(Invitrogen). Cell culture supernatants were collected at 48 or 72 hours post transfection. Gag expression was measured by a commercial ELISA kit that detects and quantifies P24 in supernatant (PerkinElmer). For assessing immunogenicity in mouse model, DNA was eluted into saline at the concentration of 0.5 mg/ml. Independent batches of DNA were prepared for immunizations. Six to eight week old female BALB/c mice (Charles River Laboratories) were housed and treated at the Laboratory Animal Research Center of The Rockefeller University in accordance with Institutional Animal Care and Use Committee guidelines. Groups of mice (4 to 5 per group) were immunized with a 25 mg of plasmid DNA vaccine in 50 ml of saline at week 0 and week 4. Serum samples were collected from individual mice at week 4 (4 weeks post first vaccination) and week 6 (2 weeks post second vaccination). Direct ELISA was used to measure serum anti-Gag antibody titers from immunized mice. Briefly, 96-well plates coated with 0.25 mg recombinant P24 protein overnight were blocked for 2 hours with References 1. Liu MA, Wahren B, Karlsson Hedestam GB (2006) DNA vaccines: recent developments and future possibilities. Hum Gene Ther 17: 1051–1061. 2. Laddy DJ, Weiner DB (2006) From plasmids to protection: a review of DNA vaccines against infectious diseases. Int Rev Immunol 25: 99–123. 3. Cullen BR (2003) Nuclear mRNA export: insights from virology. Trends Biochem Sci 28: 419–424. 4. Schneider R, Campbell M, Nasioulas G, Felber BK, Pavlakis GN (1997) Inactivation of the human immunodeficiency virus type 1 inhibitory elements Recoded HIV Vaccine PBS-T containing 5% dry milk and 0.5% BSA. Individual mouse serum samples were added in serial dilutions and incubated for 2 hours. The plates were washed five times with PBS-T and incubated for one hour with AKP-conjugated rat anti-mouse secondary antibodies. The plates were then washed six times with AMPAK washing solution, developed with AMPAK kit (DAKO Corporation). The plates were read on an ELISA reader at 490 nm. The end-point antibody titers were calculated as the reciprocal dilution of the last dilution that was at least 2-fold higher than normal mice sera controls and yields an absorbance of .0.1. Author Contributions Conceived and designed the experiments: YH MK AL DDH HR. Performed the experiments: YH YS DDH. Analyzed the data: RR DMW AL HR. Contributed reagents/materials/analysis tools: RR DMW DDH HR. Wrote the paper: HR. allows Rev-independent expression of Gag and Gag/protease and particle formation. J Virol 71: 4892–4903. 5. Robins H, Krasnitz M, Barak H, Levine AJ (2005) A relative-entropy algorithm for genomic fingerprinting captures host-phage similarities. J Bacteriol 187: 8370–8374. 6. Kotsopoulou E, Kim VN, Kingsman AJ, Kingsman SM, Mitrophanous KA (2000) A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codon-optimized HIV-1 gag-pol gene. J Virol 74: 4839–4852. PLoS ONE | www.plosone.org 4 September 2008 | Volume 3 | Issue 9 | e3214