was made through the use of an information theoretic motif- finding method called the Robins-Krasnitz algorithm, described previously[5].The algorithm identifies dozens of motifs that exhibit substantial differences in representation between the HIV-1 genome and the human coding genes. The study presented here focuses on a single motif in order to isolate its contribution to expression level in a controlled experiment. A codon-optimized version of HIV-1 consensus gag is modified, making synonymous changes to reduce the number of occurrences of AGG. Two plasmids are constructed, one with the original codon-optimized (CO) sequence of gag (AD- gag) and the other with the motif-optimized (MO) sequence with AGG significantly reduced (RK-gag). The (DNA) constructs are transfected into a human epithelial cell line (293 cells) and expression of Gag is shown to be 70% higher for the MO sequence. The two sequences of gag are also tested as DNA vaccines in a murine model for differential immune responses between the two constructs. The mice with the MO version of the vaccine have a 4.5-fold greater anti- Gag antibody response after 4 weeks. With a DNA boost at four weeks and a second readout of anti-Gag antibody titers measured at six weeks, the gap continues to widen between the MO and CO vaccines to 6-fold. Results Finding the signal and recoding Gag The Robins-Krasnitz algorithm finds short nucleotide motifs in coding regions of the human genome that are independent of amino acid order and codon usage.[5] Codon usage is defined as the distribution of synonymous codons present in a given gene. The result of the Robins-Krasnitz algorithm is a set of exact nucleotide motifs of length 2–7 bases which are under and over represented in the coding regions of the human genome. The frequency of these motifs in the HIV genome can then be assessed. See Methods for details. Beginning with the set of the 100 most under- and over- represented motifs in the human genome, our study attempts to identify the motif with the largest density difference between the HIV genome and the human genome, after accounting for A content. The motifs are restricted to the set of human genes with A content within 1% of the average HIV A content. The ratios of the densities in the HIV genome are then divided by densities in the human coding regions. If the human density is greater than that of HIV, the quantity is replaced by its reciprocal. We predict that the motif with the largest ratio of densities is responsible for nuclear isolation of HIV mRNAs. Figure 1. Gag expression in transiently transfected 293 cells. Figure 1 presents the results from four independent transfection experiments. The results are expressed as the mean P24 value (ng/ml, 6SD) of triplicates. The two different gag sequences are the codon optimized version (AD) and the motif optimized version (RK) that we created. Our (RK) version of the Gag gene has approximately two-fold higher expression than the codon optimized version. doi:10.1371/journal.pone.0003214.g001 Recoded HIV Vaccine The triplet AGG, which is significantly under represented in the coding region of the human genome, is found with a high frequency in HIV when the nucleotide bias of HIV is taken into consideration. We hypothesize that recoding the ORFs of HIV by reducing the frequency of the motif AGG will increase protein expression. For this initial study, our experimental tests focused on the Gag gene. The codon-optimized sequence of gag, referred to as AD- gag, is recoded by systematically removing all AGGs such that the amino acid sequence is not modified and very rare codons are not introduced. The result is the motif optimized RK-gag. Both the AD-gag and RK-gag sequences are found in the supplementary materials. Testing expression First, we determine whether our RK-gag has increased expression as compared to the codon optimized version, AD- gag. Since our version of Gag is undoing part of the codon optimization present in the AD-gag sequence, the protein expression levels should be expected to decrease unless the motif AGG significantly inhibits mRNA synthesis or processing or transport. To compare expression levels, human 293 cells were transfected in vitro with one of the two different versions of Gag (see methods for details). Gag protein expression was measured in the extracts of transfected cells by a quantitative P24 ELISA. RK-gag was 70% higher than the codon optimized AD-gag, with a p-value ,0.00001 calculated by a permutation test (see Figure 1). Humoral immune response To test the effect of the almost two-fold gain in expression on the immune response, we created DNA vaccines from each of the sequences. These DNA vaccines were injected into the hind leg muscle of Balb/C mice. The mice were given a booster shot after four weeks. Anti-Gag antibody titers were measured by anti-P24 ELISA at the four week and six week time points (see Methods for details). The results are found in Figure 2. The 70% increase in the expression of the GAG protein in vitro translated into more than a five-fold difference in humoral immune response in a mouse model. Two weeks post-boost, mice were sacrificed and splenocytes were prepared for measuring Gag-specific cell-mediated immune (CMI) responses by an IFN-c ELISpot assay. Although the difference between these two groups was not statistically significant, there was a trend for RK-gag immunized mice to have stronger CMI responses to both Gag-specific CD4 and CD8 peptides tested (data not shown). Discussion Recoding the Gag gene in order to reduce the occurrences of a single triplet, AGG, substantially improves immune response to an HIV DNA vaccine in a mouse model. This short sequence motif occurs less frequently in the human coding sequence than in the mouse coding sequence by about twenty percent, so it is possible that these results would be even more dramatic in humans. A set of additional steps would be required to move in the direction of a clinically viable vaccine. These include a recoding of the ENV ORF and a test of its ability to induce an improved immune response. Testing these concepts in primates would be a useful step. The goal of this study was to provide convincing evidence that recoding the HIV ORFs can improve HIV protein expression and the immune response compared to the present codon optimization schemes. It likely that including the other motifs identified by the Robins- Krasnitz algorithm in a systematic way has the potential to improve upon the large gains displayed in this study. PLoS ONE | www.plosone.org 2 September 2008 | Volume 3 | Issue 9 | e3214