@article{3ea817e6c1494721b1fd63cdb93f462c,
title = "Novel linear DNA vaccines induce protective immune responses against lethal infection with influenza virus type A/H5N1",
abstract = "Vaccine development for possible influenza pandemics has been challenging. Conventional vaccines such as inactivated and live attenuated virus preparations are limited in terms of production speed and capacity. DNA vaccination has emerged as a potential alternative to conventional vaccines against influenza pandemics. In this study, we use a novel, cell-free DNA manufacturing process (synDNA{\texttrademark}) to produce prototype linear DNA vaccines against the influenza virus type A/H5N1. This synDNA{\texttrademark} process does not require bacterial fermentation, so it avoids the use of antibiotic resistance genes and other nucleic acid sequences unrelated to the antigen gene expression in the actual therapeutic DNA construct. The efficacy of various vaccines expressing the hemagglutinin and neuraminidase proteins (H5N1 synDNA{\texttrademark}), hemagglutinin alone (H5 synDNA{\texttrademark}) or neuraminidase alone (N1 synDNA{\texttrademark}) was evaluated in mice. Two of the constructs (H5 synDNA{\texttrademark} and H5N1 synDNA{\texttrademark}) induced a robust protective immune response with up to 93% of treated mice surviving a lethal challenge of a virulent influenza A/Vietnam/1203/04 H5N1 isolate. In combination with a potent biological activity and simplified production footprint, these characteristics make DNA vaccines prepared with our synDNA{\texttrademark} process highly suitable as alternatives to other vaccine preparations.",
keywords = "Cell-free DNA vaccine production, DNA vaccine, Immune response, Influenza virus type A/H5N1, SynDNA{\texttrademark} vaccine",
author = "Fr{\'e}d{\'e}ric Kendirgi and Yun, {Nadezda E.} and Linde, {Nathaniel S.} and Zacks, {Michele A.} and Smith, {Jeanon N.} and Smith, {Jennifer K.} and Harilyn McMicken and Yin Chen and Slobodan Paessler",
note = "Funding Information: Transfection of cultured cells and Western blot- ting. A549 human lung carcinoma cells (ATCC Cat. No. CCL-185, Manassas, VA) cultured in DMEM medium supplemented with 10% FCS (Invitrogen, Carlsbad, CA) were electroporated using GenePulser XcellTM (BioRad). At 12–16 hrs following elec- troporation, cells were washed with ice cold PBS and directly lysed in SDS-PAGE loading buffer. Detection of HA and NA expression in the crude cells lysates was subsequently conducted, as follows. Lysates were resolved by SDS-PAGE and subse- quently, proteins were transferred onto a nylon membrane (GE Healthcare), blocked with 5% BSA in PBST and subsequently incubated overnight at 4°C with anti-H5N1 goat polyclonal serum (1:500 in PBS containing 2.5% bovine serum albumin) or with affinity-purified rabbit anti-NA (1:500; Biodesign Int.). Following extensive washing with PBST, membranes were incubated a 1:7000 dilution of secondary alkaline phosphatase (AP)-conjugated donkey anti-goat or AP-donkey anti-rabbit antibodies (Promega Corp.,), followed by color development according to the manufacturer{\textquoteright}s recommendations. Animal tissue isolation and viral titer assess- ment. Organs collected at the indicated time points were sagittally sectioned in half. One half of each brain was homogenized in MEM containing 10% fetal bovine serum, to generate a 10% suspension (maintained at -80°C until further processing). The titer of infectious virus was determined, as follows.49 Median tissue culture infectious dose (TCID50) assay was performed using serial ten-fold dilutions of the tissue homogenates prepared in MEM without serum. Madin Darby canine kidney (MDCK) cells (ATCC) were grown to confluency in 96-well tissue culture plates, washed twice with 100 μl of Dulbecco{\textquoteright}s Phosphate-Buffered Saline (DPBS), followed by inoculation of 100 μl of each tissue homogenate dilution into four replicate wells, or, as negative control, DPBS. Plates were incubated for 90 minutes at 37°C, 5% CO2, after which an additional 100 μl of MEM was added to each well. Plates were incubated for 4 days at 37°C, 5% CO2. HA assay was performed by removing 50 μl of supernatant from each well and transferring it to a 96-well plate, followed by addition of 50 μl per well of a 0.5% solution of horse erythrocytes suspended in DPBS sored research agreement from CytoGenix, Inc. and K08 Award w/Ca+ and Mg2+. Erythrocytes were allowed to settle and hemag-#AI059491-01 from the National Institutes of Health. We also thank glutination was documented for each replicate. For organ titrations, Jenna Linde for data entry and preparation of figures throughout the infectious virus titers were expressed in TCID50 dose per gram (g) animal studies. of tissue.{\textcopyright}2008 LANDES BIOSCIENCEReferences Acknowledgements 1. WHO. Cumulative number of confirmed human cases The authors would like to thank Drs. Malcolm Skolnick and Peiris JS, de Jong MD, Guan Y. Avian influenza virus (H5N1): a threat to human health. Reported to WHO: http://www.who.int/csr/don/en 2007. Cindee Ewell for their reviewing of the manuscript and Tera Guidry Clin Microbiol Rev 2007; 20:243-67. and John McHugh for their technical support. Dr. Slobodan Yun NE, Linde NS, Zacks MA, Barr IG, Hurt AC, Smith JN, Dziuba N, Holbrook MR, Paessler was supported by faculty start-up funding provided by the promotes survival in ferrets and mice infected with the highly virulent influenza virus, Zhang L, Kilpatrick JM, Arnold CS, Paessler S. Injectable peramivir mitigates disease and Institute for Human Infections and Immunity at UTMB, a spon-A/Vietnam/1203/04 (H5N1). Virol 2008; 373:198-209.",
year = "2008",
doi = "10.4161/hv.4.6.6177",
language = "English (US)",
volume = "4",
pages = "410--419",
journal = "Human Vaccines",
issn = "1554-8600",
publisher = "Landes Bioscience",
number = "6",
}