Journal: PLoS ONE

Article Title: MDA5 Can Be Exploited as Efficacious Genetic Adjuvant for DNA Vaccination against Lethal H5N1 Influenza Virus Infection in Chickens

doi: 10.1371/journal.pone.0049952

Figure Lengend Snippet: Groups of 6 chickens were immunized with a plasmid DNA mixture containing equal amounts of the plasmids for HA expression and for chMDA5(1-483) expression. Doses of 25 µg or 2.5 µg of each plasmid DNA per animal were applied to two groups of 6 animals/dose, as indicated. The same two doses of plasmid DNA for HA expression alone mixed with an equal amount of empty expression plasmid were applied to two other groups of 6 chickens. Two control groups of 3 chickens each received 25 µg or 2.5 µg of empty expression plasmid each (Vector). Two immunizations were applied at an interval of 23 days. 20 days after the second vaccination ( i.e. at 43 dpi), the chicken sera were tested for the presence of HA-specific antibodies. H5-specific antibodies were detected using a commercial competition ELISA. The reactivity of HA antibodies was calculated according the formula 100-[OD(Probe)/OD(Negative)x100] (a). Alternatively, the anti-HA IgY titers were determined in an indirect ELISA using immobilized recombinant HA protein and represented as reciprocal of the highest dilution that yielded an OD greater than 2.1 x of parallel preimmune serum samples (b). The HI titer was determined as the highest serum dilution resulting in complete inhibition of aggregation of chicken red blood cells caused by 8 HAU of Vac-1/04 (H5N1) virus (c). The neutralizing antibody titers in serum samples were determined with Vac-1/04 virus and plotted as reciprocal of the highest dilution resulting in 50% virus neutralization (d). The HA-specific IgA antibody content in the serum was measured by ELISA. Results are shown as absolute OD values from which the unspecific background values were subtracted (e). Each symbol represents an individual animal. The * indicates statistical significant differences calculated with the students t-test (p<0.05).

Article Snippet: The plasmids were propagated in E.coli XL1-Blue (Agilent Technologies), and endotoxin-free plasmid DNA was prepared by anion-exchange chromatography using the NucleoBond Xtra Midi EF DNA extraction and purification system (Macherey-Nagel).

Techniques: Plasmid Preparation, Expressing, Control, Enzyme-linked Immunosorbent Assay, Indirect ELISA, Recombinant, Inhibition, Virus, Neutralization

Journal: PLoS ONE

Article Title: MDA5 Can Be Exploited as Efficacious Genetic Adjuvant for DNA Vaccination against Lethal H5N1 Influenza Virus Infection in Chickens

doi: 10.1371/journal.pone.0049952

Figure Lengend Snippet: The chickens received two intramuscular immunizations with 25 µg (a and b) or 2.5 µg (c and d) of the indicated plasmid DNA at 23 days interval. After another 23 days (or at 46 dpi), the animals were challenged by intratracheal administration of the HPAIV Yamaguchi-7/04. The chickens were observed daily for clinical signs for a period of 9 days after the challenge infection. The daily clinical index was monitored as described elsewhere with minor modifications: healthy (0), reduced activity (0.25), slightly ill (0.5), ill (1), severely ill (2), severely ill and euthanized (2.5) or dead (3). The daily clinical index is represented as the mean value of all chickens per group (a and c). The number of surviving animals is plotted against the time (in days) after the challenge (b and d). Note that the two groups (25 µg and 2.5 µg dose) vaccinated with control vector plasmid (Vector) consisted of only three animals each.

Article Snippet: The plasmids were propagated in E.coli XL1-Blue (Agilent Technologies), and endotoxin-free plasmid DNA was prepared by anion-exchange chromatography using the NucleoBond Xtra Midi EF DNA extraction and purification system (Macherey-Nagel).

Techniques: Plasmid Preparation, Infection, Activity Assay, Control