POSTER 82

1. POSTER 82 CXCR4 using HIV-1 strains more abundant in PBMC than in plasma Chris Verhofstede1*, Linos Vandekerckhove2, Veerle van Eygen3, Kenny Dauwe1, Jacqueline Reynaerts1, Jean Plum1, Dirk Vogelaers2 and Lieven Stuyver3 Aids Reference Laboratory1 and Aids Reference Centre2, University Hospital, Ghent, Belgium; Virco, Mechelen, Belgium3 Results Useful clonal RNA and DNA sequences were obtained from 11 patients. Sequencing failed for RNA or DNA in 2 patients. Sequences predicted to be from X4 strains were detected in 10 of the 11 patients, including all patients with a positive MT2 culture and 2 of the 3 patients with no MT2 result available but with indication of disease progression. In all 10 patients with X4 virus, X4 strains were depicted from both plasma and PBMC, but the frequency of their occurrence was different in both blood compartments. X4 strains were significantly more frequently detected in PBMC compared to plasma. The higher abundance of X4 strains in PBMC, was reflected in a higher overall mean PSSM scores for PBMC but not in a shift in PSSM range (see Table 1). An overview of all V3 amino acid sequences that were obtained with the corresponding PSSM interpretation, PSSM score and frequency of occurrence is shown in Table 2. X4 strains are presented with a grey background. Background Current phenotypic and genotypic assays for tropism prediction use the patient’s plasma for RNA extraction and subsequent analysis. For the conventional MT2 assay on the other hand, virus was isolated from the patient’s peripheral blood mononuclear cells (PBMC). Whether R5 and X4 viruses are equally represented in both blood compartments is unknown. The study presented was designed to compare the dynamics of R5 and X4 viruses in plasma and PBMC. Material and methods Thirteen patients were selected, all males infected with a subtype B HIV-1 virus. Nine patients had a positive MT2 culture on the sample used for analysis. Four patients were included because they had viral or immunological indications of an evolved disease. Extensive clonal sequencing of the envelope gene was performed on viral RNA extracted from the plasma and proviral DNA extracted from the PBMC. The coreceptor tropism of all clonal sequences was predicted based on the V3 loop sequence using the PSSM prediction algorithm (http://ubik.microbiol.washington.edu/computing/pssm/). Below: illustration of the PSSM plots for plasma RNA and the cellular DNA clones (for patient 9601) Right: frequency of X4 strains expressed as a percentage of all clones in plasma (blue) and PBMC (red) for the 11 patients analyzed. Fisher’s exact p values for each patient are depicted on top. Overall distribution of the PSSM scores for the plasma (blue) and PBMC (red) sequences. All sequences from the 10 patients in whom X4 viruses were detected were included in this analysis. The results reveal that viral strains with a PSSM score of less than -7.0 (indicating a very close resembling with R5 viruses) are preferentially located in the plasma, while viral strain with a PSSM score of more than -3.0 (indicating a very close resemblance with known X4 viruses) preferentially reside in PBMC. The distribution of viral strains with a PSSM score between -7.0 and -3.0 seems evenly distributed between plasma and PBMC. These intermediate scores are likely to be associated with a dual-tropic phenotype. Conclusion This study illustrates the capacity of clonal sequencing and bioinformatic coreceptor prediction tool to detect X4 strains in both free plasma virus and cell associated provirus. Most importantly, this work shows that the frequency of occurrence of viruses classified as X4 based on the amino acid sequence of the envelope V3, is significantly higher in infected PBMC that in free plasma virus. These findings can have important implications for the development of future strategies for coreceptor tropism determination and for the use of coreceptor antagonists.