Characterizing Emerging Canine H3 Influenza Viruses.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned

Effectiveness of Whole, Inactivated, Low Pathogenicity Influenza A(H7N9) Vaccine against Antigenically Distinct, Highly Pathogenic H7N9 Virus

The recent emergence of highly pathogenic influenza A(H7N9) variants poses a great risk to humans. We show that ferrets vaccinated with low pathogenicity H7N9 virus vaccine do not develop severe symptoms after infection with an antigenically distinct, highly pathogenic H7N9 virus. These results demonstrate the protective benefits of this H7N9 vaccine

A Naturally Occurring Deletion in Its NS Gene Contributes to the Attenuation of an H5N1 Swine Influenza Virus in Chickens▿

In 2001 and 2003, we isolated two H5N1 viruses, A/swine/Fujian/1/01 (SW/FJ/01) and A/swine/Fujian/1/03 (SW/FJ/03), from pigs in Fujian Province, southern China. Genetically, these two viruses are similar, although the NS gene of the SW/FJ/03 virus has a 15-nucleotide deletion at coding positions 612 to 626. The SW/FJ/01 virus is highly lethal for chickens, whereas the SW/FJ/03 virus is nonpathogenic for chickens when administrated intravenously or intranasally. To understand the molecular basis for the difference in virulence, we used reverse genetics to create a series of single-gene recombinants of both viruses. We found that a recombinant virus containing the mutated NS gene from the SW/FJ/03 virus in the SW/FJ/01 virus background was completely attenuated in chickens. We also found that viruses expressing the mutant NS1 protein of SW/FJ/03 did not antagonize the induction of interferon (IFN) protein. Conversely, only the recombinant virus containing the wild-type SW/FJ/01 NS gene in the SW/FJ/03 background was lethal in chickens and antagonized IFN protein levels. Further, we proved that the NS1 genes of the two viruses differ in their stabilities in the host cells and in their abilities to interact with the chicken cleavage and polyadenylation specificity factor. These results indicate that the deletion of amino acids 191 to 195 of the NS1 protein is critical for the attenuation of the SW/FJ/03 virus in chickens and that this deletion affects the ability of the virus to antagonize IFN induction in host cells

Characterization of Immune Responses Induced by Immunization with the HA DNA Vaccines of Two Antigenically Distinctive H5N1 HPAIV Isolates

<div><p>The evolution of the H5N1 highly pathogenic avian influenza virus (HPAIV) has resulted in high sequence variations and diverse antigenic properties in circulating viral isolates. We investigated immune responses induced by HA DNA vaccines of two contemporary H5N1 HPAIV isolates, A/bar-headed goose/Qinghai/3/2005 (QH) and A/chicken/Shanxi/2/2006 (SX) respectively, against the homologous as well as the heterologous virus isolate for comparison. Characterization of antibody responses induced by immunization with QH-HA and SX-HA DNA vaccines showed that the two isolates are antigenically distinctive. Interestingly, after immunization with the QH-HA DNA vaccine, subsequent boosting with the SX-HA DNA vaccine significantly augmented antibody responses against the QH isolate but only induced low levels of antibody responses against the SX isolate. Conversely, after immunization with the SX-HA DNA vaccine, subsequent boosting with the QH-HA DNA vaccine significantly augmented antibody responses against the SX isolate but only induced low levels of antibody responses against the QH isolate. In contrast to the antibody responses, cross-reactive T cell responses are readily detected between these two isolates at similar levels. These results indicate the existence of original antigenic sin (OAS) between concurrently circulating H5N1 HPAIV strains, which may need to be taken into consideration in vaccine development against the potential H5N1 HPAIV pandemic.</p> </div

Plasticity of the Influenza Virus H5 HA Protein.

Since the emergence of highly pathogenic avian influenza viruses of the H5 subtype, the major viral antigen, hemagglutinin (HA), has undergone constant evolution, resulting in numerous genetic and antigenic (sub)clades. To explore the consequences of amino acid changes at sites that may affect the antigenicity of H5 viruses, we simultaneously mutated 17 amino acid positions of an H5 HA by using a synthetic gene library that, theoretically, encodes all combinations of the 20 amino acids at the 17 positions. All 251 mutant viruses sequenced possessed ≥13 amino acid substitutions in HA, demonstrating that the targeted sites can accommodate a substantial number of mutations. Selection with ferret sera raised against H5 viruses of different clades resulted in the isolation of 39 genotypes. Further analysis of seven variants demonstrated that they were antigenically different from the parental virus and replicated efficiently in mammalian cells. Our data demonstrate the substantial plasticity of the influenza virus H5 HA protein, which may lead to novel antigenic variants.IMPORTANCE The HA protein of influenza A viruses is the major viral antigen. In this study, we simultaneously introduced mutations at 17 amino acid positions of an H5 HA expected to affect antigenicity. Viruses with ≥13 amino acid changes in HA were viable, and some had altered antigenic properties. H5 HA can therefore accommodate many mutations in regions that affect antigenicity. The substantial plasticity of H5 HA may facilitate the emergence of novel antigenic variants

Antibody responses against the primary virus train were effectively boosted by a single heterologous HA DNA immunization.

<p>Blood samples collected from Groups QH/SX and SX/QH after the first heterologous boost immunization were analyzed for antibody responses against the primary virus strain, QH or SX viruses respectively, from which the HA DNA vaccine was used in priming immunizations. The results were compared with Groups QH or SX respectively that received homologous boosting immunizations. (<b>a</b>) Antibody responses against the QH virus. (<b>b</b>) Antibody responses against the SX virus. (<b>c</b>) HAI activity against the QH virus. (<b>d</b>) HAI activity against the SX virus. Data are presented as the mean ± standard deviation. Dashed lines indicate detection limit for HAI titer (1∶20).</p

A heterologous HA DNA boosting induced similar levels of antibodies against the second virus strain as a single immunization with the HA DNA vaccine of the second strain.

<p>Blood samples collected from Groups QH/SX and SX/QH after the first heterologous boost immunization were analyzed for antibody responses against the second virus strain, SX or QH viruses respectively, from which the HA DNA vaccine was used in boosting immunizations. The results were compared with Groups SX or QH respectively after a single immunization. (<b>a</b>) Antibody responses against the SX virus. (<b>b</b>) Antibody responses against the QH virus. Data are presented as the mean ± standard deviation.</p

Similar levels of CD8 and CD4 T cell responses against HA were induced by different DNA immunization regimens.

<p>Mice were immunized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041332#pone-0041332-g001" target="_blank">Figure 1</a>. At two weeks after the final immunization, mice were sacrificed and splenocytes were prepared for analysis of T cell responses by intracellular cytokine staining and flow cytometry. (<b>a</b>) Comparison of CD8+ T cell responses induced in mice after DNA immunizations. Splenocytes were stimulated with a peptide corresponding to known CD8+ T cell epitopes in HA for 6 h in the presence of Brefeldin A, and then stained for cell surface CD8 as well as intracellular IFN-γ, followed by flow cytometry analysis. The percentages of IFN-γ-producing CD8+ T cells in splenocytes from each individual mouse after stimulation are shown. (<b>b</b>) Comparison of CD4+ T cell responses induced in mice after DNA immunizations. Splenocytes from immunized mice were stimulated with dendritic cells that have been pulsed with different VLPs as indicated. After 6 h stimulation in the presence of Brefeldin A, the cells were stained for cell surface CD4 as well as intracellular IFN-γ, followed by flow cytometry analysis. The percentages of IFN-γ-producing CD4+ T cells in splenocytes from each individual mouse after stimulation are shown. Data are presented as the mean ± standard deviation.</p

Characterization of antibody responses induced by QH and SX H5N1 HA DNA vaccines.

<p>Blood samples from mice vaccinated with QH (Group QH) or SX (Group SX) HA DNA vaccines were collected after each immunization and analyzed for antibody responses against homologous as well as heterologous viruses. The levels of antibody responses in serum samples were determined by ELISA using purified inactivated QH or SX virus as coating antigens as indicated, and expressed as the amount of virus-specific antibodies in 1 ml of serum samples (ng/ml). The HAI activity (HAI titer) was determined as the highest serum dilution that resulted in complete inhibition of hemagglutination by inactivated QH or SX virus as indicated. (<b>a</b>) Antibody responses against the QH virus. (<b>b</b>) Antibody responses against the SX virus. (<b>c</b>) HAI activity against the QH virus. (<b>d</b>) HAI activity against the SX virus. Data are presented as the mean ± standard deviation. Dashed lines indicate detection limit for HAI titer (1∶20).</p