12th International HIV Drug Resistance Workshop


10–14 June 2003, Cabo del Sol, Los Cabos, Mexico


DETERMINANTS OF SUSCEPTIBILITY TO ENFUVIRTIDE MAP TO GP41 IN ENFUVIRTIDE-NAÏVE HIV-1

Antivir Ther. 2003; 8:S25 (abstract no. 22)

SA Stanfield-Oakley1, J Jeffrey1, CB McDanal1, S Mosier1, L Talton1, L Jin1, P Sista1, N Cammack2, TJ Matthews1 and ML Greenberg1
1Trimeris, Inc., Durham, NC; and 2Roche, Palo Alto, Calif., USA

BACKGROUND: Enfuvirtide (ENF, formerly T-20) is the first fusion inhibitor to demonstrate efficacy in controlled Phase III trials. Fusion inhibitor (FI)-naïve HIV-1 isolates exhibit a range of susceptibility to ENF in vitro, and several studies have suggested that HIV-1 envelope co-receptor tropism or affinity may contribute to this range. Co-receptor tropism and affinity are conferred by the gp120 subunit of the HIV-1 envelope glycoprotein, thus, those studies imply that determinants of susceptibility to ENF lie within gp120. Using chimeric envelope constructs and site-directed mutagenesis, we examined the envelope gp120 and gp41 subunits as loci for determinants of ENV susceptibility.

METHODS: Sensitivity of HIV-1 isolates to ENF was determined in a cMAGI infectious centre assay. HIV-1 env genes from R5- and X4-tropic isolates exhibiting a range of ENF sensitivities were cloned into an expression vector and served as the starting point for gp120- gp41 chimeric envelope constructs. Co-receptor tropism and sensitivity of envelope clones and chimeras to ENF were determined from Env-deficient reporter viruses pseudotyped with these envelopes following cotransfection of envelope expression vectors and an env-deficient NL4-3-based reporter virus construct into 293T cells. The pseudotyped reporter viruses produced were evaluated on U87 cells expressing CD4 and either CCR5 or CXCR4. Sequences of all cloned and chimeric envelope constructs were determined by dideoxy sequencing chemistries using a Beckman Coulter CEQ 2000XL system and DNAStar software.

RESULTS: Full-length functional envelopes were cloned from five R5-tropic and two X4 tropic clade B primary virus isolates of HIV-1. The cloned envelopes exhibited ENF IC50s ranging 0.04–12.6 µg/ml in the U87-based pseudotyped reporter virus assay. The cloned envelopes retained the same tropism characteristics noted with the parental virus isolates. The gp120/gp41 chimeric envelopes exhibited tropism specificity of the gp120 parental virus as expected. On the other hand, the major determinants of ENF sensitivity tracked with the gp41 donor. Chimeric env constructs exchanging either the N-terminal ectodomain (containing HR1) or the C-terminal ectodomain through the end of gp41 (containing HR2) suggested that both regions can contribute to baseline susceptibility to ENF. In support of this assertion, we found that ENF sensitivity can be modulated by SDM of gp41 amino acids 45 (within HR1) and 135 (within HR2) with amino acids observed in rare FI-naïve isolates or those more commonly found.

CONCLUSIONS: Previous studies have demonstrated that the HR1 region of the HIV-1 gp41 is the target for ENF. In addition, results from Phase III clinical studies of ENF have shown that this same region is the primary locus for development of ENF resistance. Our current results suggest that gp41 also contains the major determinants for baseline sensitivity to ENF of clade B FI-naïve virus.

PRESENTING AUTHOR: SA Stanfield-Oakley

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2003-07-08
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