Our findings show that 16–18 weeks' treatment with a standard clinical dose of valproic acid, in the setting of intensified HAART, produces a substantial decline in the frequency of replication-competent HIV in circulating resting CD4+ T cells. We measured replication-competent HIV in resting CD4+ T cells under stringent conditions: our volunteers had been successfully treated with HAART for at least 2 years; we used an additional incubation step with reverse transcriptase and integrase inhibitors to reduce to a minimum the likelihood of recovery of replication-competent HIV from recently infected cells; and we did multiple assessments in every patient before initiating treatment with enfuvirtide and valproic acid, none of which showed a consistent fall in IUPB. Depletion of HIV recovered from circulating resting CD4+ cells after treatment, however, indicated a pronounced shortening of the half-life of the latent reservoir.
An examination of the response of individual patients is revealing. Patient 1 had a single episode of transient viraemia, possibly associated with an intercurrent infection, before protocol therapy, and also had low-level viraemia that was unaffected by the addition of enfuvirtide. Resting cell IUPB did not decline much in this patient after therapy with enfuvirtide and valproic acid. Low-level viraemia continued while on intensified HAART. Perhaps these clinical characteristics explain the lack of depletion of resting cell infection seen in this individual. Patient 2 had durable and complete suppression of detectable viraemia, without known episodes of viraemia, without recoverable HIV in CD8-depleted cultures, and without detectable viraemia in the single-copy assay. IUPB of resting cells had fallen greatly by the end of the study. Patient 3 had two episodes of transient viraemia during a state of immune activation before protocol therapy, and one while on study treatment. Low-level viraemia was detected throughout the study, despite the addition of enfuvirtide to HAART. After treatment with enfuvirtide and valproic acid, resting cell IUPB declined. It is noteworthy that resting cell IUPB declined in this patient during therapy with intensified HAART and valproic acid despite continued viraemia. Finally, the IUPB of patient 4's resting CD4+ Tcells measured before protocol therapy was stable. Low-level viraemia was detected before the addition of enfuvirtide, decreased after enfuvirtide therapy, and was less than 1 copy per mL 1 month after enfuvirtide was discontinued. However, this apparent decline might be within the range of experimental variation of the one-copy assay.
Although seminal HIV RNA was not detected, HIV was initially recovered from CD8-depleted PBMCs. Consistent with the suppression of low-level replication by intensified HAART, CD8-depleted cultures were negative at the end of study. After 12 weeks of exposure to valproic acid and intensified HAART a decline of resting cell IUPB was noted.
Our pilot study is limited and leaves many questions unanswered. First, we did not note any immune activation in our patients. This finding is probably due to a combination of factors. HIV expressed in resting cells might have been too rare to induce detectable immune activation, a response of HIV-specific T cells present in these durably suppressed patients might have been too rare to detect, or immune responses could have been blunted by effective containment of new rounds of HIV replication by HAART and enfuvirtide. Second, one patient, whose background HAART included zidovudine, developed anaemia. Valproic acid inhibits glucuronidation of zidovudine and increases its bioavailability [35
], theoretically increasing the risk of zidovudine-induced anaemia. Whether this adverse effect is infrequently seen in clinical practice, or is under-reported [36
] is unclear. In our ongoing studies, we plan to avoid the coadministration of valproic acid and zidovudine. Third, treatment with valproic acid and enfuvirtide seems to have reduced latent HIV infection to an unprecedented extent. We postulate that HDAC inhibition is essential to achieve this effect. However, this study is too small to separate the effects of entry and HDAC inhibition. Furthermore, two of our patients had persistent, low-level viraemia despite the addition of enfuvirtide, an antiretroviral with a new mechanism to which they had not previously been exposed. The source and importance of this viraemia is unclear, and an important topic for future study. Nevertheless, we noted depletion of resting cell infection in one individual despite continuous low level viraemia. Finally, we have studied a small number of patients, with a single course of therapy of an arbitrary duration. The depletion of resting cell infection we noted seems much greater than that reported after intensification [7
], and at least as great as that reported after long-term therapy with interleukin 2 [11
]. However, further studies are needed to confirm, expand, and deepen our observations.
Our findings suggest that eradication of established HIV infection might be achieved in a staged approach. Patients should perhaps first be treated with standard antiretroviral regimens at an early stage of infection. For those in whom viral replication is suppressed, latent viral infection should then be tackled with HDAC inhibitors, intensified therapy, or both. Alternatively, or additionally, therapeutic vaccination might augment the antiviral immune response.