Analysis of CD4 cell responses during 48 weeks of enfuvirtide therapy after virological failure (analysis of covariance) demonstrated significant associations between V38 mutations (n = 58 subjects) and continued CD4 cell increases and between Q40 mutations (n = 8) and loss of CD4 cell benefit (+34 versus −95 cells/μl, P < 0.001). Subjects with N43 (n = 20) or other mutations (n = 48) had intermediate CD4 cell responses. These data suggest that key enfuvirtide resistance mutations may be associated with reduced viral pathogenicity in vivo.
aTrimeris Inc., Morrisville, North Carolina, USA
bRoche, Palo Alto, California, USA
cRoche, Nutley, New Jersey, USA.
Received 1 December, 2006
Revised 26 January, 2007
Accepted 14 February, 2007
Enfuvirtide is a peptide inhibitor of HIV-1 membrane fusion and the first HIV entry inhibitor approved for clinical use. Enfuvirtide resistance mutations have been shown to reduce the rate and extent of membrane fusion  and to confer reduced viral fitness in vivo and in vitro [2,3]. Continued immunological benefit after virological failure of enfuvirtide-based therapies has also been reported [4,5]. Aquaro and colleagues  recently analysed a cohort of 54 patients and found that patients developing V38A or E mutations continued to experience CD4 cell gains after virological rebound, whereas those developing Q40H mutations experienced a loss of CD4 cells. We have therefore retrospectively examined CD4 cell responses relative to specific enfuvirtide resistance mutations in the much larger enfuvirtide phase III studies [7,8].
Data were analysed post hoc for subjects in the TORO trials who met virological failure criteria through 48 weeks, continued enfuvirtide-based therapy for at least 48 additional weeks, and had gp41 genotypes available at baseline and virological failure and CD4 cell data available at virological failure and at 8, 24 and 48 weeks after virological failure (n = 134). Subjects were analysed on the basis of the mutually exclusive presence of an enfuvirtide resistance mutation at gp41 position V38, Q40, or N43 or for all other genotypes combined (‘other’; including mutations at multiple positions). CD4 cell count changes were calculated as least squares means using analysis of covariance adjusting for the baseline CD4 cell count and viral RNA, the use of lopinavir/ritonavir, emergence of the N126K mutation in gp41 and contemporaneous change from baseline in the plasma HIV-1-RNA load. Virological failure was defined per protocol and changes to the background regimen were permitted after virological failure and were not controlled for in this analysis .
Of 355 patients who met virological failure criteria through 48 weeks, 134 (38%) also met our analysis criteria. At virological failure, 58 (43%) of these patients had V38 genotypes, 48 (36%) had other genotypes, 20 (15%) had N43 genotypes and eight (6%) had Q40 genotypes; these proportions were comparable to those in phase III studies as a whole . Baseline HIV-RNA levels were comparable across the genotype groups (5.0–5.2 log10 copies/ml) whereas a non-significant trend was observed towards lower baseline CD4 cell counts for the Q40 group, particularly relative to the V38 group (see Fig. 1). At the time of virological failure, least squares mean increases in the CD4 cell count were comparable except that the Q40 group experienced a significantly greater CD4 cell count increase than either the V38 or N43 groups (both comparisons P < 0.05).
Measured at 48 weeks after virological failure, substantial differences in the calculated CD4 cell responses were observed between the mutation groups. Most strikingly, the V38 group had gained an additional 34 CD4 cells/μl whereas the Q40 group had lost 95 CD4 cells/μl (P < 0.001; Fig. 1) relative to CD4 cell counts at virological failure. CD4 cell increases for the V38 group also showed at least a trend towards superiority relative to both the other and N43 groups at 48 weeks after virological failure (P < 0.05 compared with other and P < 0.1 compared with N43). Responses in the N43 and other groups were roughly equivalent to one another and were in both cases superior to those of the Q40 group (both comparisons with Q40 P < 0.05 at virological failure plus 48 weeks). On the basis of these data, the rank order of continued CD4 cell response associated with the mutations was V38 > N43 ∼ other > Q40. As shown in Figure 1, all mutation groups maintained least squares mean CD4 cell counts above baseline through 48 weeks of additional enfuvirtide-based therapy.
Several limitations should be considered in interpreting these data. First, the mutation groups are based on genotype at the time of virological failure and additional genotyping will be required to confirm that those genotypes were maintained at later timepoints. In addition, as our analysis was retrospective, we could only study patients who continued enfuvirtide treatment after virological failure, which is likely to have selected for patients continuing to benefit from enfuvirtide. As similar proportions of patients with each mutation type discontinued treatment , however, discontinuations are unlikely to have severely biased the comparisons between mutation groups. Finally, changes to the background regimen may have impacted these results but were not adjusted for because of the difficulty of predicting their impact on the CD4 cell response. Of note is the fact that the differences in immunological response between groups in this analysis were statistically independent of the viral load response. As the viral burden did not generally return to baseline in the V38, N43 and other groups, an overall contribution from decreased viral load is also likely.
Previous reports have indicated that virus envelope-mediated fusogenicity modulates the rate of CD4 cell depletion in SHIV animal models [11–13]. Given that some enfuvirtide mutations confer reduced viral fusogenicity in vitro , it seems plausible that enfuvirtide resistance mutations may be impacting HIV pathogenicity via an impact on fusogenicity in vivo. Such an effect could occur through directly lowering the rate of infection of target cells or via an impact on envelope-mediated immune activation or bystander cell apoptosis [14–16]. Intriguingly, the rank order of the groups' baseline CD4 cell counts corresponded to the rank order of their continued CD4 cell benefit after virological failure. This serves to highlight the point that whatever the mechanism(s) for the improvements in CD4 cell counts, the mutations could be acting directly (e.g. V38 slowing fusion more than Q40) or could have been selected on the basis of their relative tolerability within a given viral context (e.g. a fast-fusing virus might better tolerate mutations that confer a greater defect in the rate of fusion).
In summary, these data indicate that V38, N43 and other genotypes were often associated with continued CD4 cell benefits during ongoing enfuvirtide-based therapy, whereas Q40 mutations were not. These results independently confirm the recent report by Aquaro and colleagues  based on a much larger dataset and extend their findings to other common enfuvirtide resistance mutations. The combined findings suggest that gp41 genotyping may be useful in assessing the potential clinical benefit of continuing enfuvirtide-based therapy in cases in which full virological suppression is not achieved.
Sponsorship: All authors were employed and funding was provided by Trimeris, Inc. and Roche Pharmaceuticals, co-developers and marketers of Fuzeon (enfuvirtide).
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