Gemma Moncunill

Evaluation of the anti-HIV activity of statins

Recent data suggest that statins block HIV-1 replication, which may have important implications for an alternative treatment for AIDS. We tested different statins in cell culture against HIV and conducted a pilot study in HIV-positive patients receiving simvastatin. No anti-HIV activity was detected at subtoxic concentrations and simvastatin did not induce a significant change in the mean viral load or CD4 cell count in study patients. We caution on the use of statins as antiretroviral agents.

HIV-1 escape to CCR5 coreceptor antagonism through selection of CXCR4-using variants in vitro.

Background: HIV-1 coreceptor switch from CCR5 to CXCR4 is associated with disease progression and AIDS. Selection of resistant HIV-1 to CCR5 agents in cell culture has often occurred in the absence of coreceptor switch. With CCR5 antagonists currently in clinical trials, their impact on coreceptor use is still in doubt. Methods: Six R5 HIV-1 strains were passaged in lymphoid cells expressing high CXCR4 and low CCR5, in the absence or presence of CCR5 inhibitors (TAK-779, mAb 2D7 and CCL5). AMD3100, zidovudine and lamivudine were used as controls. Phenotype and genotype changes as well as virus coreceptor use were evaluated. Results: In the absence of drug pressure, three out of six strains expanded their coreceptor use to CXCR4 at different times, suggesting that not all virus strains had the capacity to do so. Lowering the replication rate with a suboptimal concentration of different anti-HIV agents (reverse transcriptase inhibitors or CCR5 agents) delayed coreceptor switch. However, virus breakthrough was observed earlier in the presence of CCR5-targeting agents than in presence of reverse transcriptase inhibitors and was associated with a change in sensitivity to TAK-779 or AMD3100, virus coreceptor expansion to CXCR4 and changes in the V3 loop region of gp120. Conclusion: Our results suggest that HIV-1 may escape CCR5 drug pressure through coreceptor switch. Experimental conditions strongly determine the outcome of CCR5 drug pressure in cell culture. A cell culture model of the evolution of HIV-1 coreceptor use may be relevant to assess the propensity of clinical isolates to develop resistance through coreceptor change.

Anti-HIV Activity and Resistance Profile of the CXC Chemokine Receptor 4 Antagonist POL3026

We have studied the mechanism of action of Arg*-Arg-Nal2-Cys(1×)-Tyr-Gln-Lys-(d-Pro)-Pro-Tyr-Arg-Cit-Cys(1×)-Arg-Gly-(d-Pro)* (POL3026), a novel specific β-hairpin mimetic CXC chemokine receptor (CXCR)4 antagonist. POL3026 specifically blocked the binding of anti-CXCR4 monoclonal antibody 12G5 and the intracellular Ca2+ signal induced by CXC chemokine ligand 12. POL3026 consistently blocked the replication of human immunodeficiency virus (HIV), including a wide panel of X4 and dualtropic strains and subtypes in several culture models, with 50% effective concentrations (EC50) at the subnanomolar range, making POL3026 the most potent CXCR4 antagonist described to date. However, 1-[[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclotetradecane (AMD3100)-resistant and stromal cell-derived factor-1α-resistant HIV-1 strains were cross-resistant to POL3026. Time of addition experiments and a multiparametric evaluation of HIV envelope function in the presence of test compounds confirmed the activity of POL3026 at an early step of virus replication: interaction with the coreceptor. Generation of HIV-1 resistance to POL3026 led to the selection of viruses 12- and 25-fold less sensitive and with mutations in gp120, including the V3 loop region. However, POL3026 prevented the emergence of CXCR4-using variants from an R5 HIV-1 strain that may occur in the presence of anti-HIV agents targeting CC chemokine receptor 5.

Performance of multiplex commercial kits to quantify cytokine and chemokine responses in culture supernatants from Plasmodium falciparum stimulations.

Background Cytokines and chemokines are relevant biomarkers of pathology and immunity to infectious diseases such as malaria. Several commercially available kits based on quantitative suspension array technologies allow the profiling of multiple cytokines and chemokines in small volumes of sample. However, kits are being continuously improved and information on their performance is lacking. Methodology/Principal Findings Different cytokine/chemokine kits, two flow cytometry-based (eBioscience® FlowCytomix™ and BD™ Cytometric Bead Array Human Enhanced Sensitivity) and four Luminex®-based (Invitrogen™ Human Cytokine 25-Plex Panel, Invitrogen™ Human Cytokine Magnetic 30-Plex Panel, Bio-Rad® Bio-Plex Pro™ Human Cytokine Plex Assay and Millipore™ MILLIPLEX® MAP Plex Kit) were compared. Samples tested were supernatants of peripheral blood mononuclear cells of malaria-exposed children stimulated with Plasmodium falciparum parasite lysates. Number of responses in range that could be detected was determined and reproducibility of duplicates was evaluated by the Bland-Altman test. Luminex® kits performed better than flow cytometry kits in number of responses in range and reproducibility. Luminex® kits were more reproducible when magnetic beads were used. However, within each methodology overall performance depended on the analyte tested in each kit. Within the Luminex® kits, the Invitrogen™ with polystyrene beads had the poorer performance, whereas Invitrogen™ with magnetic beads had the higher percentage of cytokines/chemokines with both readings in range (40%), followed by Bio-Rad® with magnetic beads (35%). Regarding reproducibility, the Millipore™ kit had the highest percentage (60%) of cytokines/chemokines with acceptable limits of agreement (<30%), followed by the Invitrogen™ with magnetic beads (40%) that had tighter limits of agreement. Conclusions/Significance Currently available kits for cytokine and chemokine quantification differ in reproducibility and concentration range of accurate detection. Luminex®-based kits with magnetic beads perform the best. Data highlights the importance of testing different kits before each study to choose the most appropriate, depending on the priority of the cytokines assessed.

Low antibodies against Plasmodium falciparum and imbalanced pro-inflammatory cytokines are associated with severe malaria in Mozambican children: a case-control study.

BackgroundThe factors involved in the progression from Plasmodium falciparum infection to severe malaria (SM) are still incompletely understood. Altered antibody and cellular immunity against P. falciparum might contribute to increase the risk of developing SM.MethodsTo identify immune responses associated with SM, a sex- and age-matched case–control study was carried out in 134 Mozambican children with SM (cerebral malaria, severe anaemia, acidosis and/or respiratory distress, prostration, hypoglycaemia, multiple seizures) or uncomplicated malaria (UM). IgG and IgM against P. falciparum lysate, merozoite antigens (MSP-119, AMA-1 and EBA-175), a Duffy binding like (DBL)-α rosetting domain and antigens on the surface of infected erythrocytes were measured by ELISA or flow cytometry. Plasma concentrations of IL-12p70, IL-2, IFN-γ, IL-4, IL-5, IL-10, IL-8, IL-6, IL-1β, TNF, TNF-β and TGF-β1 were measured using fluorescent bead immunoassays. Data was analysed using McNemar’s and Signtest.ResultsCompared to UM, matched children with SM had reduced levels of IgG against DBLα (P < 0.001), IgM against MSP-119 (P = 0.050) and AMA-1 (P = 0.047), TGF-β1 (P <0.001) and IL-12 (P = 0.039). In addition, levels of IgG against P. falciparum lysate and IL-6 concentrations were increased (P = 0.004 and P = 0.047, respectively). Anti-DBLα IgG was the only antibody response associated to reduced parasite densities in a multivariate regression model (P = 0.026).ConclusionsThe lower levels of antibodies found in children with SM compared to children with UM were not attributable to lower exposure to P. falciparum in the SM group. IgM against P. falciparum and specific IgG against a rosetting Pf EMP1 domain may play a role in the control of SM, whereas an imbalanced pro-inflammatory cytokine response may exacerbate the severity of infection. A high overlap in symptoms together with a limited sample size of different SM clinical groups reduced the power to identify immunological correlates for particular forms of SM.

High Antibody Responses against Plasmodium falciparum in Immigrants after Extended Periods of Interrupted Exposure to Malaria

Background Malaria immunity is commonly believed to wane in the absence of Plasmodium falciparum exposure, based on limited epidemiological data and short-lived antibody responses in some longitudinal studies in endemic areas. Methods A cross-sectional study was conducted among sub-Saharan African adults residing in Spain for 1 up to 38 years (immigrants) with clinical malaria (n=55) or without malaria (n=37), naïve adults (travelers) with a first clinical malaria episode (n=20) and life-long malaria exposed adults from Mozambique (semi-immune adults) without malaria (n=27) or with clinical malaria (n=50). Blood samples were collected and IgG levels against the erythrocytic antigens AMA-1 and MSP-142 (3D7 and FVO strains), EBA-175 and DBL-α were determined by Luminex. IgG levels against antigens on the surface of infected erythrocytes (IEs) were measured by flow cytometry. Results Immigrants without malaria had lower IgG levels than healthy semi-immune adults regardless of the antigen tested (P≤0.026), but no correlation was found between IgG levels and time since migration. Upon reinfection, immigrants with malaria had higher levels of IgG against all antigens than immigrants without malaria. However, the magnitude of the response compared to semi-immune adults with malaria depended on the antigen tested. Thus, immigrants had higher IgG levels against AMA-1 and MSP-142 (P≤0.015), similar levels against EBA-175 and DBL-α, and lower levels against IEs (P≤0.016). Immigrants had higher IgG levels against all antigens tested compared to travelers (P≤0.001), both with malaria. Conclusions Upon cessation of malaria exposure, IgG responses to malaria-specific antigens were maintained to a large extent, although the conservation and the magnitude of the recall response depended on the nature of the antigen. Studies on immigrant populations can shed light on the factors that determine the duration of malaria specific antibody responses and its effect on protection, with important implications for future vaccine design and public health control measures.

Different selection patterns of resistance and cross-resistance to HIV-1 agents targeting CCR5

OBJECTIVES Identification of CCR5 as an antiretroviral target led to the development of several CCR5 antagonists in clinical trials and the approval of maraviroc. Evaluating the mechanism of drug resistance to CCR5 agents may have implications in the clinical development of this class of agents. We have analysed the resistance profile of two R5 HIV-1 strains [BaL and a clinical isolate (CI)] after long-term passage in cell culture in the presence of TAK-779, the first developed non-peptidic small molecule targeting CCR5. METHODS Genotypic and phenotypic tests were used to evaluate the resistance of virus isolated from cell culture in the presence of the CCR5 inhibitor TAK-779. RESULTS Mutations conferring resistance appeared in the gp120 sequence but were not confined to the V3 loop region, and both strains had a different mutation pattern. Recombination of the env gene of the BaL-derived resistant virus into the HIV-1 HXB2 wild-type backbone conferred resistance to TAK-779 and cross-resistance to maraviroc, with 63- and 11-fold changes in their EC(50) (50% effective concentration), respectively, together with an apparent reduction of the maximal plateau inhibition (MPI) of TAK-779 but not of maraviroc. Conversely, the resistant CI viruses showed an approximately 50% reduction in MPI for both TAK-779 and maraviroc. CONCLUSIONS We confirm that different pathways to the generation of CCR5 drug resistance/cross-resistance may occur that strongly depend on cell culture conditions, CCR5 availability and the genetic background of the HIV strain. Our study provides complementary information to understand the complexity of resistance to CCR5 antagonists.

Cytokine profiling in immigrants with clinical malaria after extended periods of interrupted exposure to Plasmodium falciparum.

Immunity to malaria is believed to wane with time in the absence of exposure to Plasmodium falciparum infection, but immunoepidemiological data on longevity of immunity remain controversial. We quantified serum cytokines and chemokines by suspension array technology as potential biomarkers for durability of immunity in immigrants with clinical malaria after years without parasite exposure. These were compared to serum/plasma profiles in naïve adults (travelers) and semi-immune adults under continuous exposure, with malaria, along with immigrant and traveler patients without malaria. Immigrants had higher levels of IL-2, IL-5 and IL-8 compared to semi-immune adults with malaria (P≤0.0200). Time since immigration correlated with increased IL-2 (rho=0.2738P=0.0495) and IFN-γ (rho=0.3044P=0.0282). However, immigrants did not show as high IFN-γ concentrations as travelers during a first malaria episode (P<0.0001). Immigrants and travelers with malaria had higher levels of IFN-γ, IL-6, and IL-10 (P<0.0100) than patients with other diseases, and IL-8 and IL-1β were elevated in immigrants with malaria (P<0.0500). Therefore, malaria patients had a characteristic strong pro-inflammatory/Th1 signature. Upon loss of exposure, control of pro-inflammatory responses and tolerance to P. falciparum appeared to be reduced. Understanding the mechanisms to maintain non-pathogenic effector responses is important to develop new malaria control strategies.

Cytokine and Antibody Responses to Plasmodium falciparum in Naïve Individuals during a First Malaria Episode: Effect of Age and Malaria Exposure

Age- and exposure-dependent immune responses during a malaria episode may be key to understanding the role of these factors in the acquisition of immunity to malaria. Plasma/serum samples collected from naïve Mozambican children (n = 48), European adults (naïve travelers, n = 22; expatriates with few prior malaria exposures, n = 15) and Mozambican adults with long-life malaria exposure (n = 99) during and after a malaria episode were analyzed for IgG against merozoite proteins by Luminex and against infected erythrocytes by flow cytometry. Cytokines and chemokines were analyzed in plasmas/sera by suspension array technology. No differences were detected between children and adults with a primary infection, with the exception of higher IgG levels against 3D7 MSP-142 (P = 0.030) and a P. falciparum isolate (P = 0.002), as well as higher IL-12 (P = 0.020) in children compared to other groups. Compared to malaria-exposed adults, children, travelers and expatriates had higher concentrations of IFN-γ (P≤0.0090), IL-2 (P≤0.0379) and IL-8 (P≤0.0233). Children also had higher IL-12 (P = 0.0001), IL-4 (P = 0.003), IL-1β (P = 0.024) and TNF (P = 0.006) levels compared to malaria-exposed adults. Although IL-12 was elevated in children, overall the data do not support a role of age in immune responses to a first malaria episode. A TH1/pro-inflammatory response was the hallmark of non-immune subjects.

Evaluation of the anti-HIV activity of natalizumab, an antibody against integrin alpha4.

The alpha4beta7 integrin has been shown to serve as a coreceptor for HIV. One anti-alpha4 integrin agent (natalizumab) has been approved for the treatment of multiple sclerosis and Crohns disease. We found that activation of CD4+ T cells with retinoic acid induced the upregulation of alpha4 and beta7 integrins. However, natalizumab failed to block the replication of HIV-1 strains in lymphoid MT-4 cells or CD4+ T cells at concentrations up to 125 μg/ml. Our results suggest that alpha4 integrins are not essential cofactors for HIV replication.