacunas en aves contra AH7N3

(SAGARPA 15/8/12)
Para mantener seguro el abasto, el SENASICA ha otorgado a 385 granjas libres del virus seis mil 865 certificados zoosanitarios de movilización para huevo fértil, huevo industrial, huevo para plato, harinas, yema líquida y aves vivas, entre otros derivados de ave.
El Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria (SENASICA) informó que se han aplicado 80 millones de dosis de vacuna contra la Influenza Aviar AH7N3 en la región de Los Altos de Jalisco, con lo que se cumple la primera etapa de vacunación prevista en las granjas avícolas.
A siete semanas de haber iniciado el Dispositivo Nacional de Emergencia de Sanidad Animal (DINESA) para controlar y erradicar la enfermedad se han revisado 426 granjas, ubicadas en 45 municipios de la región, de las que 385 están libres del virus; el número de positivas se mantiene en 41, igual que lo reportado desde hace dos semanas.
El Director en Jefe del SENASICA, Enrique Sánchez Cruz, manifestó que con base en el Programa Nacional de Vigilancia Epidemiológica se ha contenido el virus en la región referida.
Con la finalidad de mantener el abasto a la población, detalló, se han otorgado seis mil 865 certificados zoosanitarios de movilización para huevo fértil, huevo industrial, huevo para plato, harinas, yema líquida y aves vivas, entre otros derivados.
“Las granjas libres del virus han tenido facilidades para la movilización de sus productos hacia los principales mercados de abasto”, subrayó.
Destacó que se mantiene la cuarentena en la zona de riesgo y el aislamiento precautorio, así como el control de la movilización para evitar la diseminación del virus.
Una medida de control para evitar la diseminación de la enfermedad es el sacrificio sanitario, que hasta la fecha se mantiene en ocho millones de aves que dieron positivo al AH7N3.
Puntualizó que, a través del Programa Nacional de Vigilancia Epidemiológica, se han recabado y analizado 11 mil 253 muestras en granjas ubicadas en 20 estados del país, que han arrojado resultados negativos al aislamiento viral, lo que evidencia científicamente que el brote sigue contenido en la zona de Los Altos de Jalisco, donde se originó.
Sánchez Cruz reiteró que la presencia del virus no implica ningún riesgo para la salud humana, por lo que las medidas de control aplicadas por el DINESA tienen el propósito de proteger la producción avícola de la zona.

Web of Knowledge
Record 1 of 40
Walker, Patrick; Cauchemez, Simon; Hartemink, Nienke; Tiensin, Thanawat; Ghani, Azra C.
Outbreaks of H5N1 in poultry in Thailand: the relative role of poultry production types in sustaining transmission and the impact of active surveillance in control
H5N1, highly pathogenic avian influenza, continues to pose a public health risk in the countries of southeast Asia where it has become endemic. However, in Thailand, which experienced two of the largest recorded epidemics in 2004-2005, the disease has been successfully reduced to very low levels. We fitted a spatio-temporal model of the spread of infection to outbreak data collected during the second wave of outbreaks to assess the extent to which different poultry types were responsible for propagating infection. Our estimates suggest that the wave of outbreaks would not have been possible without the contribution of backyard flocks to the susceptibility of a sub-district. However, we also estimated that outbreaks involving commercial poultry, a much larger sector in Thailand than in neighbouring countries, were disproportionately infectious, a factor which was also crucial in sustaining the wave. As a result, implemented measures that aim to reduce the role of commercial farms in the spread of infection, such as the drive to bring aspects of the supply chain 'in house', may help to explain the subsequent success in controlling H5N1 in Thailand. We also found that periods of active surveillance substantially improved the rate of outbreak detection.
ISSN: 1742-5689

Record 2 of 40
Koopmans, M.; de Bruin, E.; Godeke, G-J; Friesema, I.; van Gageldonk, R.; Schipper, M.; Meijer, A.; van Binnendijk, R.; Rimmelzwaan, G. F.; de Jong, M. D.; Buisman, A.; van Beek, J.; van de Vijver, D.; Reimerink, J.
Profiling of humoral immune responses to influenza viruses by using protein microarray
Clin Microbiol Infect Abstract The emergence of pandemic A(H1N1) 2009 influenza showed the importance of rapid assessment of the degree of immunity in the population, the rate of asymptomatic infection, the spread of infection in households, effects of control measures, and ability of candidate vaccines to produce a response in different age groups. A limitation lies in the available assay repertoire: reference standard methods for measuring antibodies to influenza virus are haemagglutination inhibition (HI) assays and virus neutralization tests. Both assays are difficult to standardize and may be too specific to assess possible partial humoral immunity from previous exposures. Here, we describe the use of antigen-microarrays to measure antibodies to HA1 antigens from seven recent and historical seasonal H1, H2 and H3 influenza viruses, the A(H1N1) 2009 pandemic influenza virus, and three avian influenza viruses. We assessed antibody profiles in 18 adult patients infected with A(H1N1) 2009 influenza virus during the recent pandemic, and 21 children sampled before and after the pandemic, against background reactivity observed in 122 persons sampled in 2008, a season dominated by seasonal A(H1N1) influenza virus. We show that subtype-specific and variant-specific antibody responses can be measured, confirming serological responses measured by HI. Comparison of profiles from persons with similar HI response showed that the magnitude and broadness of response to individual influenza subtype antigens differs greatly between individuals. Clinical and vaccination studies, but also exposure studies, should take these findings into consideration, as they may indicate some level of humoral immunity not measured by HI assays.
ISSN: 1198-743X

Record 3 of 40
Abdelwhab, E. M.; Arafa, Abdel-Satar; Stech, Juergen; Grund, Christian; Stech, Olga; Graeber-Gerberding, Marcus; Beer, Martin; Hassan, Mohamed K.; Aly, Mona M.; Harder, Timm C.; Hafez, Hafez M.
Diversifying evolution of highly pathogenic H5N1 avian influenza virus in Egypt from 2006 to 2011
VIRUS GENES 45(1) 14-23. AUG 2012
An evolutionary analysis was conducted of 354 hemagglutinin (HA) and 208 neuraminidase (NA) genes, including newly generated sequences of 5 HA and 30 NA, of Egyptian H5N1 clade 2.2.1 viruses isolated from poultry and humans. Five distinct phylogenetically distinguishable clusters arose from a monophyletic origin since 2006. Only two clusters remained in circulation after 2009: (i) A cluster of viruses arose in 2007 in industrial-vaccinated chickens and carried multiple mutations in or adjacent to the immunogenic epitopes of the HA. Viruses within this cluster evolved with significantly elevated mutation rates indicating persisting selective pressures, e.g. to escape host immunity and (ii) The second group arose in 2008 and harboured strains from recent human infections featuring a conspicuous deletion in the HA receptor-binding domain and substitutions close to the highly conserved active site of the NA. In both sublineages, a number of positively selected amino acids, different glycosylation patterns and variations in the polybasic proteolytic cleavage site were observed. Continuous monitoring of the evolving H5N1 virus in Egypt is essential to develop new control campaigns in poultry and human population.
ISSN: 0920-8569

Record 4 of 40
Chen, Feng; Yan, Zhuan-Qiang; Liu, Jun; Ji, Jun; Chang, Shuang; Liu, Di; Qin, Jian-Ping; Ma, Jing-Yun; Bi, Ying-Zuo; Xie, Qing-Mei
Phylogenetic analysis of hemagglutinin genes of 40 H9N2 subtype avian influenza viruses isolated from poultry in China from 2010 to 2011
VIRUS GENES 45(1) 69-75. AUG 2012
Avian influenza virus (H9N2) infection is a major problem of product performance in poultry worldwide. Vaccination is used to limit spread, but more knowledge is needed on the epidemiology of virus subtypes to improve vaccine design. In this study, 40 H9N2 subtype avian influenza viruses (AIVs) were isolated from vaccinated poultry flocks in China from 2010 to 2011. Hemagglutinin (HA) from different virus strains was sequenced and analyzed. We found that the HA genes of these strains shared nucleotide and deduced amino acid homologies that ranged from 90.1 to 92.9 and 91.4 to 95.0 %, respectively, when compared with vaccine strains. Phylogenetic analysis showed that the strains tested could be divided into two major groups. Group I consisted of 24 strains isolated mainly from Eastern and Central China. Group II consisted of 20 strains isolated from Southern China. The cleavage site within the HA protein contained two basic motifs, PSRSSRa dagger"GLF for group I, and PARSSRa dagger"GLF for group II. Additional potential glycosylation sites were found at amino acid position 295 in the HA1 of the isolates in group I, compared with isolates in group II and the vaccine strains. Furthermore, 38 out of the 40 isolates had a leucine residue at position 216 (aa 226 in H3), which was characteristic of human influenza virus-like receptor specificity. In the present study we found that geographical factors play a significant role in virus evolution, and emphasize the importance of continuing surveillance of H9N2 AIVs in chickens in China.
ISSN: 0920-8569

Record 5 of 40
Vergara-Alert, Julia; Argilaguet, Jordi M.; Busquets, Nuria; Ballester, Maria; Martin-Valls, Gerard E.; Rivas, Raquel; Lopez-Soria, Sergio; Solanes, David; Majo, Natalia; Segales, Joaquim; Veljkovic, Veljko; Rodriguez, Fernando; Darji, Ayub
Conserved Synthetic Peptides from the Hemagglutinin of Influenza Viruses Induce Broad Humoral and T-Cell Responses in a Pig Model
PLOS ONE 7(7). JUL 16 2012
Outbreaks involving either H5N1 or H1N1 influenza viruses (IV) have recently become an increasing threat to cause potential pandemics. Pigs have an important role in this aspect. As reflected in the 2009 human H1N1 pandemia, they may act as a vehicle for mixing and generating new assortments of viruses potentially pathogenic to animals and humans. Lack of universal vaccines against the highly variable influenza virus forces scientists to continuously design vaccines a la carte, which is an expensive and risky practice overall when dealing with virulent strains. Therefore, we focused our efforts on developing a broadly protective influenza vaccine based on the Informational Spectrum Method (ISM). This theoretical prediction allows the selection of highly conserved peptide sequences from within the hemagglutinin subunit 1 protein (HA1) from either H5 or H1 viruses which are located in the flanking region of the HA binding site and with the potential to elicit broader immune responses than conventional vaccines. Confirming the theoretical predictions, immunization of conventional farm pigs with the synthetic peptides induced humoral responses in every single pig. The fact that the induced antibodies were able to recognize in vitro heterologous influenza viruses such as the pandemic H1N1 virus (pH1N1), two swine influenza field isolates (SwH1N1 and SwH3N2) and a H5N1 highly pathogenic avian virus, confirm the broad recognition of the antibodies induced. Unexpectedly, all pigs also showed T-cell responses that not only recognized the specific peptides, but also the pH1N1 virus. Finally, a partial effect on the kinetics of virus clearance was observed after the intranasal infection with the pH1N1 virus, setting forth the groundwork for the design of peptide-based vaccines against influenza viruses. Further insights into the understanding of the mechanisms involved in the protection afforded will be necessary to optimize future vaccine formulations.
ISSN: 1932-6203

Record 6 of 40
Jazayeri, Seyed Davoud; Ideris, Aini; Zakaria, Zunita; Shameli, Kamyar; Moeini, Hassan; Omar, Abdul Rahman
Cytotoxicity and immunological responses following oral vaccination of nanoencapsulated avian influenza virus H5 DNA vaccine with green synthesis silver nanoparticles
DNA formulations provide the basis for safe and cost effective vaccine. Low efficiency is often observed in the delivery of DNA vaccines. In order to assess a new strategy for oral DNA vaccine formulation and delivery, plasmid encoding hemagglutinin (HA) gene of avian influenza virus, A/Ck/Malaysia/5858/04 (H5N1) (pcDNA3.1/H5) was formulated using green synthesis of sliver nanoparticles (AgNP) with polyethylene glycol (PEG). AgNP were successfully synthesized uniformly dispersed with size in the range of 4 to 18 nm with an average size of 11 nm. Cytotoxicity of the prepared AgNP was investigated in vitro and in vivo using MCF-7 cells and cytokine expression, respectively. At the concentration of -5 log(10)AgNP, no cytotoxic effects were detected in MCF-7 cells with 9.5% cell death compared to the control. One-day-old specific pathogen-free (SPF) chicks immunized once by oral gavage with 10 mu l of pcDNA3.1/H5 (200 ng/ml) nanoencapsulated with 40 mu l AgNP (3.7x10(-2) mu g of Ag) showed no clinical manifestations. PCR successfully detect the AgNP/H5 plasmid from the duodenum of the inoculated chicken as early as 1 h post-immunization. Immunization of chickens with AgNP/H5 enhanced both pro inflammatory and Th1-like expressions, although no significant differences were recorded in the chickens inoculated with AgNP, AgNP/pcDNA3.1 and the control. In addition, serum samples collected from immunized chickens with AgNP/H5 showed rapidly increasing antibody against H5 on day 14 after immunization. The highest average antibody titres were detected on day 35 post-immunization at 51.2 +/- 7.5. AgNP/H5 also elicited both CD4+ and CD8+ T cells in the immunized chickens as early as day 14 after immunization, at 7.5 +/- 2.0 and 20 +/- 1.9 percentage, respectively. Hence, single oral administrations of AgNP/H5 led to induce both the antibody and cell-mediated immune responses as well as enhanced cytokine production. (C) 2012 Elsevier B.V. All rights reserved.
ISSN: 0168-3659

Record 7 of 40
Marcelin, Glendie; Sandbulte, Matthew R.; Webby, Richard J.
Contribution of antibody production against neuraminidase to the protection afforded by influenza vaccines
Vaccines are instrumental in controlling the burden of influenza virus infection in humans and animals. Antibodies raised against both major viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), can contribute to protective immunity. Vaccine-induced HA antibodies have been characterized extensively, and they generally confer protection by blocking the attachment and fusion of a homologous virus onto host cells. Although not as well characterized, some functions of NA antibodies in influenza vaccine-mediated immunity have been recognized for many years. In this review, we summarize the case for NA antibodies in influenza vaccine-mediated immunity. In the absence of well-matched HA antibodies, NA antibodies can provide varying degrees of protection against disease. NA proteins of seasonal influenza vaccines have been shown in some instances to elicit serum antibodies with cross-reactivity to avian-origin and swine-origin influenza strains, in addition to HA drift variants. NA-mediated immunity has been linked to (i) conserved NA epitopes amongst otherwise antigenically distinct strains, partly attributable to the segmented influenza viral genome; (ii) inhibition of NA enzymatic activity; and (iii) the NA content in vaccine formulations. There is a potential to enhance the effectiveness of existing and future influenza vaccines by focusing greater attention on the antigenic characteristics and potency of the NA protein. Copyright (c) 2012 John Wiley & Sons, Ltd.
ISSN: 1052-9276

Record 8 of 40
Pearce, Melissa B.; Belser, Jessica A.; Gustin, Kortney M.; Pappas, Claudia; Houser, Katherine V.; Sun, Xiangjie; Maines, Taronna R.; Pantin-Jackwood, Mary J.; Katz, Jacqueline M.; Tumpey, Terrence M.
Seasonal Trivalent Inactivated Influenza Vaccine Protects against 1918 Spanish Influenza Virus Infection in Ferrets
JOURNAL OF VIROLOGY 86(13) 7118-7125. JUL 2012
The influenza virus H1N1 pandemic of 1918 was one of the worst medical catastrophes in human history. Recent studies have demonstrated that the hemagglutinin (HA) protein of the 1918 virus and 2009 H1N1 pandemic virus [A(H1N1)pdm09], the latter now a component of the seasonal trivalent inactivated influenza vaccine (TIV), share cross-reactive antigenic determinants. In this study, we demonstrate that immunization with the 2010-2011 seasonal TIV induces neutralizing antibodies that cross-react with the reconstructed 1918 pandemic virus in ferrets. TIV-immunized ferrets subsequently challenged with the 1918 virus displayed significant reductions in fever, weight loss, and virus shedding compared to these parameters in nonimmune control ferrets. Seasonal TIV was also effective in protecting against the lung infection and severe lung pathology associated with 1918 virus infection. Our data demonstrate that prior immunization with contemporary TIV provides cross-protection against the 1918 virus in ferrets. These findings suggest that exposure to A(H1N1)pdm09 through immunization may provide protection against the reconstructed 1918 virus which, as a select agent, is considered to pose both biosafety and biosecurity threats.
ISSN: 0022-538X

Record 9 of 40
Sakoda, Yoshihiro; Okamatsu, Masatoshi; Isoda, Norikazu; Yamamoto, Naoki; Ozaki, Koichi; Umeda, Yasuto; Aoyama, Shigeyuki; Kida, Hiroshi
Purification of human and avian influenza viruses using cellulose sulfate ester (Cellufine Sulfate) in the process of vaccine production
Affinity chromatography using sulfated, spherical cellulose beads (Cellufine Sulfate) was assessed for purification of influenza A and influenza B viruses. Recovery rates of viruses eluted from the beads were high for all tested virus strains. This method was also useful for removing chicken egg-derived impurities from allantoic fluids containing influenza viruses; the hemagglutination activity per amount of protein in the eluted sample was significantly higher than that in the applied sample. These results suggest that use of Cellufine Sulfate is a practical method for primary purification of influenza viruses in the process of influenza vaccine production.
ISSN: 0385-5600

Record 10 of 40
Wang ZhaoGuo; Jiang WenMing; Liu Shuo; Hou GuangYu; Li JinPing; Wang ZhiYu; Chen JiMing
Increased substitution rate in H5N1 avian influenza viruses during mass vaccination of poultry
CHINESE SCIENCE BULLETIN 57(19) 2419-2424. JUL 2012
As a means of heated debate, mass vaccination of poultry has been used in some countries to control H5N1 highly pathogenic avian influenza (HPAI), which remains of global economic and public health significance. Theoretically, mass vaccination can act as an evolutionary selective force facilitating the emergence of vaccine-resistant viruses, similar to that widespread use of antibiotics facilitates the emergence of antibiotic-resistant bacteria. To support the hypothesis, the substitution rates in the two subunits, HA1 and HA2, of the viral hemagglutinin gene, were calculated using a Bayesian Markov Chain Monte Carlo (MCMC) approach. It was found that the rate in the HA1 subunit, but not in the HA2 subunit, increased significantly during periods of mass vaccination (2005-2010 in China and 2003-2009 in Indonesia), in contrast to the periods when no mass vaccination programs took place (1996-2004 in China and 2004-2008 in Thailand). Because substitutions in the HA1 subunit rather than in the HA2 subunit can lead to vaccine-resistant viruses, the results support that mass vaccination programs facilitate the emergence of vaccine-resistant viruses, which, in turn, will render mass vaccination programs less effective. Therefore, caution must be taken when adopting mass vaccination as a long-term strategy to control HPAI.
ISSN: 1001-6538

Record 11 of 40
Chittaganpitch, Malinee; Supawat, Krongkaew; Olsen, Sonja J.; Waicharoen, Sunthareeya; Patthamadilok, Sirima; Yingyong, Thitipong; Brammer, Lynnette; Epperson, Scott P.; Akrasewi, Passakorn; Sawanpanyalert, Pathom
Influenza viruses in Thailand: 7 years of sentinel surveillance data, 2004-2010
Please cite this paper as: Chittaganpitch et similar to al. (2012) Influenza viruses in Thailand: 7 years of sentinel surveillance data, 20042010. Influenza and Other Respiratory Viruses 6(4), 276283. Background The re-emergence of avian influenza A (H5N1) in 2004 and the pandemic of influenza A (H1N1) in 2009 highlight the need for routine surveillance systems to monitor influenza viruses, particularly in Southeast Asia where H5N1 is endemic in poultry. In 2004, the Thai National Institute of Health, in collaboration with the US Centers for Disease Control and Prevention, established influenza sentinel surveillance throughout Thailand. Objectives To review routine epidemiologic and virologic surveillance for influenza viruses for public health action. Methods Throat swabs from persons with influenza-like illness and severe acute respiratory illness were collected at 11 sentinel sites during 20042010. Influenza viruses were identified using the standard protocol for polymerase chain reaction. Viruses were cultured and identified by immunofluorescence assay; strains were identified by hemagglutination inhibition assay. Data were analyzed to describe frequency, seasonality, and distribution of circulating strains. Results Of the 19 457 throat swabs, 3967 (20%) were positive for influenza viruses: 2663 (67%) were influenza A and able to be subtyped [21% H1N1, 25% H3N2, 21% pandemic (pdm) H1N1] and 1304 (33%) were influenza B. During 20092010, the surveillance system detected three waves of pdm H1N1. Influenza annually presents two peaks, a major peak during the rainy season (June August) and a minor peak in winter (Octobe February). Conclusions These data suggest that March April may be the most appropriate months for seasonal influenza vaccination in Thailand. This system provides a robust profile of the epidemiology of influenza viruses in Thailand and has proven useful for public health planning.
ISSN: 1750-2640

Record 12 of 40
Wodal, Walter; Falkner, Falko G.; Kerschbaum, Astrid; Gaiswinkler, Claudia; Fritz, Richard; Kiermayr, Stefan; Portsmouth, Daniel; Savidis-Dacho, Helga; Coulibaly, Sogue; Piskernik, Christina; Hohenadl, Christine; Howard, M. Keith; Kistner, Otfried; Barrett, P. Noel; Kreil, Thomas R.
A cell culture-derived whole-virus H9N2 vaccine induces high titer antibodies against hemagglutinin and neuraminidase and protects mice from severe lung pathology and weight loss after challenge with a highly virulent H9N2 isolate
VACCINE 30(31) 4625-4631. JUL 2012
Background: Influenza viruses of subtype A/H9N2 are enzootic in poultry across Asia and the Middle East and are considered to have pandemic potential. The development of new vaccine manufacturing technologies is a cornerstone of influenza pandemic preparedness.
Methods: A non-adjuvanted whole-virus H9N2 vaccine was developed using Vero cell culture manufacturing technology. The induction of hemagglutination inhibition (HI) and virus-neutralizing antibodies was assessed in CD1 mice and guinea pigs. A highly sensitive enzyme-linked lectin assay was used to investigate the induction of antibodies capable of inhibiting the enzymatic activity of the H9N2 neuraminidase. Protective efficacy against virus replication in the lung after challenge with the homologous virus was evaluated in BALB/c mice by a TCID50 assay, and prevention of virus replication in the lung and associated pathology were evaluated by histology and immunohistochemistry. To investigate the ability of the vaccine to prevent severe disease, BALB/c mice were challenged with a highly virulent mouse-adapted H9N2 isolate which was generated by multiple lung-to-lung passage of wild-type virus.
Results: The vaccine elicited high titers of functional H9N2-specific HA antibodies in both mice and guinea pigs, as determined by HI and virus neutralization assays. High titer H9N2-specific neuraminidase inhibiting (NAi) antibodies were also induced in both species. Vaccinated mice were protected from lung virus replication in a dose-dependent manner after challenge with the homologous H9N2 virus. Immunohistochemical analyses confirmed the lack of virus replication in the lung and an associated substantial reduction in lung pathology. Dose-dependent protection from severe weight loss was also provided after challenge with the highly virulent mouse-adapted H9N2 virus. Conclusions: The induction of high titers of H9N2-specific HI, virus-neutralizing and NAi antibodies and dose-dependent protection from virus replication and severe disease in animal models suggest that the Vero cell culture-derived whole-virus vaccine will provide an effective intervention in the event of a H9N2 pandemic situation. (C) 2012 Elsevier Ltd. All rights reserved.
ISSN: 0264-410X

Record 13 of 40
Herfst, Sander; Schrauwen, Eefje J. A.; Linster, Martin; Chutinimitkul, Salin; de Wit, Emmie; Munster, Vincent J.; Sorrell, Erin M.; Bestebroer, Theo M.; Burke, David F.; Smith, Derek J.; Rimmelzwaan, Guus F.; Osterhaus, Albert D. M. E.; Fouchier, Ron A. M.
Airborne Transmission of Influenza A/H5N1 Virus Between Ferrets
SCIENCE 336(6088) 1534-1541. JUN 22 2012
Highly pathogenic avian influenza A/H5N1 virus can cause morbidity and mortality in humans but thus far has not acquired the ability to be transmitted by aerosol or respiratory droplet ("airborne transmission") between humans. To address the concern that the virus could acquire this ability under natural conditions, we genetically modified A/H5N1 virus by site-directed mutagenesis and subsequent serial passage in ferrets. The genetically modified A/H5N1 virus acquired mutations during passage in ferrets, ultimately becoming airborne transmissible in ferrets. None of the recipient ferrets died after airborne infection with the mutant A/H5N1 viruses. Four amino acid substitutions in the host receptor-binding protein hemagglutinin, and one in the polymerase complex protein basic polymerase 2, were consistently present in airborne-transmitted viruses. The transmissible viruses were sensitive to the antiviral drug oseltamivir and reacted well with antisera raised against H5 influenza vaccine strains. Thus, avian A/H5N1 influenza viruses can acquire the capacity for airborne transmission between mammals without recombination in an intermediate host and therefore constitute a risk for human pandemic influenza.
ISSN: 0036-8075

Record 14 of 40
Hou, Yanxia; Guo, Yingying; Wu, Chunyan; Shen, Nan; Jiang, Yongping; Wang, Jingfei
Prediction and Identification of T Cell Epitopes in the H5N1 Influenza Virus Nucleoprotein in Chicken
PLOS ONE 7(6). JUN 20 2012
T cell epitopes can be used for the accurate monitoring of avian influenza virus (AIV) immune responses and the rational design of vaccines. No T cell epitopes have been previously identified in the H5N1 AIV virus nucleoprotein (NP) in chickens. For the first time, this study used homology modelling techniques to construct three-dimensional structures of the peptide-binding domains of chicken MHC class I molecules for four commonly encountered unique haplotypes, i.e., B4, B12, B15, and B19. H5N1 AIV NP was computationally parsed into octapeptides or nonapeptides according to the peptide-binding motifs of MHC class I molecules of the B4, B12, B15 and B19 haplotypes. Seventy-five peptide sequences were modelled and their MHC class I molecule-binding abilities were analysed by molecular docking. Twenty-five peptides (Ten for B4, six for B12, two for B15, and seven for B19) were predicted to be potential T cell epitopes in chicken. Nine of these peptides and one unrelated peptide were manually synthesized and their T cell responses were tested in vitro. Spleen lymphocytes were collected from SPF chickens that had been immunised with a NP-expression plasmid, pCAGGS-NP, and they were stimulated using the synthesized peptides. The secretion of chicken IFN-gamma and the proliferation of CD8(+) T cells were tested using an ELISA kit and flow cytometry, respectively. The significant secretion of chicken IFN-gamma and proliferation of CD8(+) T lymphocytes increased by 13.7% and 11.9% were monitored in cells stimulated with peptides NP89-97 and NP198-206, respectively. The results indicate that peptides NP89-97 (PKKTGGPIY) and NP198-206 (KRGINDRNF) are NP T cell epitopes in chicken of certain haplotypes. The method used in this investigation is applicable to predicting T cell epitopes for other antigens in chicken, while this study also extends our understanding of the mechanisms of the immune response to AIV in chicken.
ISSN: 1932-6203

Record 15 of 40
Ehrlich, Hartmut J.; Berezuk, Gregory; Fritsch, Sandor; Aichingere, Gerald; Singer, Julia; Portsmouth, Daniel; Hart, Mary Kate; El-Amin, Wael; Kistner, Otfried; Barrett, P. Noel
Clinical development of a Vero cell culture-derived seasonal influenza vaccine
VACCINE 30(29) 4337-4386. JUN 19 2012
Background: Cell culture technologies have the potential to improve the robustness and flexibility of influenza vaccine supply and to substantially shorten manufacturing timelines. We investigated the safety, immunogenicity and efficacy of a Vero cell culture-derived seasonal influenza vaccine and utilized these studies to establish a serological correlate of vaccine protection. Methods: Two multicenter, randomized, double-blind phase Ill trials were undertaken in the US during the 2008-2009 Northern hemisphere influenza season, in young (18-49 years) and older (50-64 years and >= 65 years) adult subjects. 7250 young adults were randomized 1:1 to receive either Vero-derived vaccine or placebo. 3210 older adult subjects were randomized 8:1 to receive either Vero-derived vaccine or a licensed egg-derived vaccine. Serum hemagglutination inhibition antibody titers were assessed 21 days post-vaccination. Vaccine efficacy in preventing cell culture-confirmed influenza infection was determined for the young adult population. Local and systemic adverse events were recorded in both studies.
Results: The Vero-derived vaccine was safe and well tolerated in both young and older adults. All US and European immunological licensing thresholds were comfortably met in both populations. Vaccine efficacy in young adults was 79% against A/H1N1 viruses antigenically matching the corresponding vaccine strain and 78.5% for all antigenically matched influenza viruses. A hemagglutination inhibition antibody titer of >= 1:15 provided a reliable correlate of protection for the Vero-derived influenza vaccine, with no additional benefit at titers >1:30. Bridging of the correlate of protection established in the young adult population to the older adult immunogenicity data demonstrated the likely effectiveness of the Vero-derived vaccine in the older adult population. Conclusions: A Vero cell culture-derived seasonal influenza vaccine is safe, immunogenic and protects against infection with influenza virus. The novel vaccine technology has the potential to make a substantial contribution to improving influenza vaccine supply. Clinical trial registration: The studies are registered with, numbers NCT00566345 and NCT00782431. (C) 2011 Elsevier Ltd. All rights reserved.
Conference 5th Semmering Vaccine Symposium APR 28-30, 2011 Baden, AUSTRIA
ISSN: 0264-410X

Record 16 of 40
Rahman, Md Masudur; Uyangaa, Erdenebileg; Han, Young Woo; Kim, Seong Bum; Kim, Jin Hyoung; Choi, Jin Young; Eo, Seong Kug
Oral co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing chicken interferon-alpha and interleukin-18 enhances the alleviation of clinical signs caused by respiratory infection with avian influenza virus H9N2
VETERINARY MICROBIOLOGY 157(3-4) 448-455. JUN 15 2012
The combined use of cytokines has shown synergistic and/or additive effects in controlling several viral infections of livestock animals. However, little is known concerning the practical use of chicken cytokine combinations to control avian diseases. Here, we investigated the antiviral efficacy of oral co-administration of chicken interferon-alpha (chIFN-alpha) and chicken interleukin-18 (chIL-18) using attenuated Salmonella enterica serovar Typhimurium in chickens infected with avian influenza virus (AIV) H9N2. Our results demonstrate that oral co-administration of S. enterica serovar Typhimurium expressing chIFN-alpha and chIL-18 produced a greater alleviation of clinical signs caused by respiratory infection with AIV H9N2 in chickens, when compared to administration of S. enterica serovar Typhimurium expressing either chIFN-alpha or chIL-18 alone. Mortality, clinical symptom severity, and feed and water intake were used to access treatment effectiveness. This enhancement of antiviral immunity was further confirmed by evidence of reduced rectal shedding and decreased replication of AIV H9N2 in several different tissues of challenged chickens including trachea, lung, cecal tonsil, and brain. Furthermore, oral co-administration of chIFN-alpha and chIL-18 more efficiently modulated the immune responses of chickens against AIV H9N2 by enhancing both humoral and Th1-biased cell-mediated immunity, compared to single administration of either construct. Therefore, our results suggest that the combined administration of two chicken cytokines, chIFN-alpha and chIL-18, using attenuated S. enterica serovar Typhimurium as an oral carrier, provides an effective means for controlling respiratory disease caused by AIV H9N2 infection. (C) 2011 Elsevier B.V. All rights reserved.
ISSN: 0378-1135

Record 17 of 40
Jing, Xianghong; Phy, Kathryn; Li, Xing; Ye, Zhiping
Increased hemagglutinin content in a reassortant 2009 pandemic H1N1 influenza virus with chimeric neuraminidase containing donor A/Puerto Rico/8/34 virus transmembrane and stalk domains
VACCINE 30(28) 4144-4152. JUN 13 2012
The glycoproteins, heamagglutinin (HA) and neuraminidase (NA) of influenza virus confer host protective immune responses during vaccination, which is the most effective approach for preventing influenza-associated morbidity and mortality. Since the functional balance between the HA and NA proteins may affect viral receptor binding and replication, a pandemic influenza A virus (H1N1 pdm09), strain A/Texas/05/2009, was optimized to elevate its HA antigen content by modifying the NA gene. In this study, we have constructed two 2:6 reassortant viruses between pdmH1N1 (A/Texas/05/2009) and A/Puerto Rico/8/34 (PR8), in which the NA gene of A/Texas/05/2009 was modified to contain part of the NA gene from PR8. One chimeric NA virus has the PR8 transmembrane (TM) region (HNtm 2:6) and the other contains both the PR8 NA TM and stem regions (HNst 2:6). Using quantitative reverse phase-HPLC (RP-HPLC) analysis, we observed that the HNst2:6 virus contains a higher HA1 content than HN2:6 wild type. In addition, this mutant virus displays a higher HA1 to nucleoprotein (NP) ratio, based on gel electrophoresis densitometry analysis. Furthermore, the neuraminidase activity of purified HNst 2:6 virus is approximately 30% lower than that of HN2:6 virus, which is suggestive of a lower incorporation of NA into the viral envelope. Therefore, we propose that the reduction of NA packaging in the virion may lead to a compensatory increase of HA. Such an improvement in HA yield is possibly beneficial to H1N1 pdm09 vaccine production. Published by Elsevier Ltd.
ISSN: 0264-410X

Record 18 of 40
Lin, Shih-Chang; Lin, Yu-Fen; Chong, Pele; Wu, Suh-Chin
Broader Neutralizing Antibodies against H5N1 Viruses Using Prime-Boost Immunization of Hyperglycosylated Hemagglutinin DNA and Virus-Like Particles
PLOS ONE7(6). JUN 13 2012
Background: Highly pathogenic avian influenza (HPAI) H5N1 viruses and their transmission capability from birds to humans have raised global concerns about a potential human pandemic. The inherent nature of antigenic changes in influenza viruses has not been sufficiently taken into account in immunogen designs for broadly protective HPAI H5N1 vaccines. Methods: We designed a hyperglycosylated HA vaccine using N-linked glycan masking on highly variable sequences in the HA1 globular head. Immunization of these hyperglycosylated HA DNA vaccines followed by a flagellin-containing virus-like particle booster in mice was conducted to evaluate neutralizing antibody responses against various clades of HPAI H5N1 viruses. Results: We introduced nine N-X-S/T motifs in five HA1 regions: 83NNT, 86NNT, 94NFT, 127NSS, 138NRT, 156NTT, 161NRS, 182NDT, and 252NAT according to sequence alignment analyses from 163 HPAI H5N1 human isolates. Although no significant differences of anti-HA total IgG titers were found with these hyperglycosyalted HA compared to the wild-type control, the 83NNT and 127NSS mutants elicited significantly potent cross-clade neutralizing antibodies against HPAI H5N1 viruses.
Conclusions: This finding may have value in terms of novel immunogen design for developing cross-protective H5N1 vaccines.
ISSN: 1932-6203

Record 19 of 40
Banner, David; Kelvin, Alyson A.
The current state of H5N1 vaccines and the use of the ferret model for influenza therapeutic and prophylactic development
Highly pathogenic avian influenza H5N1 is a threat to global public health as a natural pandemic causing agent but has recently been considered a bioterrorism concern. The evolving view of the H5N1 virus necessitates the re-evaluation of the current status of H5N1 therapeutics and prophylactics, in particular the preparation of viable H5N1 vaccination strategies as well as the use of ferrets in influenza research. Here the highly pathogenic H5N1 virus dilemma is discussed in context with the current H5N1 vaccine status and the use of the ferret model. Previously, the development of various H5N1 vaccine platforms have been attempted, many of them tested in the ferret model, including vector vaccines, adjuvant vaccines, DNA vaccines, and reverse engineered vaccines. Moreover, as ferrets are a superlative animal model for influenza investigation and vaccine testing, it is imperative that this model is recognized for its uses in prophylactic development and not only as an agent for creating transmissible influenza viruses. Elucidating the ferret immune response and creating ferret immune reagents remain important goals in conjunction with the development and manufacture of H5N1 vaccines. In summary, an efficacious H5N1 vaccine is urgently needed and the ferret model remains an appropriate model for its development.
ISSN: 1972-2680

Record 20 of 40
Song, Min-Suk; Moon, Ho-Jin; Kwon, Hyeok-il; Pascua, Philippe Noriel Q.; Lee, Jun Han; Baek, Yun Hee; Woo, Kyu-Jin; Choi, Juhee; Lee, Sangho; Yoo, Hyunseung; Oh, In Gyeong; Yoon, Yeup; Rho, Jong-Bok; Sung, Moon-Hee; Hong, Seung-Pyo; Kim, Chul-Joong; Choi, Young Ki
Evaluation of the Efficacy of a Pre-pandemic H5N1 Vaccine (MG1109) in Mouse and Ferret Models
The threat of a highly pathogenic avian influenza (HPAI) H5N1 virus causing the next pandemic remains a major concern. In this study, we evaluated the immunogenicity and efficacy of an inactivated whole-virus H5N1 pre-pandemic vaccine (MG1109) formulated by Green Cross Co., Ltd containing the hemagglutinin (HA) and neuraminidase (NA) genes of the clade 1 A/Vietnam/1194/04 virus in the backbone of A/Puerto Rico/8/34 (RgVietNam/04xPR8/34). Administration of the MG1109 vaccine (2-doses) in mice and ferrets elicited high HI and SN titers in a dose-dependent manner against the homologous (RgVietNam/04xPR8/34) and various heterologous H5N1 strains, (RgKor/W149/06xPR8/34, RgCambodia/04xPR8/34, RgGuangxi/05xPR8/34), including a heterosubtypic H5N2 (A/Aquatic bird/orea/W81/05) virus. However, efficient cross-reactivity was not observed against heterosubtypic H9N2 (A/Ck/Korea/H0802/08) and H1N1 (PR/8/34) viruses. Mice immunized with 1.9 mu g HA/dose of MG1109 were completely protected from lethal challenge with heterologous wild-type HPAI H5N1 A/EM/Korea/W149/06 (clade 2.2) and mouse-adapted H5N2 viruses. Furthermore, ferrets administered at least 3.8 mu g HA/dose efficiently suppressed virus growth in the upper respiratory tract and lungs. Vaccinated mice and ferrets also demonstrated attenuation of clinical disease signs and limited virus spread to other organs. Thus, this vaccine provided immunogenic responses in mouse and ferret models even against challenge with heterologous HPAI H5N1 and H5N2 viruses. Since the specific strain of HPAI H5N1 virus that would potentially cause the next outbreak is unknown, pre-pandemic vaccine preparation that could provide cross-protection against various H5 strains could be a useful approach in the selection of promising candidate vaccines in the future.
ISSN: 1225-8873

Record 21 of 40
Kim, Yeu-Chun; Song, Jae-Min; Lipatov, Aleksandr S.; Choi, Seong-O; Lee, Jeong Woo; Donis, Ruben O.; Compans, Richard W.; Kang, Sang-Moo; Prausnitz, Mark R.
Increased immunogenicity of avian influenza DNA vaccine delivered to the skin using a microneedle patch
Effective public health responses to an influenza pandemic require an effective vaccine that can be manufactured and administered to large populations in the shortest possible time. In this study, we evaluated a method for vaccination against avian influenza virus that uses a DNA vaccine for rapid manufacturing and delivered by a microneedle skin patch for simplified administration and increased immunogenicity. We prepared patches containing 700-mu m long microneedles coated with an avian H5 influenza hemagglutinin DNA vaccine from A/Viet Nam/1203/04 influenza virus. The coating DNA dose increased with DNA concentration in the coating solution and the number of dip-coating cycles. Coated DNA was released into the skin tissue by dissolution within minutes. Vaccination of mice using microneedles induced higher levels of antibody responses and hemagglutination inhibition titers, and improved protection against lethal infection with avian influenza as compared to conventional intramuscular delivery of the same dose of the DNA vaccine. Additional analysis showed that the microneedle coating solution containing carboxymethylcellulose and a surfactant may have negatively affected the immunogenicity of the DNA vaccine. Overall, this study shows that DNA vaccine delivery by microneedles can be a promising approach for improved vaccination to mitigate an influenza pandemic. (C) 2012 Elsevier B.V. All rights reserved.
ISSN: 0939-6411

Record 22 of 40
Hu, Zhi-peng; Yin, Juan; Zhang, Yuan-yuan; Jia, Shu-ya; Chen, Zuo-jia; Zhong, Jiang
Characterization of the immune responses elicited by baculovirus-based vector vaccines against influenza virus hemagglutinin
Aim: To compare the specific immune responses elicited by different baculovirus vectors in immunized mice.
Methods: We constructed and characterized two recombinant baculoviruses carrying the expression cassette for the H5N1 influenza virus hemagglutinin (HA) gene driven by either an insect cell promoter (vAc-HA) or a dual-promoter active both in insect and mammalian cells (vAc-HA-DUAL). Virus without the HA gene (vAc-EGFP) was used as a control. These viruses were used to immunize mice subcutaneously and intraperitoneally. The production of total and specific antibodies was determined by ELISA and competitive ELISA. Cytokine production by the spleen cells of immunized mice was studied using the ELISPOT assay.
Results: Both the vAc-HA and vAc-HA-DUAL vectors expressed HA proteins in insect Sf9 cells, and HA antigen was present in progeny virions. The vAc-HA-DUAL vector also mediated HA expression in virus-transduced mammalian cell lines (BHK and A547). Both vAcHA and vAc-HA-DUAL exhibited higher transduction efficiencies than vAc-EGFP in mammalian cells, as shown by the expression of the reporter gene egfp. Additionally, both vAc-HA and vAc-HA-DUAL induced high levels of HA-specific antibody production in immunized mice; vAc-HA-DUAL was more efficient in inducing IFN-gamma and IL-2 upon stimulation with specific antigen, whereas vAc-HA was more efficient in inducing IL-4 and IL-6. Conclusion: Baculovirus vectors elicited efficient, specific immune responses in immunized mice. The vector displaying the HA antigen on the virion surface (vAc-HA) elicited a Th2-biased immune response, whereas the vector displaying HA and mediating HA expression in the cell (vAc-HA-DUAL) elicited a Th1-biased immune response.
ISSN: 1671-4083

Record 23 of 40
Ngunjiri, John M.; Lee, Chang-Won; Ali, Ahmed; Marcus, Philip I.
Influenza Virus Interferon-Inducing Particle Efficiency Is Reversed in Avian and Mammalian Cells, and Enhanced in Cells Co-Infected with Defective-Interfering Particles
Naturally selected variants of influenza virus encoding truncated NS1 proteins were tested in chickens as candidate live-attenuated influenza vaccines. Their effectiveness correlated with the amount of interferon (IFN) induced in chicken cells. Effective variants induced large amounts of IFN and contained subpopulations with high ratios of defective-interfering particles: IFN-inducing particles (DIP: IFP). Ineffective variants induced less IFN and contained lower ratios of DIP: IFP. Unexpectedly, there was a reversal of phenotypes in mammalian cells. Variants that induced low amounts of IFN and had low DIP: IFP ratios in chicken cells were excellent IFN inducers with high DIP: IFP ratios in mammalian cells, and vice versa. The high DIP: IFP ratios and computer-simulated dynamics of infection suggested that DIP, as an individual particle, did not function as an IFP. The higher efficiency of IFPs in the presence of DIPs was attributed to reduced amounts of newly synthesized viral polymerase known to result from out-competition by defective-interfering RNAs, and the subsequent failure of that polymerase to turn-off cellular mRNA transcription-including IFN-mRNA.
ISSN: 1079-9907

Record 24 of 40
Zhou, Fan; Wang, Guiqin; Buchy, Philippe; Cai, Zhipeng; Chen, Honglin; Chen, Zhiwei; Cheng, Genhong; Wan, Xiu-Feng; Deubel, Vincent; Zhou, Paul
A Triclade DNA Vaccine Designed on the Basis of a Comprehensive Serologic Study Elicits Neutralizing Antibody Responses against All Clades and Subclades of Highly Pathogenic Avian Influenza H5N1 Viruses
JOURNAL OF VIROLOGY86(12) 6970-6978. JUN 2012
Because of their rapid evolution, genetic diversity, broad host range, ongoing circulation in birds, and potential human-to-human transmission, H5N1 influenza viruses remain a major global health concern. Their high degree of genetic diversity also poses enormous burdens and uncertainties in developing effective vaccines. To overcome this, we took a new approach, i.e., the development of immunogens based on a comprehensive serologic study. We constructed DNA plasmids encoding codon-optimized hemagglutinin (HA) from 17 representative strains covering all reported clades and subclades of highly pathogenic avian influenza H5N1 viruses. Using DNA plasmids, we generated the corresponding H5N1 pseudotypes and immune sera. We performed an across-the-board pseudotype-based neutralization assay and determined antigenic clusters by cartography. We then designed a triclade DNA vaccine and evaluated its immunogenicity and protection in mice. We report here that (sub)clades 0, 1, 3, 4, 5, 6, 7.1, and 9 were grouped into antigenic cluster 1, (sub)clades, 2.3.4, 2.4, 2.5, and 8 were grouped into another antigenic cluster, with subclade 2.2.1 loosely connected to it, and each of subclades and 7.2 was by itself. Importantly, the triclade DNA vaccine encoding HAs of (sub)clades 0,, and 7.2 elicited broadly neutralizing antibody responses against all H5 clades and subclades and protected mice against high-lethal-dose heterologous H5N1 challenge. Thus, we conclude that broadly neutralizing antibodies against all H5 clades and subclades can indeed be elicited with immunogens on the basis of a comprehensive serologic study. Further evaluation and optimization of such an approach in ferrets and in humans is warranted.
ISSN: 0022-538X

Record 25 of 40
Woodland, David L.
Cutting-Edge Articles on the Host Response to Viral Infections
VIRAL IMMUNOLOGY 25(3) 173-173. JUN 2012
ISSN: 0882-8245

Record 26 of 40
Mallick, Amirul I.; Kulkarni, Raveendra R.; St Paul, Michael; Parvizi, Payvand; Nagy, Eva; Behboudi, Shahriar; Sharif, Shayan
Vaccination with CpG-Adjuvanted Avian Influenza Virosomes Promotes Antiviral Immune Responses and Reduces Virus Shedding in Chickens
VIRAL IMMUNOLOGY 25(3) 226-231. JUN 2012
The use of virosomes as a vaccine platform has proven successful against several viruses. Here we examined the protective efficacy of a virosome-based vaccine consisting of avian influenza virus (AIV) A/Duck/Czech/56/H4N6 in chickens against a homologous AIV challenge. Virosomes adjuvanted with CpG-ODN or recombinant chicken interferon (IFN)-gamma significantly reduced virus shedding after virus challenge. Furthermore, immunization with virosomes adjuvanted with CpG-ODN increased hemagglutination inhibition (HI) and virus-specific neutralizing serum antibodies, as well as virus-specific serum IgG and mucosal IgA responses. We also found a significant increase in the expression of type I and II interferon genes in the protected birds following virus challenge. In summary, this study demonstrated the ability of virosomes adjuvanted with CpG-ODN to reduce AIV shedding, and elicit virus-specific protective antibody responses in vaccinated birds.
ISSN: 0882-8245

Record 27 of 40
Tan, Gene S.; Krammer, Florian; Eggink, Dirk; Kongchanagul, Alita; Moran, Thomas M.; Palese, Peter
A Pan-H1 Anti-Hemagglutinin Monoclonal Antibody with Potent Broad-Spectrum Efficacy In Vivo
JOURNAL OF VIROLOGY 86(11) 6179-6188. JUN 2012
Seasonal epidemics caused by antigenic variations in influenza A virus remain a public health concern and an economic burden. The isolation and characterization of broadly neutralizing anti-hemagglutinin monoclonal antibodies (MAb) have highlighted the presence of highly conserved epitopes in divergent influenza A viruses. Here, we describe the generation and characterization of a mouse monoclonal antibody designed to target the conserved regions of the hemagglutinin of influenza A H1 viruses, a subtype that has caused pandemics in the human population in both the 20th and 21st centuries. By sequentially immunizing mice with plasmid DNA encoding the hemagglutinin of antigenically different HI influenza A viruses (A/South Carolina/1/1918, A/USSR/92/1977, and A/California/4/2009), we isolated and identified MAb 6F12. Similar to other broadly neutralizing MAb previously described, MAb 6F12 has no hemagglutination inhibition activity against influenza A viruses and targets the stalk region of hemagglutinins. As designed, it has neutralizing activity against a divergent panel of H1 viruses in vitro, representing 79 years of antigenic drift. Most notably, MAb 6F12 prevented gross weight loss against divergent H1 viruses in passive transfer experiments in mice, both in pre- and postexposure prophylaxis regimens. The broad but specific activity of MAb 6F12 highlights the potent efficacy of monoclonal antibodies directed against a single subtype of influenza A virus.
ISSN: 0022-538X

Record 28 of 40
Liu, Kun; Yao, Zhidong; Zhang, Liangyan; Li, Junli; Xing, Li; Wang, Xiliang
MDCK cell-cultured influenza virus vaccine protects mice from lethal challenge with different influenza viruses
Influenza epidemics are major health concern worldwide. Vaccination is the major strategy to protect the general population from a pandemic. Currently, most influenza vaccines are manufactured using chicken embroynated eggs, but this manufacturing method has potential limitations, and cell-based vaccines offer a number of advantages over the traditional method. We reported here using the scalable bioreactor to produce pandemic influenza virus vaccine in a Madin-Darby canine kidney cell culture system. In the 7.5-L bioreactor, the cell concentration reached to 3.2 x 10(6) cells/mL and the highest virus titers of 256 HAU/50 mu L and 1 x 10(7) TCID50/mL. The HA concentration was found to be 11.2 mu g/mL. The vaccines produced by the cell-cultured system induced neutralization antibodies, cross-reactive T-cell responses, and were protective in a mouse model against different lethal influenza virus challenge. These data indicate that microcarrier-based cell-cultured influenza virus vaccine manufacture system in scalable bioreactor could be used to produce effective pandemic influenza virus vaccines.
ISSN: 0175-7598

Record 29 of 40
Cao, Zhiliang; Meng, Jiazi; Li, Xingxing; Wu, Ruiping; Huang, Yanxin; He, Yuxian
The Epitope and Neutralization Mechanism of AVFluIgG01, a Broad-Reactive Human Monoclonal Antibody against H5N1 Influenza Virus
PLOS ONE 7(5). MAY 25 2012
The continued spread of highly pathogenic avian influenza (HPAI) H5N1 virus underscores the importance of effective antiviral approaches. AVFluIgG01 is a potent and broad-reactive H5N1-neutralizing human monoclonal antibody (mAb) showing great potential for use either for therapeutic purposes or as a basis of vaccine development, but its antigenic epitope and neutralization mechanism have not been finely characterized. In this study, we first demonstrated that AVFluIgG01 targets a novel conformation-dependent epitope in the globular head region of H5N1 hemagglutinin (HA). By selecting mimotopes from a random peptide library in combination with computational algorithms and site-directed mutagenesis, the epitope was mapped to three conserved discontinuous sites (I-III) that are located closely at the three-dimensional structure of HA. Further, we found that this HA1-specific human mAb can efficiently block both virus-receptor binding and post-attachment steps, while its Fab fragment exerts the post-attachment inhibition only. Consistently, AVFluIgG01 could inhibit HA-mediated cell-cell membrane fusion at a dose-dependent manner and block the acquisition of pH-induced protease sensitivity. These results suggest a neutralization mechanism of AVFluIgG01 by simultaneously blocking viral attachment to the receptors on host cells and interfering with HA conformational rearrangements associated with membrane fusion. The presented data provide critical information for developing novel antiviral therapeutics and vaccines against HPAI H5N1 virus.
ISSN: 1932-6203

Record 30 of 40
Chaung, Hso-Chi; Cheng, Li-Ting; Hung, Li-Hsiang; Tsai, Pei-Chun; Skountzou, Ioanna; Wang, Baozhong; Compans, Richard W.; Lien, Yi-Yang
Salmonella flagellin enhances mucosal immunity of avian influenza vaccine in chickens
VETERINARY MICROBIOLOGY 157(1-2) 69-77. MAY 25 2012
Flagellin, a bioactive Toll-like receptor (TLR) 5 ligand, may trigger the innate immunity that in turn is important for subsequent adaptive immune responses. In the present study, the adjuvant effects of the monomeric and polymeric forms of Salmonella flagellin (mFliC and pFliC, respectively) were examined in specific-pathogen free (SPF) chickens immunized intramuscularly (i.m.) or intranasally (i.n.) with formalin-inactivated avian influenza virus (AIV) H5N2 vaccines. Results showed that mFliC cooperating with the 64CpG adjuvant significantly induced influenza-specific antibody titers of plasma IgA in the i.m.-vaccinated animals. The nasal IgA levels in the i.n.-mFliC-coadministrated AIV vaccinated chickens were significantly elevated compared to levels observed in the control group (H5N2 vaccine alone). The pFliC cooperating with the 64CpG adjuvant significantly enhanced cell proliferation of splenocytes in the i.m.-vaccinated animals. TLR3 and TLR5 expressions were activated by flagellin stimulation in vitro and in vivo. These results suggest that flagellin can be used as an adjuvant in an AIV H5N2 vaccine, especially for mucosal immunity. (C) 2011 Elsevier B.V. All rights reserved.
ISSN: 0378-1135

Record 31 of 40
Arikata, Masahiko; Itoh, Yasushi; Okamatsu, Masatoshi; Maeda, Toshinaga; Shiina, Takashi; Tanaka, Keiko; Suzuki, Shingo; Nakayama, Misako; Sakoda, Yoshihiro; Ishigaki, Hirohito; Takada, Ayato; Ishida, Hideaki; Soda, Kosuke; Van Loi Pham; Tsuchiya, Hideaki; Nakamura, Shinichiro; Torii, Ryuzo; Shimizu, Takeshi; Inoko, Hidetoshi; Ohkubo, Iwao; Kida, Hiroshi; Ogasawara, Kazumasa
Memory Immune Responses against Pandemic (H1N1) 2009 Influenza Virus Induced by a Whole Particle Vaccine in Cynomolgus Monkeys Carrying Mafa-A1*052:02
PLOS ONE 7(5). MAY 18 2012
We made an H1N1 vaccine candidate from a virus library consisting of 144 (= 16 HAx9 NA) non-pathogenic influenza A viruses and examined its protective effects against a pandemic (2009) H1N1 strain using immunologically naive cynomolgus macaques to exclude preexisting immunity and to employ a preclinical study since preexisting immunity in humans previously vaccinated or infected with influenza virus might make comparison of vaccine efficacy difficult. Furthermore, macaques carrying a major histocompatibility complex class I molecule, Mafa-A1*052:02, were used to analyze peptide-specific CD8(+) T cell responses. Sera of macaques immunized with an inactivated whole particle formulation without addition of an adjuvant showed higher neutralization titers against the vaccine strain A/Hokkaido/2/1981 (H1N1) than did sera of macaques immunized with a split formulation. Neutralization activities against the pandemic strain A/Narita/1/2009 (H1N1) in sera of macaques immunized twice with the split vaccine reached levels similar to those in sera of macaques immunized once with the whole particle vaccine. After inoculation with the pandemic virus, the virus was detected in nasal samples of unvaccinated macaques for 6 days after infection and for 2.67 days and 5.33 days on average in macaques vaccinated with the whole particle vaccine and the split vaccine, respectively. After the challenge infection, recall neutralizing antibody responses against the pandemic virus and CD8(+) T cell responses specific for nucleoprotein peptide NP262-270 bound to Mafa-A1*052:02 in macaques vaccinated with the whole particle vaccine were observed more promptly or more vigorously than those in macaques vaccinated with the split vaccine. These findings demonstrated that the vaccine derived from our virus library was effective for pandemic virus infection in macaques and that the whole particle vaccine conferred more effective memory and broader cross-reactive immune responses to macaques against pandemic influenza virus infection than did the split vaccine.
ISSN: 1932-6203

Record 32 of 40
Zhou, Fangye; Zhou, Jian; Ma, Lei; Song, Shaohui; Zhang, Xinwen; Li, Weidong; Jiang, Shude; Wang, Yue; Liao, Guoyang
High-yield production of a stable Vero cell-based vaccine candidate against the highly pathogenic avian influenza virus H5N1
Highly pathogenic avian influenza (HPAI) viruses pose a global pandemic threat, for which rapid large-scale vaccine production technology is critical for prevention and control. Because chickens are highly susceptible to HPAI viruses, the supply of chicken embryos for vaccine production might be depleted during a virus outbreak. Therefore, developing HPAI virus vaccines using other technologies is critical. Meeting vaccine demand using the Vero cell-based fermentation process has been hindered by low stability and yield. In this study, a Vero cell-based HPAI H5N1 vaccine candidate (H5N1/YNVa) with stable high yield was achieved by reassortment of the Vero-adapted (Va) high growth A/Yunnan/1/2005(H3N2) (YNVa) virus with the A/Anhui/1/2005(H5N1) attenuated influenza vaccine strain (H5N1delta) using the 6/2 method. The reassorted H5N1/YNVa vaccine maintained a high hemagglutination (HA) titer of 1024. Furthermore, H5N1/YNVa displayed low pathogenicity and uniform immunogenicity compared to that of the parent virus. (C) 2012 Elsevier Inc. All rights reserved.
ISSN: 0006-291X

Record 33 of 40
Giles, Brendan M.; Crevar, Corey J.; Carter, Donald M.; Bissel, Stephanie J.; Schultz-Cherry, Stacey; Wiley, Clayton A.; Ross, Ted M.
A Computationally Optimized Hemagglutinin Virus-Like Particle Vaccine Elicits Broadly Reactive Antibodies that Protect Nonhuman Primates from H5N1 Infection
JOURNAL OF INFECTIOUS DISEASES 205(10)1562-1570. MAY 15 2012
Highly pathogenic H5N1 avian influenza viruses continue to spread via waterfowl, causing lethal infections in humans. Vaccines can prevent the morbidity and mortality associated with pandemic influenza isolates. Predicting the specific isolate that may emerge from the 10 different H5N1 clades is a tremendous challenge for vaccine design.
In this study, we generated a synthetic hemagglutinin (HA) on the basis of a new method, computationally optimized broadly reactive antigen (COBRA), which uses worldwide sequencing and surveillance efforts that are specifically focused on sequences from H5N1 clade 2 human isolates. Cynomolgus macaques vaccinated with COBRA clade 2 HA H5N1 virus-like particles (VLPs) had hemagglutination-inhibition antibody titers that recognized a broader number of representative isolates from divergent clades as compared to nonhuman primates vaccinated with clade 2.2 HA VLPs. Furthermore, all vaccinated animals were protected from A/Whooper Swan/Mongolia/244/2005 (WS/05) clade 2.2 challenge, with no virus detected in the nasal or tracheal washes. However, COBRA VLP-vaccinated nonhuman primates had reduced lung inflammation and pathologic effects as compared to those that received WS/05 VLP vaccines. The COBRA clade 2 HA H5N1 VLP elicits broad humoral immunity against multiple H5N1 isolates from different clades. In addition, the COBRA VLP vaccine is more effective than a homologous vaccine against a highly pathogenic avian influenza virus challenge.
ISSN: 0022-1899

Record 34 of 40
Meliopoulos, Victoria A.; Andersen, Lauren E.; Brooks, Paula; Yan, Xiuzhen; Bakre, Abhijeet; Coleman, J. Keegan; Tompkins, S. Mark; Tripp, Ralph A.
MicroRNA Regulation of Human Protease Genes Essential for Influenza Virus Replication
PLOS ONE 7(5) MAY 14 2012
Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-kappa B), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies.
ISSN: 1932-6203

Record 35 of 40
Schmeisser, Falko; Adamo, Joan E.; Blumberg, Benjamin; Friedman, Rachel; Muller, Jacqueline; Soto, Jackeline; Weir, Jerry P.
Production and characterization of mammalian virus-like particles from modified vaccinia virus Ankara vectors expressing influenza H5N1 hemagglutinin and neuraminidase
VACCINE 30(23) 3413-3422. MAY 14 2012
Several studies have described the production of influenza virus-like particles (VLP) using a variety of platform systems. These VLPs are non-replicating particles that spontaneously self-assemble from expressed influenza virus proteins and have been proposed as vaccine candidates for both seasonal and pandemic influenza. Although still in the early stages of development and evaluation as influenza vaccines, influenza VLPs have a variety of other valuable uses such as examining and understanding correlates of protection against influenza and investigating virus-cell interactions. The most common production system for influenza VLPs is the baculovirus-insect cell expression which has several attractive features including the ease in which new gene combinations can be constructed, the immunogenicity elicited and protection afforded by the produced VLPs, and the scalability offered by the system. However, there are differences between the influenza VLPs produced by baculovirus expression systems in insect cells and the influenza viruses produced for use as current vaccines or the virus produced during a productive clinical infection. We describe here the development of a modified vaccinia virus Ankara (MVA) system to generate mammalian influenza VLPs containing influenza H5N1 proteins. The MVA vector system is flexible for manipulating and generating various VLP constructs, expresses high level of influenza hemagglutinin (HA), neuraminidase (NA), and matrix (M) proteins, and can be scaled up to produce VLPs in quantities sufficient for in vivo studies. We show that mammalian VLPs are generated from recombinant MVA vectors expressing H5N1 HA alone, but that increased VLP production can be achieved if NA is co-expressed. These mammalian H5N1 influenza VLPs have properties in common with live virus, as shown by electron microscopy analysis, their ability to hemagglutinate red blood cells, express neuraminidase activity, and to bind influenza specific antibodies. Importantly, these VLPs are able to elicit a protective immune response in a mouse challenge model, suggesting their utility in dissecting the correlates of immunity in such models. Mammalian derived VLPs may also provide a useful tool for studying virus-cell interactions and may have potential for development as pandemic vaccines. Published by Elsevier Ltd.
ISSN: 0264-410X

Record 36 of 40
Read, A. J.; Arzey, K. E.; Finlaison, D. S.; Gu, X.; Davis, R. J.; Ritchie, L.; Kirkland, P. D.
A prospective longitudinal study of naturally infected horses to evaluate the performance characteristics of rapid diagnostic tests for equine influenza virus
VETERINARY MICROBIOLOGY 156(3-4) 246-255. MAY 4 2012
An outbreak of equine influenza (EI) occurred in Australia in 2007. During the laboratory support for this outbreak, real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assays and a blocking enzyme linked immunosorbent assay (bELISA) were used as testing methods to detect infection with the virus. The qRT-PCR and bELISA tests had not been used for El diagnosis before, so it was not known how soon after infection these tests would yield positive results, or for how long these results would remain positive. To answer these questions, nasal swabs and blood samples were collected daily from a group of 36 naturally infected horses. El viral RNA was detected in all horses by qRT-PCR from the first to tenth day after clinical signs were evident, and was detected in some horses for up to 34 days. Antibody was detected in the bELISA in some horses by day 3, with a median time to seroconversion of 5 days. The results from this study indicate that viral RNA can be detected from nasal swabs for much longer than infectious virus is thought to be shed from horses. The bELISA detected antibodies against El virus in all horses for 139 days following infection, but only detected approximately 50% of horses 12 months following infection. Haemagglutination inhibition testing detected antibodies against H3 antigens in all horses for 28 days following infection, but 2 were negative by 35 days following infection. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.
ISSN: 0378-1135

Record 37 of 40
Desvaux, S.; Garcia, J. M.; Nguyen, T. D.; Reid, S. A.; Bui, N. A.; Roger, F.; Fenwick, S.; Peiris, J. S. M.; Ellis, T.
Evaluation of serological tests for H5N1 avian influenza on field samples from domestic poultry populations in Vietnam: Consequences for surveillance
VETERINARY MICROBIOLOGY 156(3-4) 277-284. MAY 4 2012
In Vietnam, serological post H5N1 vaccination surveillance using the HI test is applied to assess the efficiency of the vaccination in addition to virological monitoring. In this paper we report on the evaluations of the performances of the haemagglutination inhibition (HI) test and of a H5-ELISA, using chicken and duck field samples. The evaluations were conducted by comparison with a pseudotyped-based virus neutralization test (H5pp VNT) performed in a reference laboratory and considered as a "gold standard" and also by using methods developed for imperfect reference test. Their global accuracy and best cut-offs were also estimated. Results from the HI test for several haemagglutinin subtypes and from a commercial type A influenza competition ELISA were also compared. The results showed that performance of the HI test was very good in comparison with the H5pp VNT. Data also clearly supported the cut-off of >4 log, used for the HI test for chickens but, a 3 log(2) positivity cut-off would be more appropriate for ducks. When compared with the VNT, the H5-ELISA showed poor specificity when using the positivity cut-off specified by the manufacturer but could be used as a screening test if confirmed by the HI test or the H5ppVNT which presents some interests for large scale testing (no need for biosafety level 3 conditions and high performance). A general and highly sensitive pre-screening can also be achieved using the detection of NP-specific antibodies with a competition ELISA. This appears of little interest in a context of high subtypes diversity where only a subtype is targeted for surveillance and control. (C) 2011 Elseviel B.V. All rights reserved.
ISSN: 0378-1135

Record 38 of 40
Zhang, Yi; Yin, Yanbo; Bi, Yuhai; Wang, Shouchun; Xu, Shouzhen; Wang, Jianlin; Zhou, Shun; Sun, Tingting; Yoon, Kyoung-Jin
Molecular and antigenic characterization of H9N2 avian influenza virus isolates from chicken flocks between 1998 and 2007 in China
VETERINARY MICROBIOLOGY 156(3-4) 285-293. MAY 4 2012
Despite extensive vaccination, H9N2 subtype influenza A viruses have prevailed in chicken populations in China. H9N2 IAVs have been a major cause of respiratory disease and reduced egg production, resulting in great economic losses to the Chinese poultry industry. In attempt to find reasons for lack of adequate protection by commercial vaccines, 41 H9N2 viruses isolated from chicken flocks in various regions of China through surveillance between 1998 and 2007 were systemically analyzed using molecular and serological methods in comparison to IAV Ck/Shandong/6/96 and Ck/Shanghai/F/98 that have been used in a majority of commercial vaccines for H9N2 in China since 1998. The analyses showed that the field isolates were predominantly of Beijing/94 lineage and underwent rapid genetic and antigenic changes, forming several antigenic groups. Comparisons between the field isolates and vaccine strains revealed that a majority of the field isolates examined were antigenically distinct from the vaccine strains to some extent. Therefore, the rapid antigenic evolution of H9N2 IAV and resulting antigenic difference from the earlier vaccine strains appears to be a key factor for sub optimal control of H9N2 IAV in China, emphasizing that the vaccine strain should be updated in a timely manner through surveillance and accompanying laboratory evaluation of contemporary viruses for antigenic similarity with existing vaccine strains. (C) 2011 Elsevier B.V. All rights reserved.
ISSN: 0378-1135

Record 39 of 40
Bi-Hung, Peng; Nadezhda, Yun; Olga, Chumakova; Michele, Zacks; Gerald, Campbell; Jeanon, Smith; Jennifer, Smith; Seth, Linde; Jenna, Linde; Slobodan, Paessler
Neuropathology of H5N1 virus infection in ferrets
VETERINARY MICROBIOLOGY 156(3-4) 294-304. MAY 4 2012
Highly pathogenic H5N1 virus remains a potential threat to humans. Over 289 fatalities have been reported in WHO confirmed human cases since 2003, and lack of effective vaccines and early treatments contribute to increasing numbers of cases and fatalities. H5N1 encephalitis is a recognized cause of death in Vietnamese cases, and brain pathology is described in other human cases and naturally infected animals. However, neither pathogenesis of H5N1 viral infection in human brain nor post-infection effects in survivors have been fully investigated. We report the brain pathology in a ferret model for active infection and 18-day survival stages. This model closely resembles the infection pattern and progression in human cases of influenza A, and our report is the first description of brain pathology for longer term (18-day) survival in ferrets. We analyzed viral replication, type and severity of meningoencephalitis, infected cell types, and cellular responses to infection. We found viral replication to very high titers in ferret brain, closely correlating with severity of meningoencephalitis. Viral antigens were detected predominantly in neurons, correlating with inflammatory lesions, and less frequently in astrocytes and ependymal cells during active infection. Mononuclear cell infiltrates were observed in early stages predominantly in cerebral cortex, brainstem, and leptomeninges, and less commonly in cerebellum and other areas. Astrogliosis was mild at day 4 post-infection, but robust by day 18. Early and continuous treatment with an antiviral agent (peramivir) inhibited virus production to non-detectable levels, reduced severity of brain injury, and promoted higher survival rates. (C) 2011 Elsevier B.V. All rights reserved.
ISSN: 0378-1135

Record 40 of 40
Montomoli, Emanuele; Khadang, Baharak; Piccirella, Simona; Trombetta, Claudia; Mennitto, Elisa; Manini, Ilaria; Stanzani, Valerio; Lapini, Giulia
Cell culture-derived influenza vaccines from Vero cells: a new horizon for vaccine production
In the 20th century, three influenza pandemics killed approximately 100 million people. The traditional method of influenza vaccine manufacturing is based on using chicken eggs. However, the necessity of the availability of millions of fertile eggs in the event of a pandemic has led research to focus on the development of cell culture-derived vaccines, which offer shorter lead-in times and greater flexibility of production. So far, the cell substrates being evaluated and in use include Vero, Madin-Darby canine kidney, PER. C6 and insect cells. However, Vero cells are the most widely accepted among others. This review introduces briefly the concepts of advanced cell culture-derived influenza vaccine production and highlights the advantages of these vaccines in terms of efficiency, speed and immunogenicity based on the clinical data obtained from different studies.
ISSN: 1476-0584

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