Abstract
A tropism test is required prior to initiation of CCR5 antagonist therapy in HIV-1 infected individuals, as these agents are not effective in patients harboring CXCR4 (X4) coreceptor-using viral variants. We developed a clinical laboratory-based genotypic tropism test for detection of CCR5-using (R5) or X4 variants that utilizes triplicate population sequencing (TPS) followed by ultradeep sequencing (UDS) for samples classified as R5. Tropism was inferred using the bioinformatic algorithms geno2pheno[coreceptor] and PSSMx4r5. Virologic response as a function of tropism readout was retrospectively assessed using blinded samples from treatment-experienced subjects who received maraviroc (N = 327) in the MOTIVATE and A4001029 clinical trials. MOTIVATE patients were classified as R5 and A4001029 patients were classified as non-R5 by the original Trofile test. Virologic response was compared between the R5 and non-R5 groups determined by TPS, UDS alone, the reflex strategy and the Trofile Enhanced Sensitivity (TF-ES) test. UDS had greater sensitivity than TPS to detect minority non-R5 variants. The median log10 viral load change at week 8 was −2.4 for R5 subjects, regardless of the method used for classification; for subjects with non-R5 virus, median changes were −1.2 for TF-ES or the Reflex Test and −1.0 for UDS. The differences between R5 and non-R5 groups were highly significant in all 3 cases (p<0.0001). At week 8, the positive predictive value was 66% for TF-ES and 65% for both the Reflex test and UDS. Negative predictive values were 59% for TF-ES, 58% for the Reflex Test and 61% for UDS. In conclusion, genotypic tropism testing using UDS alone or a reflex strategy separated maraviroc responders and non-responders as well as a sensitive phenotypic test, and both assays showed improved performance compared to TPS alone. Genotypic tropism tests may provide an alternative to phenotypic testing with similar discriminating ability.
Highlights
In order for the human immunodeficiency virus type 1 (HIV-1) to infect cells, its gp120 envelope glycoprotein must interact with the cellular CD4 receptor and one of two chemokine coreceptors: CCR5 or CXCR4 [1,2,3]
The baseline median viral load was similar regardless of tropism results the baseline CD4(+) T cell count was lower for subjects predicted to have nonR5 virus by both assays (Table 1, X4/X4 group) or by Trofile Enhanced Sensitivity (TF-ES) alone (Table 1, R5/X4 group)
We have presented an analysis of a genotypic reflex strategy for tropism testing
Summary
In order for the human immunodeficiency virus type 1 (HIV-1) to infect cells, its gp120 envelope glycoprotein must interact with the cellular CD4 receptor and one of two chemokine coreceptors: CCR5 or CXCR4 [1,2,3]. Tropism can be determined by phenotypic or genotypic testing Phenotypic assays such as the original Trofile and the more recently offered Trofile Enhanced Sensitivity (TF-ES) from Monogram Biosciences measure the ability of pseudoviruses carrying the entire cloned envelope gene from a patient’s virus to infect CD4(+)/CCR5(+) and CD4(+)/CXCR4(+) indicator cells [16,17]. This approach has proven to be sensitive and correlates well to clinical outcomes [10,14], phenotypic testing is expensive to perform and requires a relatively long turnaround time
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