Franco Lori

Latent infection of CD4 + T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy

Combination therapy for HIV-1 infection can reduce plasma virus to undetectable levels, indicating that prolonged treatment might eradicate the infection. However, HIV-1 can persist in a latent form in resting CD4+ T cells. We measured the decay rate of this latent reservoir in 34 treated adults whose plasma virus levels were undetectable. The mean half-life of the latent reservoir was very long (43.9 months). If the latent reservoir consists of only 1 × 105 cells, eradication could take as long as 60 years. Thus, latent infection of resting CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective anti-retroviral therapy.

Mutations in the pol Gene of Human Immunodeficiency Virus Type 1 in Infected Patients Receiving Didanosine and Hydroxyurea Combination Therapy

The pattern of mutations in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) strains that confer resistance to didanosine (ddI) was analyzed in 2 groups of patients receiving either ddI monotherapy or ddI plus hydroxyurea (HU) combination therapy. Twelve patients receiving combination therapy and 8 receiving monotherapy were tested. Combinations of ddI plus HU did not prevent the onset of mutations, which emerged in 50% of the patients in this group compared with 25% of the ddI monotherapy group. In addition, in 1 patient from the combination therapy arm, who had a limited response to the therapy, an unusual pattern of mutations was found: the insertion of 2 amino acids between residues 69 and 70, a region critical for resistance to nucleoside analogs. The higher efficacy of the combination of HU and ddI compared with that of ddI monotherapy cannot be attributed to a delayed or decreased onset of resistance to ddI.

Treatment of Human Immunodeficiency Virus Infection with Hydroxyurea, Didanosine, and a Protease Inhibitor before Seroconversion Is Associated with Normalized Immune Parameters and Limited Viral Reservoir

Current treatments for human immunodeficiency virus (HIV) require uninterrupted drug administration because they are unable to reconstitute the immune response and do not affect the viral reservoir. Ten patients were treated during acute HIV infection before complete Western blot (WB) seroconversion with the combination of hydroxyurea, didanosine, and indinavir. This treatment was associated with the normalization of some immune parameters and functions. No loss of naive CD4 T lymphocytes was observed, and recovery of up to 35% of naive CD8 T lymphocytes occurred in several weeks. A vigorous HIV-specific T helper response (stimulation index >8) was observed in 7 of 8 patients treated before complete WB seroconversion but in only 1 of 5 controls treated after seroconversion. In addition, a limited latent viral reservoir (<0.02-0.5 infectious units/106 cells) was documented in quiescent peripheral blood lymphocytes after treatment initiated before complete WB seroconversion.

Structured treatment interruptions to control HIV-1 infection

Structured treatment interruptions progressively lowered the rate of viral rebound in some HIV-1 infected patients. This approach should be explored as an alternative to continuous antiretroviral therapies.

Direct analysis of mitochondrial toxicity of antiretroviral drugs

ObjectivesMitochondrial toxicity is a serious side-effect of antiretroviral drugs, especially nucleoside reverse transcriptase inhibitors (NRTI). An in vitro assay to predict mithocondrial toxicity of in-use and developmental NRTI would be invaluable. To test the ability of a cytofluorimetric technique to predict the mitochondrial-dependent pancreatic and hepatic toxicity we used didanosine (ddI) alone or in combination with hydroxyurea (HU). MethodsThe technique is based on the ability of the lipophilic cation JC-1 to enter selectively into mitochondria and change its colour as the membrane potential changes due to toxicity. Mitochondrial toxicity by HU and ddI was evaluated in pancreatic and hepatic human cell lines. The results were expressed as mitochondrial toxicity index (MTI), ranging from 0 to 100: the negative control was 0, and 100 indicating maximal toxicity. ResultsDose-dependent pancreatic toxicity of ddI was evident after 14 days of culture (MTI 34 ± 4 at 100 μM, 10 ± 4 at 10 μM, 2 ± 3 at 1 μM ddI). HU alone was not toxic (MTI 7 ± 10 at 100 μM, 2 ± 2 at 50 μM and 2 ± 4 at 10 μM HU); however, HU increased the toxicity of high, but not low, concentrations of ddI. For example, the MTI of 10 μM ddI plus 50 μM HU was 54 ± 9. Negligible mitochondrial toxicity was observed in the hepatic cell line exposed to ddI alone or in combination with HU. ConclusionsThis in vitro assay might have in vivo relevance. First, ddI-related pancreatitis is dose dependent, and is reported more frequently than hepatic failure, consistent with our in vitro results. Second, patients who developed pancreatitis during randomized, controlled trials were treated with HU in combination with 400 mg ddI once daily (high peak concentration of ddI in the blood). In contrast, no pancreatitis was observed when HU was combined with 200 mg ddI twice daily (low peak concentration of ddI). These in vivo results are consistent with our in vitro observation that HU increases pancreatic cell toxicity in the presence of high concentrations of ddI. The in vitro assay described here might be used to predict the mitochondrial toxicity of other NRTI, alone or in combination.

Control of viral rebound through therapeutic immunization with DermaVir.

Objective:To reconstitute immune responses capable of eliminating infected cells and suppressing viral load during chronic retroviral infection. Design:A topical, DNA-based therapeutic immunization (DermaVir) was designed to express most of the regulatory and structural viral genes in dendritic cells. Methods:DermaVir alone and in combination with antiretroviral drugs was tested in chronically SIV-infected macaques. Results:DermaVir provided virological, immunological and clinical benefit for SIV-infected macaques during chronic infection and AIDS. In combination with antiretroviral drugs, DermaVir augmented SIV-specific T-cell responses and enhanced control of viral load rebound during treatment interruptions. Conclusions:The results indicate the feasibility of therapeutic immunization even in immune compromised hosts, and suggest that DermaVir can complement antiretroviral drugs to sustain suppression of HIV-1 replication.

Induction of Potent Human Immunodeficiency Virus Type 1-Specific T-Cell-Restricted Immunity by Genetically Modified Dendritic Cells

ABSTRACT A novel technology combining replication- and integration-defective human immunodeficiency virus type 1 (HIV-1) vectors with genetically modified dendritic cells was developed in order to induce T-cell immunity. We introduced the vector into dendritic cells as a plasmid DNA using polyethylenimine as the gene delivery system, thereby circumventing the problem of obtaining viral vector expression in the absence of integration. Genetically modified dendritic cells (GMDC) presented viral epitopes efficiently, secreted interleukin 12, and primed both CD4+ and CD8+ HIV-specific T cells capable of producing gamma interferon and exerting potent HIV-1-specific cytotoxicity in vitro. In nonhuman primates, subcutaneously injected GMDC migrated into the draining lymph node at an unprecedentedly high rate and expressed the plasmid DNA. The animals presented a vigorous HIV-specific effector cytotoxic-T-lymphocyte (CTL) response as early as 3 weeks after a single immunization, which later developed into a memory CTL response. Interestingly, antibodies did not accompany these CTL responses, indicating that GMDC can induce a pure Th1 type of immune response. Successful induction of a broad and long-lasting HIV-specific cellular immunity is expected to control virus replication in infected individuals.

HIV-1 suppression by early treatment with hydroxyurea, didanosine, and a protease inhibitor

Most HIV-1 production occurs in dividing (activated) Tlymphocytes. Non-dividing resting lymphocytes and macrophages, however, might be long-term reservoirs for HIV-1. We wanted to increase the antiviral efficacy of combination therapies by targeting simultaneously reservoir cells and the virus. Hydroxyurea blocks cellular activation necessary for viral replication in CD4 Tlymphocytes, protease-inhibitors inhibit HIV-1 replication in dividing cells, and hydroxyurea with didanosine is effective in non-dividing cells. 1,2 Eleven individuals were treated within 2 months after the onset of symptoms of primary HIV-1 infection and before complete serconversion, with a combination of hydroxyurea, didanosine, and indinavir. No greater than grade 2 toxicity was found, except for grade 3 episodes of nephrolithiasis in two patients; toxic events were resolved by switching from indinavir to nelfinavir. CD4 counts increased significantly by an average of 207 whereas CD8 counts decreased by an average of 399. CD4/CD8 ratio returned to normal in nine of eleven treated patients. This increase of CD4 counts was unexpected because the combination of hydroxyurea with didanosine, despite synergistic HIV-1 inhibition, in symptom-free individuals with CD4 counts between 200 and 500/mL, had not resulted in a significant increase of CD4 T-lymphocytes. 3

Single DermaVir immunization: Dose-dependent expansion of precursor/memory T cells against all HIV antigens in HIV-1 infected individuals

Background The GIHU004 study was designed to evaluate the safety and immunogenicity of three doses of DermaVir immunization in HIV-infected subjects on fully suppressive combination antiretroviral therapy (cART). Methodology/Principal Findings This first-in-human dose escalation study was conducted with three topical DermaVir doses targeted to epidermal Langerhans cells to express fifteen HIV antigens in draining lymph nodes: 0.1 mg DNA targeted to two, 0.4 mg and 0.8 mg DNA targeted to four lymph nodes. Particularly, in the medium dose cohort 0.1 mg DNA was targeted per draining lymph node via ∼8 million Langerhans cells located in 80 cm2 epidermis area. The 28-days study with 48-week safety follow-up evaluated HIV-specific T cell responses against Gag p17, Gag p24 and Gag p15, Tat and Rev antigens. DermaVir-associated side effects were mild, transient and not dose-dependent. Boosting of HIV-specific effector CD4+ and CD8+ T cells expressing IFN-gamma and IL-2 was detected against several antigens in every subject of the medium dose cohort. The striking result was the dose-dependent expansion of HIV-specific precursor/memory T cells with high proliferation capacity. In low, medium and high dose cohorts this HIV-specific T cell population increased by 325-, 136,202 and 50,759 counts after 4 weeks, and by 3,899, 9,878 and 18,382 counts after one year, respectively, compared to baseline. Conclusions/Significance Single immunization with the DermaVir candidate therapeutic vaccine was safe and immunogenic in HIV-infected individuals. Based on the potent induction of Gag, Tat and Rev-specific memory T cells, especially in the medium dose cohort, we speculate that DermaVir boost T cell responses specific to all the 15 HIV antigens expressed from the single DNA. For durable immune reactivity repeated DermaVir immunization might be required since the frequency of DermaVir-boosted HIV-specific memory T cells decreased during the 48-week follow up. Trial Registration ClinicalTrial.gov NCT00712530.

Expansion of CD57 and CD62L-CD45RA+ CD8 T lymphocytes correlates with reduced viral plasma RNA after primary HIV infection.

OBJECTIVE CD8 T cells, expressing cell surface molecules distinct from those on resting and naive T cells, are increased in HIV infection. The association of increased CD38 and human leukocyte antigen DR (HLA-DR) CD8 T cells with poor prognosis has suggested that activated CD8 T cells may aggravate HIV infection. We examined whether other immunological parameters might influence the viral setpoint. DESIGN Peripheral T cells from nine untreated patients, obtained after primary HIV infection when plasma HIV had stabilized, were examined for proteins expressed in activated versus resting, memory versus naive, and cytolytic versus non-cytolytic T cells. METHODS The proportion of CD8 T cells that stain for CD38 and HLA-DR, CD28 and CD57 was compared with plasma viraemia and CD4 cell count. These parameters were also compared with the proportion of CD4 and CD8 T cells that express CD62L and CD45RA, present on naive cells and down-modulated in memory cells. Internal staining for the cytotoxic protein granzyme A was also examined. RESULTS An increase in CD38 and CD38 HLA-DR CD8 T cells correlated with increased plasma viral RNA (P < 0.00002, P < 0.03, respectively). An increase in CD8 T cells expressing granzyme A was associated with lower CD4 cell counts (P < 0.04). However, the expansion of CD57 and CD62L CD45RA+ CD8 T cells was associated with a lower viral setpoint (P < 0.01, P < 0.02, respectively). CONCLUSION Phenotypically defined activated CD8 T cells may have different functions in HIV infection. Activated CD8 T cells that are CD57 or CD62L(-)CD45RA+ may be beneficial, because their expansion in untreated patients correlates with a reduced viral setpoint after primary infection.