Construction and immunogenicity evaluation of recombinant influenza A viruses containing chimeric hemagglutinin genes derived from genetically divergent influenza A H1N1 subtype viruses

Citation: McCormick, K., Jiang, Z., Zhu, L., Lawson, S. R., Langenhorst, R., Ransburgh, R., . . . Fang, Y. (2015). Construction and immunogenicity evaluation of recombinant influenza A viruses containing chimeric hemagglutinin genes derived from genetically divergent influenza A H1N1 subtype viruses. Plos One, 10(6). doi:10.1371/journal.pone.0127649Background and Objectives: Influenza A viruses cause highly contagious diseases in a variety of hosts, including humans and pigs. To develop a vaccine that can be broadly effective against genetically divergent strains of the virus, in this study we employed molecular breeding (DNA shuffling) technology to create a panel of chimeric HA genes. Methods and Results: Each chimeric HA gene contained genetic elements from parental swine influenza A viruses that had a history of zoonotic transmission, and also from a 2009 pandemic virus. Each parental virus represents a major phylogenetic clade of influenza A H1N1 viruses. Nine shuffled HA constructs were initially screened for immunogenicity in mice by DNA immunization, and one chimeric HA (HA-129) was expressed on both a A/Puerto Rico/8/34 backbone with mutations associated with a live, attenuated phenotype (PR8LAIV- 129) and a A/swine/Texas/4199-2/98 backbone (TX98-129). When delivered to mice, the PR8LAIV- 129 induced antibodies against all four parental viruses, which was similar to the breadth of immunity observed when HA-129 was delivered as a DNA vaccine. This chimeric HA was then tested as a candidate vaccine in a nursery pig model, using inactivated TX98-129 virus as the backbone. The results demonstrate that pigs immunized with HA-129 developed antibodies against all four parental viruses, as well as additional primary swine H1N1 influenza virus field isolates. Conclusion: This study established a platform for creating novel genes of influenza viruses using a molecular breeding approach, which will have important applications toward future development of broadly protective influenza virus vaccines. © 2015 McCormick et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Nonstructural protein 2 of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene 15

Molecular basis of virus replication, viral pathogenesis and antiviral strategie

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Not AvailablePorcine epidemic diarrhea virus (PEDV) is an economically devastating enteric disease in the swine industry. The virus infects pigs of all ages, but it cause severe clinical disease in neonatal suckling pigs with up to 100% mortality. Currently, available vaccines are not completely effective and feedback methods utilizing PEDV infected material has variable success in preventing reinfection. Comprehensive information on the levels and duration of effector/memory IgA and IgG antibody secreting B cell response in the intestines and lymphoid organs of PEDV-infected sows, and their association with specific antibody levels in clinical samples such as plasma, oral fluid, and feces is important. Therefore, our goal in this study was to quantify PEDV specific IgA and IgG B cell responses in sows at approximately 1 and 6 months post-infection in commercial swine herds, including parity one and higher sows. Our data indicated that evaluation of both PEDV specific IgA and IgG antibody levels in the plasma and oral fluid (but not feces) samples is beneficial in disease diagnosis. PEDV specific B cell response in the intestine and spleen of infected sows decline by 6 months, and this associates with specific antibody levels in the plasma and oral fluid samples; but the virus neutralization titers in plasma remains high beyond 6 months post-infection. In conclusion, in sows infected with PEDV the presence of effector/memory B cell response and strong virus neutralization titers in plasma up to 6 months post-infection, suggests their potential to protect sows from reinfection and provide maternal immunity to neonates, but challenge studies are required to confirm such responses.Not Availabl

Schematic diagram of DNA shuffled chimeric HA gene sequences.

<p>Alignment of HA genes from chimeric constructs and parental viruses was performed using clustal W (MEGA 6) and the assignment of homology between each construct and the parental viruses was determined by a DNA shuffling alignment analysis tool (Salanto, version 2.0.2; <a href="https://bitbucket.org/benderc/salanto/wiki/Home" target="_blank">https://bitbucket.org/benderc/salanto/wiki/Home</a>). Different colors represent different HA gene elements from parental virus.</p

Antibody reactivity against viruses expressing parental or non-parental HAs using serum samples from pigs immunized with the TX98-129 IIV.

<p>Sera were collected at 14 days after secondary inoculation of pigs with the candidate TX98-129 IIV vaccine. (A) Serum antibody HAI titers are defined as the reciprocal of the final serum dilution where inhibition of hemagglutination was observed. (B) Serum antibody MN titers are defined as the reciprocal of the final serum dilution where OD₄₉₀ was below 50% of positive control wells, using 100 TCID₅₀ virus inoculum (confirmed by back-titration). In both panels, serum samples with a titer below the detectable limit of the assay (initial serum dilution of 1:10) were assigned a value of 5 for graphical representation and statistical analyses. Viruses expressing non-parental HA proteins are abbreviated (A/North Carolina/18161/2002: NC02; A/swine/Iowa/1/1985: IA85; A/swine/Iowa/40766/1992: IA92; A/swine/Germany/2/1981: GE81; A/New Caledonia/20/99: NC99) and shown with clade representation. Significance between vaccinated vs. unvaccinated for all viruses was determined using a Mann Whitney nonparametric test (*p<0.05).</p

Phylogenetic comparison of swine H1 influenza hemagglutinins used to create the chimeric HAs.

<p>Parental viruses included in the DNA shuffling of chimeric HA genes are identified with colored boxes in each phylogenic clade. The phylogenetic tree was constructed using the Neighbor-Joining method by MEGA software version 6.0. The numbers on branches are bootstrap values from 1000 replicates.</p

Comparison of growth kinetics of wild type virus with recombinant viruses expressing HA-129.

<p>(<b>A</b>) MDCK monolayers were inoculated with 0.01 MOI of either wild type virus PR8_LAIV or recombinant virus PR8_LAIV-129. (<b>B</b>) MDCK monolayers were infected with wild type virus A/swine/Texas/4199-2/98 (H3N2) or recombinant virus TX98-129. At the 12-hour time points indicated, cell culture supernatants were collected, and virus titers were determined using TCID₅₀ quantitation. Error bars represent SEM, with significance between paired viruses at time points denoted by asterisks (*p<0.05 and **p<0.01, using two-way repeated measures ANOVA with Bonferroni post-test).</p

IgG antibody response in mice immunized with plasmid DNAs expressing chimeric HA.

<p>Mice (n = 4) were vaccinated with plasmid DNAs of chimeric HA, delivered by gene gun. Serum antibody (IgG) titers after third vaccination were evaluated by ELISA, with samples considered positive if their initial serum OD₄₀₅ was at least three times greater than the OD₄₀₅ of negative control sera. Samples with antibody titers below the detectable limit of the assay were assigned a titer of 50 for the purpose of generating graphs. Horizontal bars show mean values, and vertical error bars indicate standard deviation.</p

Serum antibody HAI titers from mice inoculated with recombinant virus PR8_LAIV-129 vaccine.

<p>Balb/c mice (n = 7) were vaccinated intranasally with PR8_LAIV-129. Serum antibody titers were analyzed using the HAI assay against the parental viruses and PR8_LAIV-129 itself. HAI titers are defined as the reciprocal of the final serum dilution where inhibition of hemagglutination was observed. Serum samples with a titer below the detectable limit of the assay (initial serum dilution of 1:10) were assigned a value of 5 for graphical representation and statistical analyses. HAI titers from vaccinated (color bars) and unvaccinated (black bars) groups are presented for each HA tested (PR8_LAIV-129, OH07, TN09, NJ76, and IA06). Reactivity of antibodies induced by PR8_LAIV-129 from vaccinated mice was compared with that of unvaccinated mice (n = 7) using Mann Whitney nonparametric test (*p<0.05). Bars represent mean values for the indicated groups, with vertical error bars indicating standard deviation.</p