Teresa Gallart

Dynamics of viral load rebound and immunological changes after stopping effective antiretroviral therapy

BACKGROUND This study addresses the dynamic of viral load rebound and immune system changes in a cohort of eight consecutive HIV-1-infected patients in very early stages [all the patients were taking highly active antiretroviral therapy (HAART} and were recruited in the coordinating center from a larger study] who decided to discontinue HAART after 1 year of treatment and effective virologic response. The safety of this procedure and the outcome with reintroduction of the same treatment was also investigated. METHODS Plasma, cerebrospinal fluid (CSF), and lymphatic tissue viral loads were measured at baseline; lymphocyte immunophenotyping and CD4 lymphocyte proliferative responses to mitogens and specific antigens were assessed. The same antiretroviral therapy was reintroduced as soon as plasma viral load became detectable (above 200 copies/ml). RESULTS At day 0, plasma viral load was below 20 copies/ml in all eight patients (and below 5 copies/ml in five of eight patients). A rebound in plasma viral load was detected in all patients from day 3 to day 31 with a mean doubling time of 2.01 (SE 0.29) days. Three out of eight patients achieved a peak plasma viral load at least 0.5 log10 above baseline, pretreatment values. Mutations associated with resistance to reverse transcriptase or protease inhibitors were not detected. After 31 days off therapy, CD4 lymphocytes decreased [mean 45% (SE 4) to 37% (SE 3); P = 0.04], CD8+CD28+ lymphocytes decreased [mean 59% (SE 5) to 43% (SE 4); P = 0.03], and CD8+CD38+ lymphocytes increased [mean 55% (SE 3) to 66% (SE 4); P = 0.009]. Mean stimulation indices of lymphocytes treated with phytohemagglutinin (PHA) and CD3 decreased from day 0 to day 31 from 34% (SE 8) to 17% (SE 9) (P = 0.06) and from 24% (SE 8) to 5% (SE 2) (P = 0.02), respectively. These changes were mainly contributed by the group of five patients with plasma viral load below 5 copies/ml at day 0. Viral load dropped below 20 copies/ml in all patients after 1 month of restarting the same antiretroviral regimen. CONCLUSIONS Discontinuation of HAART after 1 year of successful treatment is followed by a rapid rebound of viral load; this rapidly returns to undetectable levels following reintroduction of the same treatment. In patients with more effective control of virus replication (viremia below 5 copise/ml), discontinuation of treatment was associated with more severe impairment of immunologic parameters.

The virological and immunological consequences of structured treatment interruptions in chronic HIV-1 infection

BackgroundSome individuals with chronic HIV-1 infection have discontinued their drug therapy with consequent plasma virus rebound. In a small number of patients, a delayed or absent rebound in plasma virus load has been noted after drug cessation, apparently associated with prior drug interruptions and autologous boosting of HIV-1 specific immune responses. We hypothesized that cyclic structured treatment interruptions structured treatment interruptions (STI) could augment HIV-1 specific immune responses in chronic HIV-1 infection, which might help to control HIV-1 replication off therapy. MethodsWe initiated an STI pilot study in 10 antiretroviral treatment-naive HIV-1 chronically infected subjects with baseline CD4 T-cell counts > 500 × 106 cells/l and plasma viral load > 5000 copies/ml who received highly active antiretroviral therapy (HAART) for 1 year with good response (plasma viral load < 20 copies/ml for at least 32 weeks). Three cycles of HAART interruption were performed. ResultsIn all of the patients viral load rebounded, but doubling times increased significantly between the first and third stops (P = 0.008), and by the third stop, six out of nine subjects had a virological set-point after a median 12 months off therapy that was lower than baseline before starting HAART (ranging from 0.6 log10 to 1.3 log10 lower than baseline) and in four it remained stable below 5000 copies/ml. Those subjects who controlled viral replication developed significantly stronger HIV-1 specific cellular immune responses than subjects lacking spontaneous decline (P < 0.05). During viral rebounds no genotypic or phenotypic changes conferring resistance to reverse trancriptase inhibitors or protease inhibitors was detected, but mean absolute CD4 T-cell counts declined significantly, although never below 450 × 106/l and the mean value at 12 months off therapy was significantly higher than the pre-treatment level (P = 0.004). ConclusionsOur findings suggest that STI in chronic HIV-1 infection might augment HIV-1-specific cellular immune responses associated with a spontaneous and sustained drop in plasma viral load in some subjects but at the potential cost of lower CD4 T-cell counts.

Human hepatic stellate cells show features of antigen‐presenting cells and stimulate lymphocyte proliferation

Following cell activation, hepatic stellate cells (HSCs) acquire proinflammatory and profibrogenic properties. We investigated whether activated HSCs also display immune properties. Here we show that cultured human HSCs express membrane proteins involved in antigen presentation, including members of the HLA family (HLA-I and HLA-II), lipid-presenting molecules (CD1b and CD1c), and factors involved in T-cell activation (CD40 and CD80). Exposure of HSCs to proinflammatory cytokines markedly up-regulates these molecules. Importantly, cells freshly isolated from human cirrhotic livers (in vivo activated HSCs) highly express HLA-II and CD40, suggesting that HSCs can act as antigen-presenting cells (APCs) in human fibrogenesis. We also explored whether human HSCs can efficiently process exogenous antigens. Activated HSCs internalize low- and high-molecular-weight dextran and transferrin, indicating that they can perform fluid-phase and receptor-mediated endocytosis. Moreover, HSCs can perform phagocytosis of macromolecules because they internalize latex particles as well as bacteria. Interestingly, both culture-activated and in vivo activated HSCs express high levels of CD68, a protein involved in antigen trafficking. Finally, we studied whether HSCs modulate T-lymphocyte proliferation. In basal conditions, coculture of irradiated HSCs barely induces allogeneic T-lymphocyte proliferation. However, cytokine-stimulated HSCs stimulate the allogeneic T-lymphocyte response in an HLA-II-dependent manner. In conclusion, human activated HSCs express molecules for antigen presentation, internalize macromolecules, and modulate T-lymphocyte proliferation. These results suggest that HSCs may play a role in the immune function of the liver.

Long-term CD4+ T-cell response to highly active antiretroviral therapy according to baseline CD4+ T-cell count.

Current treatment guidelines for HIV infection recommend a relatively late initiation of highly active antiretroviral therapy (HAART). Nevertheless, there is still a concern that immune recovery may not be as complete once CD4+ T cells have decreased below a certain threshold. This study addressed the long-term response of CD4+ T-cell counts in patients on HAART and analyzed the influence of baseline CD4+ T-cell counts, baseline viral load, and age. An observational analysis of evolution of CD4+ T cells in 861 antiretroviral therapy–naive chronic HIV-1–infected patients who started treatment consisting of at least 3 drugs in or after 1996 was performed. Patients were classified in 4 groups according to baseline CD4+ T cells: <200 cells/mm3, 200–349 cells/mm3, 350–499 cells/mm3, and ≥500 cells/mm3. The main outcome measures were proportion of patients with CD4+ T cells <200/mm3 and >500/mm3 at last determination and rate of CD4+ T-cell recovery. Patients were followed-up for a median of 173 weeks (interquartile range [IQR], 100–234). There were no differences in follow-up between the 4 groups. CD4+ T cells increased in the whole cohort from a median of 214 cells/mm3 (IQR, 90–355) to 499 cells/mm3 (IQR, 312–733) (P < 0.001). Compared with the group with a baseline CD4+ T-cell count of ≥500/mm3, the relative risk of having a last determination of CD4+ T-cell counts >200 cells/mm3 was 0.79 (95% CI, 0.75–0.83), 0.92 (95% CI, 0.89–0.96) and 1 for baseline CD4+ T cells <200 cells/mm3, 200–349 cells/mm3, and 350–499 cells/mm3, respectively. The relative risk of having a last determination of CD4+ T-cell counts >500 cells/mm3 was 0.32 (95% CI, 0.27–0.39, P < 0.001), 0.69 (95% CI, 0.60–0.79, P < 0.001), and 0.94 (95% CI, 0.83–1.06, P = 0.38) for baseline CD4+ T-cell counts <200 cells/mm3, 200–349 cells/mm3, and 350–499 cells/mm3, respectively, compared with a baseline CD4+ T-cell count of ≥500 cells/mm3. The increase in CD4+ T cells from baseline was statistically significant and was maintained for up to 4 years of follow-up. This increase seemed to slow down after approximately 3 years and reached a plateau after 4–5 years of follow-up even in patients who achieved and maintained viral suppression in plasma. Long-term immune recovery is possible regardless of baseline CD4+ T-cell count. However, patients who start therapy with a CD4+ T-cell count <200 cells/mm3 have poorer immunologic outcome as measured by the proportion of patients with CD4+ T cells <200/mm3 or >500/mm3 at last determination. It seems that the immune recovery slows down after approximately 3 years of HAART and reaches a plateau after 4–5 years of HAART.

Lack of T-cell proliferative response to HIV-1 antigens after 1 year of highly active antiretroviral treatment in early HIV-1 disease

included as a control group (figure). All people on triple therapy had a plasma RNA viraemia of less than 20 copies/mL for up to 8 months before the assays. Lymphoid tissue RNA viral load of these patients was undetectable. People on double therapy had a median RNA viraemia (range) at 4, 8, and 12 months of 254 (200–44 000), 517 (200–47 771) and 447 (200–19 512), respectively. The proportion of people below detectable levels (200 copies/mL) at 4, 8, and 12 months was 50%, 39%, 44%, respectively. None had an RNA viraemia below 20 copies/mL.

A Dendritic Cell–Based Vaccine Elicits T Cell Responses Associated with Control of HIV-1 Replication

Therapeutic DC vaccines elicit HIV-1–specific immune responses that change viral set point in patients of cART. Putting the Vaccine Before the cART Combination antiretroviral therapy has turned HIV infection from a death sentence to a manageable disease. However, current treatment requires “cART for life,” a less than ideal situation for HIV-infected individuals because of drug cost and worries about resistance. New vaccine strategies are attempting to control viral replication after infection, thus allowing discontinuation of cART and a “functional cure.” Garcia et al. report a dendritic cell (DC)–based vaccine that elicits an HIV-1–specific immune response and may change the setpoint of viral load. The authors pulsed the patient’s own DCs with heat-inactivated whole HIV and then used these DCs as a therapeutic vaccine. The vaccine was safe and well tolerated. They observed a decrease in viral setpoint after cART interruption in vaccinated patients with a concomitant increase in HIV-1–specific T cell responses. Although not yet a functional cure, these results support future studies optimizing a therapeutic vaccine to maintain HIV-1–infected patients. Combination antiretroviral therapy (cART) greatly improves survival and quality of life of HIV-1–infected patients; however, cART must be continued indefinitely to prevent viral rebound and associated disease progression. Inducing HIV-1–specific immune responses with a therapeutic immunization has been proposed to control viral replication after discontinuation of cART as an alternative to “cART for life.” We report safety, tolerability, and immunogenicity results associated with a control of viral replication for a therapeutic vaccine using autologous monocyte-derived dendritic cells (MD-DCs) pulsed with autologous heat-inactivated whole HIV. Patients on cART with CD4+ >450 cells/mm3 were randomized to receive three immunizations with MD-DCs or with nonpulsed MD-DCs. Vaccination was feasible, safe, and well tolerated and shifted the virus/host balance. At weeks 12 and 24 after cART interruption, a decrease of plasma viral load setpoint ≥1 log was observed in 12 of 22 (55%) versus 1 of 11 (9%) and in 7 of 20 (35%) versus 0 of 10 (0%) patients in the DC–HIV-1 and DC-control groups, respectively. This significant decrease in plasma viral load observed in immunized recipients was associated with a consistent increase in HIV-1–specific T cell responses. These data suggest that HIV-1–specific immune responses elicited by therapeutic DC vaccines could significantly change plasma viral load setpoint after cART interruption in chronic HIV-1–infected patients treated in early stages. This proof of concept supports further investigation of new candidates and/or new optimized strategies of vaccination with the final objective of obtaining a functional cure as an alternative to cART for life.

Glutamate Released by Dendritic Cells as a Novel Modulator of T Cell Activation

Adaptive immune responses begin after productive immunosynaptic contacts formation established in secondary lymphoid organs by dendritic cells (DC) presenting the Ag to T lymphocytes. Despite its resemblance to the neurosynapse, the participation of soluble small nonpeptidic mediators in the intercellular cross-talk taking place during T cell–DC interactions remains poorly studied. In this study, we show that human DC undergoing maturation and in contact with T cells release significant amounts of glutamate, which is the main excitatory neurotransmitter in mammalians. The release of glutamate is nonvesicular and mediated by the DC-expressed Xc− cystine/glutamate antiporter. DC-derived glutamate stimulating the constitutively expressed metabotropic glutamate receptor 5 impairs T cell activation. However, after productive Ag presentation, metabotropic glutamate receptor 1 is expressed in T cells to mediate enhanced T cell proliferation and secretion of Th1 and proinflammatory cytokines. These data suggest that, during T cell–DC interaction, glutamate is a novel and highly effective regulator in the initiation of T cell-mediated immune responses.

Role of glutamate on T-cell mediated immunity.

The pivotal role that glutamate plays in the functioning of the central nervous system is well established. Several glutamate receptors and glutamate transporters have been extensively described in the central nervous system where they, respectively mediate glutamate effects and regulates extracellular glutamate levels. Recent studies have shown that glutamate not only has a role as neurotransmitter, but also as an important immunomodulator. In this regard, several glutamate receptors have recently been described on the T-cell surface, whereas glutamate transporters have reportedly been expressed in antigen presenting cells such as dendritic cells and macrophages. On the other hand, an increasing number of reports have described a protective autoimmune mechanism in which autoantigen specific T cells in the central nervous system protect neurons against glutamate neurotoxicity. This review integrates and summarises different findings in this emerging area. A role of glutamate as a key immunomodulator in the initiation and development of T-cell-mediated immunity in peripheral tissues as well as in the central nervous system is suggested.

Distinct Gene Expression Profiling after Infection of Immature Human Monocyte-Derived Dendritic Cells by the Attenuated Poxvirus Vectors MVA and NYVAC

ABSTRACT Although recombinants based on the attenuated poxvirus vectors MVA and NYVAC are currently in clinical trials, the nature of the genes triggered by these vectors in antigen-presenting cells is poorly characterized. Using microarray technology and various analysis conditions, we compared specific changes in gene expression profiling following MVA and NYVAC infection of immature human monocyte-derived dendritic cells (MDDC). Microarray analysis was performed at 6 h postinfection, since these viruses induced extensive cytopathic effects, rRNA breakdown, and apoptosis at late times postinfection. MVA- and NYVAC-infected MDDC shared upregulation of 195 genes compared to uninfected cells: MVA specifically upregulated 359 genes, and NYVAC upregulated 165 genes. Microarray comparison of NYVAC and MVA infection revealed 544 genes with distinct expression patterns after poxvirus infection and 283 genes specifically upregulated after MVA infection. Both vectors upregulated genes for cytokines, cytokine receptors, chemokines, chemokine receptors, and molecules involved in antigen uptake and processing, including major histocompatibility complex genes. mRNA levels for interleukin 12β (IL-12β), beta interferon, and tumor necrosis factor alpha were higher after MVA infection than after NYVAC infection. The expression profiles of transcription factors such as NF-κB/Rel and STAT were regulated similarly by both viruses; in contrast, OASL, MDA5, and IRIG-I expression increased only during MVA infection. Type I interferon, IL-6, and Toll-like receptor pathways were specifically induced after MVA infection. Following MVA or NYVAC infection in MDDC, we found similarities as well as differences between these virus strains in the expression of cellular genes with immunological function, which should have an impact when these vectors are used as recombinant vaccines.

A Therapeutic Dendritic Cell-Based Vaccine for HIV-1 Infection

A double-blinded, controlled study of vaccination of untreated patients with chronic human immunodeficiency virus type 1 (HIV-1) infection with 3 doses of autologous monocyte-derived dendritic cells (MD-DCs) pulsed with heat inactivated autologous HIV-1 was performed. Therapeutic vaccinations were feasible, safe, and well tolerated. At week 24 after first vaccination (primary end point), a modest significant decrease in plasma viral load was observed in vaccine recipients, compared with control subjects (P = .03). In addition, the change in plasma viral load after vaccination tended to be inversely associated with the increase in HIV-specific T cell responses in vaccinated patients but tended to be directly correlated with HIV-specific T cell responses in control subjects.