Expanded cellular clones carrying replication-competent HIV-1 persist, wax, and wane.

The latent reservoir for HIV-1 in resting CD4+ T cells is a major barrier to cure. Several lines of evidence suggest that the latent reservoir is maintained through cellular proliferation. Analysis of this proliferative process is complicated by the fact that most infected cells carry defective proviruses. Additional complications are that stimuli that drive T cell proliferation can also induce virus production from latently infected cells and productively infected cells have a short in vivo half-life. In this ex vivo study, we show that latently infected cells containing replication-competent HIV-1 can proliferate in response to T cell receptor agonists or cytokines that are known to induce homeostatic proliferation and that this can occur without virus production. Some cells that have proliferated in response to these stimuli can survive for 7 d while retaining the ability to produce virus. This finding supports the hypothesis that both antigen-driven and cytokine-induced proliferation may contribute to the stability of the latent reservoir. Sequencing of replication-competent proviruses isolated from patients at different time points confirmed the presence of expanded clones and demonstrated that while some clones harboring replication-competent virus persist longitudinally on a scale of years, others wax and wane. A similar pattern is observed in longitudinal sampling of residual viremia in patients. The observed patterns are not consistent with a continuous, cell-autonomous, proliferative process related to the HIV-1 integration site. The fact that the latent reservoir can be maintained, in part, by cellular proliferation without viral reactivation poses challenges to cure.

Control of HIV-1 in elite suppressors despite ongoing replication and evolution in plasma virus.

A subset of HIV-1-infected patients known as elite controllers or suppressors (ES) control the virus naturally. We have previously demonstrated sequence discordance between proviral and plasma gag clones in ES, much of which can be attributed to selective pressure from the host (J. R. Bailey, T. M. Williams, R. F. Siliciano, and J. N. Blankson, J. Exp. Med. 203:1357-1369, 2006). However, it is not clear whether ongoing viral replication continues in ES once the control of viremia has been established or whether selective pressure impacts this evolution. The cytotoxic T-lymphocyte (CTL) response in ES often targets Gag and frequently is superior to that of HIV-1 progressors, partially due to the HLA class I alleles B*57/5801 and B*27, which are overrepresented in ES. We therefore examined longitudinal plasma and proviral gag sequences from HLA-B*57/5801 and -B*27 ES. Despite the highly conserved nature of gag, we observed clear evidence of evolution in the plasma virus, largely due to synonymous substitutions. In contrast, evolution was rare in proviral clones, suggesting that ongoing replication in ES does not permit the significant reseeding of the latent reservoir. Interestingly, there was little continual evolution in CTL epitopes, and we detected de novo CTL responses to autologous viral mutants. Thus, some ES control viremia despite ongoing replication and evolution.

Evolution of the HIV-1 nef gene in HLA-B*57 positive elite suppressors.

Elite controllers or suppressors (ES) are HIV-1 infected patients who maintain viral loads of < 50 copies/ml without antiretroviral therapy. CD8+ T cells are thought to play a key role in the control of viral replication and exert selective pressure on gag and nef in HLA-B*57 positive ES. We previously showed evolution in the gag gene of ES which surprisingly was mostly due to synonymous mutations rather than non-synonymous mutation in targeted CTL epitopes. This finding could be the result of structural constraints on Gag, and we therefore examined the less conserved nef gene. We found slow evolution of nef in plasma virus in some ES. This evolution is mostly due to synonymous mutations and occurs at a rate similar to that seen in the gag gene in the same patients. The results provide further evidence of ongoing viral replication in ES and suggest that the nef and gag genes in these patients respond similarly to selective pressure from the host.

The HBV drug entecavir - effects on HIV-1 replication and resistance.

Entecavir, a drug approved by the Food and Drug Administration for the treatment of chronic hepatitis B virus (HBV) infection, is not believed to inhibit replication of human immunodeficiency virus type 1 (HIV-1) at clinically relevant doses. We observed that entecavir led to a consistent 1-log(10) decrease in HIV-1 RNA in three persons with HIV-1 and HBV coinfection, and we obtained supportive in vitro evidence that entecavir is a potent partial inhibitor of HIV-1 replication. Detailed analysis showed that in one of these patients, entecavir monotherapy led to an accumulation of HIV-1 variants with the lamivudine-resistant mutation, M184V. In vitro experiments showed that M184V confers resistance to entecavir. Until more is known about HIV-1-resistance patterns and their selection by entecavir, caution is needed with the use of entecavir in persons with HIV-1 and HBV coinfection who are not receiving fully suppressive antiretroviral regimens.

Evidence of CD8+ T-cell-mediated selective pressure on human immunodeficiency virus type 1 nef in HLA-B*57+ elite suppressors.

Elite suppressors (ES) are human immunodeficiency virus type 1 (HIV-1)-infected patients who maintain viral loads of <50 copies/ml without treatment. The observation that the HLA-B*57 allele is overrepresented in these patients implies that HIV-1-specific CD8(+) T cells play a key role in suppressing viral replication. We have previously shown that while CD8(+) T-cell escape mutations are rarely seen in proviral Gag sequences in resting CD4(+) T cells from peripheral blood, they are present in every clone amplified from the low levels of free virus in the plasma of HLA-B*57(+) ES. In this study, we compared the pattern of mutations in Nef sequences amplified from peripheral blood CD4(+) T cells and from plasma virus. We show that Nef mutations are present in plasma virus but are rare in the cellular sequences and provide evidence that these plasma Nef variants represent novel escape mutants. The results provide further evidence of CD8(+) T-cell-mediated selective pressure on plasma virus in ES and suggest that there must be ongoing HIV-1 replication in spite of the very low viral loads seen for these patients.

Analysis of human immunodeficiency virus type 1 viremia and provirus in resting CD4+ T cells reveals a novel source of residual viremia in patients on antiretroviral therapy.

Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4(+) T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure--analysis of molecular variance and the Slatkin-Maddison test--to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4(+) T cells but that proviruses in resting and activated CD4(+) T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4(+) T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4(+) T cells has implications for eradication efforts.

Limits on replenishment of the resting CD4+ T cell reservoir for HIV in patients on HAART.

Whereas cells productively infected with human immunodeficiency virus type 1 (HIV-1) decay rapidly in the setting of highly active antiretroviral therapy (HAART), latently infected resting CD4(+) T cells decay very slowly, persisting for the lifetime of the patient and thus forming a stable reservoir for HIV-1. It has been suggested that the stability of the latent reservoir is due to low-level viral replication that continuously replenishes the reservoir despite HAART. Here, we offer the first quantitative study to our knowledge of inflow of newly infected cells into the latent reservoir due to viral replication in the setting of HAART. We make use of a previous observation that in some patients on HAART, the residual viremia is dominated by a predominant plasma clone (PPC) of HIV-1 not found in the latent reservoir. The unique sequence of the PPC serves as a functional label for new entries into the reservoir. We employ a simple mathematical model for the dynamics of the latent reservoir to constrain the inflow rate to between 0 and as few as 70 cells per day. The magnitude of the maximum daily inflow rate is small compared to the size of the latent reservoir, and therefore any inflow that occurs in patients on HAART is unlikely to significantly influence the decay rate of the reservoir. These results suggest that the stability of the latent reservoir is unlikely to arise from ongoing replication during HAART. Thus, intensification of standard HAART regimens should have minimal effects on the decay of the latent reservoir.

Development of an aciclovir implant for the effective long-term control of herpes simplex virus type-1 infection in Vero cells and in experimentally infected SKH-1 mice.

Human herpes simplex virus type-1 (HSV-1) is treatable with oral doses of an antiviral agent such as aciclovir (ACV), a drug that has poor bioavailability. An alternative for delivering ACV would employ a long-lived subcutaneous implant that would allow for near zero-order drug delivery kinetics. This study aimed to develop an implant composed of a matrix of silicone and ACV that is capable of sustained long-term release of ACV. Once the implants had been created, release of ACV from the implants was determined and quantified in vitro using a spectrophotometric assay for the drug. Solvent-exposed surface area of the implant (2.86 mm(2), 6.28 mm(2), 34.62 mm(2) and 100.48 mm(2)) had a significant effect on release kinetics, whereas temperature (37 degrees C, 25 degrees C and 4 degrees C) and pH (6.0, 7.0 and 8.0) did not. The implants were also used successfully to suppress HSV-1 (KOS)-induced cytopathic effect in cultured Vero cells. The implants protected HSV-1-infected SKH-1 mice from viral reactivation (n = 37; P = 0.0367) via ultraviolet light compared with mice that were untreated (n = 37). Furthermore, mice that received silicone-only implants had no lowered risk of reactivation (n = 34; P = 0.7268), demonstrating the antiviral efficacy of the ACV implants.