Purging the latent reservoir for HIV-1 requires reactivation of latent virus and then elimination of infected host cells. Recent studies have identified several pharmacologic agents that reverse latency without inducting global T cell activation (Ylisastigui et al., 2004
; Contreras et al., 2009
; Archin et al., 2009
; Yang et al., 2009
and Xing et al., 2011
). In our study, SAHA was tested for the reactivation of latent virus and was shown to be as effective as general T cell activators in our primary cell model, although with slower kinetics. SAHA is currently used in the treatment of cutaneous T-cell lymphoma but is associated with common gastrointestinal and hematologic adverse effects. Following the development of several human primary cell models of HIV-1 latency (Sahu et al., 2006
; Marini et al., 2008
; Bosque and Planelles, 2009
; Yang et al., 2009
and Tyagi et al., 2010
), other agents that reactivate latent virus have been identified (Yang et al., 2009
and Xing et al., 2011
), and it is likely that many additional agents will be found in these screens. Mechanisms of HIV-1 latency are also being extensively studied in these primary cell models and cell line models (Jordan et al., 2003
). Eventually, combination of agents affecting different pathways will likely be used to maximally reactivate latent viruses in vivo
. However, the reactivation of latent virus is only the first step to viral eradication. Elimination of the infected cells after virus reactivation has not been previously studied.
It is often assumed that the latently infected CD4+
T cells will die after the virus is reactivated because of viral CPE or host immunity, or both. Here we demonstrate that neither viral CPE nor CD8+
T cells from patients on HAART are sufficient to cause immediate death of latently infected resting CD4+
T cells after virus reactivation with agents that do not cause T cell activation. In our in vitro
model of HIV-1 latency, Bcl-2 is over-expressed in order to promote long-term survival of primary CD4+
T cells. Bcl-2 over-expression in our system imposes some limitations and could potentially interfere with the study of viral CPE or the CTL response. To address this concern, freshly isolated primary resting CD4+
T cells were also used for the study of viral CPE and similar effects were observed. Specifically, we showed that reactivation of latent HIV-1 in freshly isolated, un-transduced resting CD4+ T cells from patients on HAART does not result in death to the infected cells. With regard to studies of CTL-mediated killing of infected cells, we showed that over-expression of Bcl-2 does not protect infected target cells from cytolytic effects of CD8+
T cells, which was consistent with other studies (Zhang et al., 2001
; Packard et al., 2007
and Goping et al., 2008
). Therefore, the lack of cell death after virus reactivation in our in vitro
model is unlikely to be due to over-expression of Bcl-2.
Resting memory CD4+
T cells may be resistant to viral CPE for several reasons. First, viral gene transcription and translation are much less efficient in resting cells. Second, resting memory CD4+
T cells are in general less prone to cell death than activated CD4+
T cells (Stockinger et al., 2006
; Surh et al., 2006
; van Leeuwen et al., 2009
and Taylor and Jenkins, 2011
). Third, resting memory CD4+
T cells remain at a quiescent G0
state after treatment with agents like SAHA, and in this state are not affected by the viral proteins Vpr and Vif (Roshal et al., 2001
; Sakai et al., 2006
and Shedlock et al., 2008
), which cause cell cycle arrest and cell death in activated cells. Thus reversing latency with agents that do not induce global T cell activation will not eliminate the latent reservoir. Finding a way to eliminate the cellular reservoir after virus reactivation is thus an important step to viral eradication.
While HIV-1-specific CTL are presumed to be capable of eliminating latently infected cells after the reversal of latency, we found that freshly isolated CD8+
T cells from patients on HAART were only effective at high E:T ratios and with a prolonged period of coculture. Little killing of infected target cells was observed within the first two days of coculture. One explanation is that the frequency of HIV-1-specific CD8+
T cells in patients on HAART is diminished as a result of the lack of antigen stimulation. In addition, the CTL dysfunction seen in patients with progressive disease is not fully restored on HAART (Kalams et al., 1999
; Saez-Cirion et al., 2007
; Migueles et al., 2008
; Migueles et al., 2009
and Hersperger et al., 2010
CD8+ T cells from one of eight patients (HAART patient 06) on HAART retained strong ability to kill infected target cells without in vitro stimulation. We used Gag peptides to stimulate CD8+ T cells from all eight patients on HAART and found that this patient had more Gag-specific CD8+ T cells and/or better antigen-driven proliferation than other patients. We also found that this patient had higher Granzyme B and INF-γ production than a representative control patient after in vitro stimulation with Gag peptides. This patient also had a notable CTL response against other HIV-1 proteins. It appears that this patient has a larger number of HIV-1-specific CD8+ T cells and has broader recognition of viral epitopes. Unfortunately, the majority of the patients on HAART does not have such strong CTL response and may not be able to eliminate the latent reservoir after virus reactivation.
Here we demonstrated that CTL activity could be restored through in vitro stimulation with Gag peptides for every patient on HAART studied. This suggests that HIV-1-specific CTL responses in patients on HAART are defective but can be restored to effectively eliminate latently infected resting CD4+ T cells after virus reactivation. Boosting CTL responses and then reactivating latent HIV-1 may be an efficient strategy to eradicate HIV-1. Stimulation with other viral proteins could provide a broader CTL response in some of the patients. Since reactivation strategies will likely be implemented with the presence of HAART, further rounds of viral replication are inhibited and de novo CTL escape mutations cannot arise. The pre-existing CTL escape variants archived in latent reservoir can be overcome by inducing strong and broad CTL responses against multiple viral epitopes, and are not an obstacle to elimination.
Other strategies for promoting the death of infected cells have been proposed. For example, elegant studies have shown that cells expressing the HIV-1 Env protein could be targeted using antibodies conjugated bacterial toxins (Brooks et al., 2003
). The application of this approach may be limited because of the high variability of the Env protein, drug bioavailability, and potential adverse effects.
In conclusion, resting CD4+ T cells latently infected with HIV-1 will not be efficiently killed by either viral CPE, or host CTL responses after virus reactivation. Our study strongly suggests that boosting CTL responses through vaccination prior to virus reactivation may be essential for eradication of HIV-1 infection.