A Recoding Method to Improve the Humoral Immune Response to an HIV DNA Vaccine Yaoxing Huang1., Michael Krasnitz2., Raul Rabadan2., Daniela M. Witten3., Yang Song 1 , Arnold J. Levine2, David D. Ho 1 , Harlan Robins4* 1 Aaron Diamond AIDS Research Center, The Rockefeller University, New York, New York, United States of America, 2 Institute for Advanced Study, Princeton, New Jersey, United States of America, 3 Department of Statistics, Stanford University, Stanford, California, United States of America, 4 Computational Biology Group, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America Abstract This manuscript describes a novel strategy to improve HIV DNA vaccine design. Employing a new information theory based bioinformatic algorithm, we identify a set of nucleotide motifs which are common in the coding region of HIV, but are under-represented in genes that are highly expressed in the human genome. We hypothesize that these motifs contribute to the poor protein expression of gag, pol, and env genes from the c-DNAs of HIV clinical isolates. Using this approach and beginning with a codon optimized consensus gag gene, we recode the nucleotide sequence so as to remove these motifs without modifying the amino acid sequence. Transfecting the recoded DNA sequence into a human kidney cell line results in doubling the gag protein expression level compared to the codon optimized version. We then turn both sequences into DNA vaccines and compare induced antibody response in a murine model. Our sequence, which has the motifs removed, induces a five-fold increase in gag antibody response compared to the codon optimized vaccine. Citation: Huang Y, Krasnitz M, Rabadan R, Witten DM, Song Y, et al. (2008) A Recoding Method to Improve the Humoral Immune Response to an HIV DNA Vaccine. PLoS ONE 3(9): e3214. doi:10.1371/journal.pone.0003214 Editor: Douglas F. Nixon, University of California San Francisco, United States of America Received March 4, 2008; Accepted August 26, 2008; Published September 15, 2008 Copyright: 2008 Huang 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. Funding: This work was supported in part by the Simons Foundation, the Ambrose Monell Foundation, and the Leon Levy Foundation. YH, YS, and DH are supported in part by the Irene Diamond Fund. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hrobins@fhcrc.org . These authors contributed equally to this work. Introduction In the effort to create a vaccine for human immunodeficiency virus type 1 (HIV-1), poor immune response to the HIV proteins is a fundamental problem. For a DNA vaccine, the immune response is correlated with protein expression levels, so an increase in expression of these proteins could alleviate a significant road block to the construction of a viable DNA vaccine.[1,2] Transcription of the HIV DNA copy is an inefficient process that is aided by the addition of an HIV protein (TAT). In addition the large mRNAs from HIV have inherent RNA processing problems and are not efficiently exported from the nucleus in the absence of a helper protein (REV). These m-RNA synthesis and transport problems are presumably due to a set of RNA sequences or structures encoded in HIV DNA and RNAs.[3,4] We hypothesize that identifying and removing these signals, which cause the poor synthesis and nuclear confinement of HIV RNA, should significantly increase expression levels of these proteins and improve the immune response. The genome of HIV-1 contains nine open reading frames (ORFs), all of which are expressed from a single promoter through alternative splicing. The splice forms for the six ORFs Gag, Pol, Env, Vpu, Vif, and Vpr, along with the full length mRNA, contain Rev response elements (RREs) encoded in their RNA. In the absence of the Rev protein, these six ORFs are poorly expressed. The remaining three ORFs, Tat, Rev, and Nef, are expressed efficiently independently of the Rev protein.[3] The mRNAs which contain RREs likely also contain an as yet unidentified signal or set of signals which prevents normal expression.[4] A primary cause of the poor expression of these ORFs is nuclear confinement.[3] The genome of HIV-1 has an anomalous nucleotide distribution as compared with the set of known coding genes in the human genome. Only 314 of the approximately 25000 genes in the human genome have a higher percentage of adenine (A) than the average clinical isolate of HIV- 1. Similar A content can be found in other retrotranscribing viruses (e.g. LINE elements, lentiviruses, spumaviruses); this suggests that retroviruses undergo different selection pressures than ones directing the evolution of the human genome. In the early 90’s, the Pavlakis lab showed experimentally that synony- mous changes to the Gag ORF, which decrease the A content, significantly increase expression of Gag in human cells.[4] Codon- optimized strains, which are widely used in present experiments and vaccine trials, can increase the protein expression level of Gag transfected into human cells between 500 and 1000 fold.[1,2] However, the substantial increase in expression due to codon optimization can, at best, indirectly address the problem of poor synthesis and nuclear isolation. We identify multiple nucleotide motifs from a systematic comparison of the HIV-1 genome and the human genome, which we conjecture to play a causative role in poor synthesis and nuclear confinement. In this study, the short motif, AGG, is found to have the maximal differential representation between the coding genes in the human genome and the HIV-1 genome. This identification PLoS ONE | www.plosone.org 1 September 2008 | Volume 3 | Issue 9 | e3214