Real-world experience of intravenous iron sucrose supplementation and dynamics of soluble transferrin receptor and hepcidin in a Spanish cohort of absolute iron deficient patients.

The study evaluated the safety and effectiveness of the generic intravenous (IV) iron treatment (Feriv®), in a Spanish cohort with absolute iron deficiency (ID) (serum ferritin <50 ng/ml, with or without anaemia) (n = 122; 91% women; median age of 44 years [IQR: 33.7-54]). Iron-related biomarkers were measured before treatment (baseline), 2 weeks after beginning the protocol (intermediate control, IC) and between 7 and 10 days after treatment completion (final time-point). Primary efficacy endpoints were ferritin levels ≥ 50 ng/ml, anaemia restoration or an increase in haemoglobin (Hb) of at least one point in patients without baseline anaemia. After treatment, iron-related biomarkers improved, including ferritin, Hb, sideremia, transferrin, transferrin saturation index, soluble transferrin receptor (sTfR), and hepcidin. Baseline ferritin concentration (13.5 ng/ml [IQR: 8-24.2]) increased at the IC and continued rising at the final time-point, reaching a median ferritin of 222 ng/ml and 97.3% of patients ≥ 50 ng/ml. At the final time-point, anaemia prevalence decreased from 26.2% to 5%, while the 34.1% without baseline anaemia showed an increase in Hb of at least one point. Headache was the only drug-adverse event recorded in 2.3% of patients. At a late time-point (27.5 median weeks after ending therapy [IQR: 22-40]), evaluated in a subgroup of 66 patients, 18% had ferritin levels < 50 ng/ml. Multivariate analysis showed that low baseline ferritin and high sTfR/hepcidin ratio tended to be independently associated with ID recurrence. Feriv® is a safe, effective first-line treatment for absolute ID, with improvement of serum ferritin and Hb. ID recurrence was associated with the baseline degree of iron stores depletion, indicated by serum ferritin, and sTfR/hepcidin ratio.

G2-S16 Polyanionic Carbosilane Dendrimer Can Reduce HIV-1 Reservoir Formation by Inhibiting Macrophage Cell to Cell Transmission.

Human immunodeficiency virus (HIV-1) is still a major problem, not only in developing countries but is also re-emerging in several developed countries, thus the development of new compounds able to inhibit the virus, either for prophylaxis or treatment, is still needed. Nanotechnology has provided the science community with several new tools for biomedical applications. G2-S16 is a polyanionic carbosilane dendrimer capable of inhibiting HIV-1 in vitro and in vivo by interacting directly with viral particles. One of the main barriers for HIV-1 eradication is the reservoirs created in primoinfection. These reservoirs, mainly in T cells, are untargetable by actual drugs or immune system. Thus, one approach is inhibiting HIV-1 from reaching these reservoir cells. In this context, macrophages play a main role as they can deliver viral particles to T cells establishing reservoirs. We showed that G2-S16 dendrimer is capable of inhibiting the infection from infected macrophages to healthy T CD4/CD8 lymphocytes by eliminating HIV-1 infectivity inside macrophages, so they are not able to carry infectious particles to other body locations, thus preventing the reservoirs from forming.

DBP rs16846876 and rs12512631 polymorphisms are associated with progression to AIDS naïve HIV-infected patients: a retrospective study.

BACKGROUND: Most of the circulating Vitamin D (VitD) is transported bound to vitamin D-binding protein (DBP), and several DBP single nucleotide polymorphisms (SNPs) have been related to circulating VitD concentration and disease. In this study, we evaluated the association among DBP SNPs and AIDS progression in antiretroviral treatment (ART)-naïve-HIV-infected patients. METHODS: We performed a retrospective study in 667 patients who were classified according to their pattern of AIDS progression (183 long-term non-progressors (LTNPs), 334 moderate progressors (MPs), and 150 rapid progressors (RPs)) and 113 healthy blood donors (HIV, HCV, and HBV negative subjects). We genotyped seven DBP SNPs (rs16846876, rs12512631, rs2070741, rs2282679, rs7041, rs1155563, rs2298849) using Agena Bioscience's MassARRAY platform. The genetic association was evaluated by Generalized Linear Models adjusted by age at the moment of HIV diagnosis, gender, risk group, and VDR rs2228570 SNP. Multiple testing correction was performed by the false discovery rate (Benjamini and Hochberg procedure; q-value). RESULTS: All SNPs were in HWE (p > 0.05) and had similar genotypic frequencies for DBP SNPs in healthy-controls and HIV-infected patients. In unadjusted GLMs, we only found significant association with AIDS progression in rs16846876 and rs12512631 SNPs. In adjusted GLMs, DBP rs16846876 SNP showed significant association under the recessive inheritance model [LTNPs vs. RPs (adjusted odds ratio (aOR) = 3.53; q-value = 0.044) and LTNPs vs. MPs (aOR = 3.28; q-value = 0.030)] and codominant [LTNPs vs. RPs (aOR = 4.92; q-value = 0.030) and LTNPs vs. MPs (aOR = 3.15; q-value = 0.030)]. Also, we found DBP rs12512631 SNP showed significant association in the inheritance model dominant [LTNPs vs. RPs (aOR = 0.49; q-value = 0.031) and LTNPs vs. MPs (aOR = 0.6; q-value = 0.047)], additive [LTNPs vs. RPs (aOR = 0.61; q-value = 0.031)], overdominant [LTNPs vs. MPs (aOR = 0.55; q-value = 0.032)], and codominant [LTNPs vs. RPs (aOR = 0.52; q-value = 0.036) and LTNPs vs. MPs (aOR = 0.55; q-value = 0.032)]. Additionally, we found a significant association between DBP haplotypes (composed by rs16846876 and rs12512631) and AIDS progression (LTNPs vs RPs): DBP haplotype AC (aOR = 0.63; q-value = 0.028) and the DBP haplotype TT (aOR = 1.64; q-value = 0.028). CONCLUSIONS: DBP rs16846876 and rs12512631 SNPs are related to the patterns of clinical AIDS progression (LTNP, MP, and RP) in ART-naïve HIV-infected patients. Our findings provide new knowledge about AIDS progression that may be relevant to understanding the pathogenesis of HIV infection.

Analysis of the dysregulation between regulatory B and T cells (Breg and Treg) in human immunodeficiency virus (HIV)-infected patients.

This study examines the relationship between regulatory B (Breg) and T (Treg) compartments, which play crucial roles in the maintenance of immune homeostasis in the context of HIV. Using flow cytometry, the phenotypes of different Breg and Treg subsets from HIV-infected and healthy individuals were analyzed, along with the suppressive capacity of Breg. Peripheral blood samples of thirteen HIV+ treatment-naïve individuals, fourteen treated-HIV+ individuals with undetectable viral load and twelve healthy individuals were analyzed. The absolute counts of Breg and Treg subsets were decreased in HIV+ treatment-naïve individuals in comparison to treated-HIV+ and healthy individuals. Interestingly, correlations between Breg subsets (CD24hiCD27+ and PD-L1+ B cells) and IL-10-producing Breg observed in healthy individuals were lost in HIV+ treatment-naïve individuals. However, a correlation between frequencies of CD24hiCD38hi or TIM-1+-Breg subsets and Treg was observed in HIV+ treatment-naïve individuals and not in healthy individuals. Therefore, we hypothesized that various Breg subsets might have different functions during B and T-cell homeostasis during HIV-1 infection. In parallel, stimulated Breg from HIV-infected treatment-naïve individuals presented a decreased ability to suppress CD4+ T-cell proliferation in comparison to the stimulated Breg from treated-HIV+ or healthy individuals. We demonstrate a dysregulation between Breg and Treg subsets in HIV-infected individuals, which might participate in the hyper-activation and exhaustion of the immune system that occurs in such patients.

In silico search, chemical characterization and immunogenic evaluation of amino-terminated G4-PAMAM-HIV peptide complexes using three-dimensional models of the HIV-1 gp120 protein.

Peptide epitopes have been widely used to develop synthetic vaccines and immunotherapies. However, peptide epitopes may exhibit poor absorption or immunogenicity due to their low molecular weights. Conversely, fourth-generation polyamidoamine (G4-PAMAM) dendrimers are nonimmunogenic and relatively nontoxic synthetic nanoparticles that have been used as adjuvants and nanocarriers of small peptides and to improve nasal absorption. Based on this information, we hypothesized that the combination of intranasal immunization and G4-PAMAM dendrimers would be useful for enhancing the antibody responses of HIV-1 gp120 peptide epitopes. Therefore, we first used structural data, peptide epitope predictors and docking and MD simulations on MHC-II to identify two peptide epitopes on the CD4 binding site of HIV-1 gp120. The formation of G4-PAMAM-peptide complexes was evaluated in silico (molecular docking studies using different G4-PAMAM conformations retrieved from MD simulations as well as the MMGBSA approach) and validated experimentally (electrophoresis, 1H NMR and cryo-TEM). Next, the G4-PAMAM dendrimer-peptide complexes were administered intranasally to groups of female BALB/cJ mice. The results showed that both peptides were immunogenic at the systemic and mucosal levels (nasal and vaginal), and G4-PAMAM dendrimer-peptide complexes improved IgG and IgA responses in serum and nasal washes. Thus, G4-PAMAM dendrimers have potential for use as adjuvants and nanocarriers of peptides.

Monocyte Phenotype and Polyfunctionality Are Associated With Elevated Soluble Inflammatory Markers, Cytomegalovirus Infection, and Functional and Cognitive Decline in Elderly Adults.

Monocytes are mediators of the inflammatory response and include three subsets: classical, intermediate, and nonclassical. Little is known about the phenotypical and functional age-related changes in monocytes and their association with soluble inflammatory biomarkers, cytomegalovirus infection, and functional and mental decline. We assayed the activation ex vivo and the responsiveness to TLR2 and TLR4 agonists in vitro in the three subsets and assessed the intracellular production of IL1-alpha (α), IL1-beta (ß), IL-6, IL-8, TNF-α, and IL-10 of elderly adults (median 83 [67-90] years old;n= 20) compared with young controls (median 35 [27-40] years old;n= 20). Ex vivo, the elderly adults showed a higher percentage of classical monocytes that expressed intracellular IL1-α (p= .001), IL1-ß (p= .001), IL-6 (p= .002), and IL-8 (p= .007). Similar results were obtained both for the intermediate and nonclassical subsets and in vitro. Polyfunctionality was higher in the elderly adults. The functionality ex vivo was strongly associated with soluble inflammatory markers. The activation phenotype was independently associated with the anti-cytomegalovirus IgG levels and with functional and cognitive decline. These data demonstrate that monocytes are key cell candidates for the source of the high soluble inflammatory levels. Our findings suggest that cytomegalovirus infection might be a driving force in the activation of monocytes and is associated with the functional and cognitive decline.

Synergistic Activation of Latent HIV-1 Expression by Novel Histone Deacetylase Inhibitors and Bryostatin-1.

Viral reactivation from latently infected cells has become a promising therapeutic approach to eradicate HIV. Due to the complexity of the viral latency, combinations of efficient and available drugs targeting different pathways of latency are needed. In this work, we evaluated the effect of various combinations of bryostatin-1 (BRY) and novel histone deacetylase inhibitors (HDACIs) on HIV-reactivation and on cellular phenotype. The lymphocyte (J89GFP) or monocyte/macrophage (THP89GFP) latently infected cell lines were treated with BRY, panobinostat (PNB) and romidepsin (RMD) either alone or in combination. Thus, the effect on the viral reactivation was evaluated. We calculated the combination index for each drug combination; the BRY/HDACIs showed a synergistic HIV-reactivation profile in the majority of the combinations tested, whereas non-synergistic effects were observed when PNB was mixed with RMD. Indeed, the 75% effective concentrations of BRY, PNB and RMD were reduced in these combinations. Moreover, primary CD4 T cells treated with such drug combinations presented similar activation and proliferation profiles in comparison with single drug treated cells. Summing up, combinations between BRY, PNB and/or RMD presented a synergistic profile by inducing virus expression in HIV-latently infected cells, rendering these combinations an attractive novel and safe option for future clinical trials.

Anticancer siRNA cocktails as a novel tool to treat cancer cells. Part (B). Efficiency of pharmacological action.

This paper examines a perspective to use newly engineered nanomaterials as effective and safe carriers for gene therapy of cancer. Three different groups of cationic dendrimers (PAMAM, phosphorus, and carbosilane) were complexed with anticancer siRNA and the biophysical properties of the dendriplexes created were analyzed. The potential of the dendrimers as nanocarriers for anticancer Bcl-xl, Bcl-2, Mcl-1 siRNAs and additionally a scrambled sequence siRNA has been explored. Dendrimer/siRNA complexes were characterised by various methods including fluorescence, zeta potential, dynamic light scattering, circular dichroism, gel electrophoresis and transmission electron microscopy. In this part of study, the transfection of complexes in HeLa and HL-60 cells was analyzed using both single apoptotic siRNAs and a mixture (cocktail) of them. Cocktails were more effective than single siRNAs, allowing one to decrease siRNAs concentration in treating cells. The dendrimers were compared as siRNA carriers, the most effective being the phosphorus-based ones. However, they were also the most cytotoxic on their own, so that in this regard the application of all dendrimers in anticancer therapy will be discussed.

Safety and immunogenicity of a modified vaccinia Ankara-based HIV-1 vaccine (MVA-B) in HIV-1-infected patients alone or in combination with a drug to reactivate latent HIV-1.

OBJECTIVES: The safety, immunogenicity, impact on the latent reservoir and rebound of viral load after therapeutic HIV-1 vaccination with recombinant modified vaccinia Ankara-based (MVA-B) HIV-1 vaccine expressing monomeric gp120 and the fused Gag-Pol-Nef polyprotein of clade B with or without a drug to reactivate latent HIV-1 (disulfiram) were assessed. METHODS: HIV-1-infected patients were randomized to receive three injections of MVA-B (n = 20) or placebo (n = 10). Twelve patients (eight who received vaccine and four who were given placebo) received a fourth dose of MVA-B followed by 3 months of disulfiram. Combined ART (cART) was discontinued 8 weeks after the last dose of MVA-B. Clinical Trials.gov identifier: NCT01571466. RESULTS: MVA-B was safe and well tolerated. A minor, but significant, increase in the T cell responses targeting vaccine inserts of Gag was observed [a median of 290, 403 and 435 spot-forming-cells/10(6) PBMCs at baseline, after two vaccinations and after three vaccinations, respectively; P = 0.02 and P = 0.04]. After interruption of cART, a modest delay in the rebound of the plasma viral load in participants receiving vaccine but not disulfiram was observed compared with placebo recipients (P = 0.01). The dynamics of the viral load rebound did not change in patients receiving MVA-B/disulfiram. No changes in the proviral reservoir were observed after disulfiram treatment. CONCLUSIONS: MVA-B vaccination was a safe strategy to increase Gag-specific T cell responses in chronically HIV-1-infected individuals, but it did not have a major impact on the latent reservoir or the rebound of plasma viral load after interruption of cART when given alone or in combination with disulfiram.

Carbosilane dendrimers as gene delivery agents for the treatment of HIV infection.

Despite the use of siRNA in the downregulation of HIV-1 replication which has been reported, CD4 T lymphocytes are difficult to transfect with non-viral vectors. We determined whether second generation carbosilane dendrimers (2G-NN16 and 2G-03NN24) may be efficient transfectants in CD4 T lymphocytes. Dendrimers were also tested on macrophages to determine whether they can modify macrophage phenotype and induce an inflammatory response. The nanoconjugate formed by 2G-03NN24/siRNA-Nef presents the highest inhibition of HIV-1 replication. Dendrimers presented safety properties because they did not induce proliferation on CD4 T lymphocytes and decrease the release of TNFα and IL-12p40 by macrophages. Both dendrimers also decrease the phagocytosis activity. Additionally, 2G-03NN24 dendrimer decreases the CCL2 and CCR2 expression in macrophages. Carbosilane dendrimers 2G-NN16 and 2G-03NN24 can be used as efficient non-viral vectors for gene therapy applications, mainly in the treatment of HIV infection.

Prostaglandin E2 reduces the release and infectivity of new cell-free virions and cell-to-cell HIV-1 transfer.

BACKGROUND: The course of human immunodeficiency virus type-1 (HIV-1) infection is influenced by a complex interplay between viral and host factors. HIV infection stimulates several proinflammatory genes, such as cyclooxigense-2 (COX-2), which leads to an increase in prostaglandin (PG) levels in the plasma of HIV-1-infected patients. These genes play an indeterminate role in HIV replication and pathogenesis. The effect of prostaglandin E2 (PGE2) on HIV infection is quite controversial and even contradictory, so we sought to determine the role of PGE2 and the signal transduction pathways involved in HIV infection to elucidate possible new targets for antiretrovirals. RESULTS: Our results suggest that PGE2 post-infection treatment acts in the late stages of the viral cycle to reduce HIV replication. Interestingly, viral protein synthesis was not affected, but a loss of progeny virus production was observed. No modulation of CD4 CXCR4 and CCR5 receptor expression, cell proliferation, or activation after PGE2 treatment was detected. Moreover, PGE2 induced an increase in intracellular cAMP (cyclic AMP) levels through the EP2/EP4 receptors. PGE2 effects were mimicked by dbcAMP and by a specific Epac (exchange protein directly activated by cyclic AMP) agonist, 8-Cpt-cAMP. Treatment with PGE2 increased Rap1 activity, decreased RhoA activity and subsequently reduced the polymerization of actin by approximately 30% compared with untreated cells. In connection with this finding, polarized viral assembly platforms enriched in Gag were disrupted, altering HIV cell-to-cell transfer and the infectivity of new virions. CONCLUSIONS: Our results demonstrate that PGE2, through Epac and Rap activation, alters the transport of newly synthesized HIV-1 components to the assembly site, reducing the release and infectivity of new cell-free virions and cell-to-cell HIV-1 transfer.

Cardiovascular biomarkers in vertically HIV-infected children without metabolic abnormalities.

Early cardiovascular disease is a major concern for ART-suppressed vertically HIV-infected children; however, evidence is lacking regarding specific preventive measures. In this study, a complete panel of biomarkers was determined together with carotid intima-media thickness (IMT), in a cohort of 64 HIV-infected children and 30 controls. Mean age of participants was 14.1±5 years. HIV-infected patients showed normal lipid profile, with only slightly higher triglycerides, and no differences between groups were found regarding IMT. HIV-infected patients displayed higher levels of soluble CD14 (sCD14) and soluble vascular cell adhesion molecule-1 (sVCAM) (all p<0.05). However, levels of C-reactive protein, interleukin-6, myeloperoxidase, monocyte chemoattractant protein-1, P-selectin and tissue plasminogen activator were similar between groups. Vertically HIV-infected subjects on ART with no significant metabolic disturbances displayed increased sCD14 and sVCAM but not up-regulation of proinflammatory pathways. Larger studies are warranted to assess the impact of a strict metabolic control on cardiovascular risk and to define specific cardiovascular disease preventive strategies in this population.

Subclinical atherosclerosis and markers of immune activation in HIV-infected children and adolescents: the CaroVIH Study.

BACKGROUND: HIV-infected adults display increased cardiovascular disease, probably driven by inflammation and immune activation. These relationships have not been addressed in vertically HIV-infected children and adolescents, a population at very high risk for long-term non-AIDS complications. METHODS: Carotid intima media thickness (IMT) was measured in a cohort of HIV-infected children and adolescents and healthy controls. C-reactive protein and markers of immune activation (CD38⁺HLA-DR⁺) and immune senescence (CD28⁻CD57⁺) were determined. RESULTS: One hundred fifty HIV-infected patients and 150 controls were included, 64.8% female. IMT was thicker in HIV-infected patients (0.434 mm ± 0.025 vs. 0.424 mm ± 0.018, P < 0.001). After adjustment by age, sex, body mass index, and smoking status, HIV infection was independently associated with thicker IMT (odds ratio, 2.28; 95% confidence interval: 1.25 to 4.13; P = 0.007). Among HIV-related variables, a low CD4 nadir was related to an increased IMT. Although HIV-infected subjects presented higher frequencies of activated CD4⁺ and CD8⁺ T cells (P = 0.002 and P = 0.087, respectively), no relation was found between IMT and inflammation, immune activation, or senescence. CONCLUSIONS: Structural changes of the vasculature present early in vertically HIV-infected subjects as well as immune activation and senescence. These patients should be carefully monitored for the prompt detection and early treatment of cardiovascular disease.

Toxicity and proapoptotic activity of poly(propylene imine) glycodendrimers in vitro: considering their contrary potential as biocompatible entity and drug molecule in cancer.

Since the first dendrimers were synthesized, scientists around the world have studied their properties and potential applications. Cationic dendrimers are characterized by significant toxicity due to their interactions with cells components. The replacement of all cationic surface groups by neutral ones and therefore diminishing positive charge reduces the toxicity but may also lead to loss of dendrimers' desirable properties and restrict their biomedical applications. We have compared the cytotoxicity, as well as proapoptotic and antiproliferative activity of unmodified fourth generation PPI dendrimer (PPI-G4) and dendrimers modified with maltose (Mal) or maltotriose (Mal-III) - for full (dense shell - DS) or partial (open shell - OS) surface modifications. We have proved that among glycodendrimers, the OS-Mal PPI-G4 dendrimer is the most toxic, whereas DS-Mal-III molecule shows relatively weak or even no effect. We have also confirmed that OS dendrimers, both maltotriose and maltose modified, not only reduce cancer cells viability by inducing apoptosis but also inhibit their proliferation. The use of dendrimers as an active substance, which may be a drug per se is one of the most exciting and clinically important applications of cancer nanotechnology, therefore a partial modification of the surface appears to be a perfect solution for this purpose.

Ni(II)-NTA modified poly(ethylene imine) glycopolymers: physicochemical properties and first in vitro study of polyplexes formed with HIV-derived peptides.

Alternative delivery entities are desirable in immunotherapies in which polyplexes are widely formed by electrostatic interactions to induce cellular uptake processes for bioactive molecules. In our study, biocompatible Ni(II)-nitrilo(triacetic acid)-modified poly(ethylene imine)-maltose (Ni-NTA-DG) is realized and evaluated as complexation agent against His-tagged peptides using fluorescence polarization and dynamic light scattering. The polyplexes are stable until a pH of 6.5-6.0, and also up to 50 mM of imidazole. A first uptake approach shows that polyplexes lead to an increase in peptide uptake in monocyte-derived immature dendritic cells. In summary, Ni-NTA-DG represents a promising (delivery) platform for forthcoming in vitro applications.

Complexation of HIV derived peptides with carbosilane dendrimers.

Dendrimers have been proposed as new carriers for selected HIV-1 peptides. This paper reports on the complexation behaviour of the three HIV-derived-peptides: Gp160, NH-EIDNYTNTIYTLLEE-COOH; P24, NH-DTINEEAAEW-COOH and Nef, NHGMDDPEREVLEWRFDSRLAF-COOH with second generation cationic carbosilane dendrimers (CBD) branched with carbonsilicon bonds (CBD-CS) or oxygensilicon bonds (CBD-OS). Studies on the formation of complexes between HIV peptides and CBDs by fluorescence polarization, zeta-potential, electrophoresis and transmission electron microscopy have shown that both studied dendrimers form complexes with HIV peptides. At a molar ratio of (2.5-3):1 (dendrimer:peptide), the complexes formed were in the size range of 180-275 nm and with significant positive surface charge. The results suggest that interactions between dendrimers and HIV peptides have electrostatic nature due to the negative charge of peptides backbone and positive charge of dendrimer functional groups. Dendriplex stability depended on the type of studied dendrimers. Time of peptides release from the complexes ranged from 1 (CBD-OS) to ~36 (CBD-CS)h. Basing on the obtained results, we propose that the water-soluble cationic carbosilane dendrimers can be considered for delivery of HIV peptides to dendritic cells.

Vector replication and expression of HIV-1 antigens by the HIV/AIDS vaccine candidate MVA-B is not affected by HIV-1 protease inhibitors.

MVA-B is an attenuated poxvirus vector expressing human immunodeficiency virus type 1 Env, Gag, Pol, and Nef antigens from clade B, and is considered a promising HIV/AIDS vaccine candidate. Recently, a phase I clinical trial in human healthy volunteers has shown that MVA-B is safe and highly immunogenic, inducing broad, polyfunctional, and long-lasting CD4(+) and CD8(+) T cell responses to HIV-1 antigens, with preference for effector memory T cells; and it also triggers the induction of specific antibodies to Env in most of the vaccines. While MVA recombinants expressing HIV-1 antigens are being used or plan to use in therapeutic clinical trials, little is known on the effect of HIV-1 highly active antiretroviral therapy in MVA life cycle. To define this role, here we have evaluated in established cell cultures and human dendritic cells to what extent different HIV-1 protease inhibitors affect virus replication and expression of HIV-1 antigens during MVA-B infection. The results obtained revealed that the most commonly used HIV-1 protease inhibitors (atazanavir, ritonavir, and lopinavir) had no effect on MVA-B virus growth kinetics, even at higher concentrations than those normally used on HAART. Furthermore, expression of gp120 and the fused Gag-Pol-Nef polyprotein in permissive and non-permissive cells infected with MVA-B were also not affected. These findings are relevant information for the therapeutic use of MVA-B as an HIV-1/AIDS vaccine.

The PDZ-adaptor protein syntenin-1 regulates HIV-1 entry.

Syntenin-1 is a cytosolic adaptor protein involved in several cellular processes requiring polarization. Human immunodeficiency virus type 1 (HIV-1) attachment to target CD4(+) T-cells induces polarization of the viral receptor and coreceptor, CD4/CXCR4, and cellular structures toward the virus contact area, and triggers local actin polymerization and phosphatidylinositol 4,5-bisphosphate (PIP(2)) production, which are needed for successful HIV infection. We show that syntenin-1 is recruited to the plasma membrane during HIV-1 attachment and associates with CD4, the main HIV-1 receptor. Syntenin-1 overexpression inhibits HIV-1 production and HIV-mediated cell fusion, while syntenin depletion specifically increases HIV-1 entry. Down-regulation of syntenin-1 expression reduces F-actin polymerization in response to HIV-1. Moreover, HIV-induced PIP(2) accumulation is increased in syntenin-1-depleted cells. Once the virus has entered the target cell, syntenin-1 polarization toward the viral nucleocapsid is lost, suggesting a spatiotemporal regulatory role of syntenin-1 in actin remodeling, PIP(2) production, and the dynamics of HIV-1 entry.

PEGylated poly(ethylene imine) copolymer-delivered siRNA inhibits HIV replication in vitro.

RNA interference is increasingly being utilized for the specific targeting and down-regulation of disease-causing genes, including targeting viral infections such as HIV. T lymphocytes, the primary target for HIV, are very difficult to treat with gene therapy applications such as RNA interference because of issues with drug delivery. To circumvent these problems, we investigated poly(ethylene imine) (PEI) as a method of improving transfection efficiency of siRNA to T lymphocytes. Additionally, polyethylene glycol (PEG) moieties were engrafted to the PEI polymers with the goals of improving stability and reducing cytotoxicity. Initial studies on PEG-PEI/siRNA polyplex formation, size and their interaction with cell membranes demonstrated their feasibility as drug delivery agents. Assays with lymphocytes revealed low cytotoxicity profiles of the polyplexes at pharmacologically relevant concentrations with PEGylated copolymers obtaining the best results. Successful transfection of a T cell line or primary T cells with siRNA was observed via flow cytometry and confocal microscopy. Finally, the biological effect of copolymer-delivered siRNA was measured. Of particular significance, siRNA targeted to the HIV gene nef and delivered by one of the PEG-PEI copolymers in repetitive treatments every 2-3 days was observed to inhibit HIV replication to the same extent as azidothymidine over the course of 15 days.

The HIV/AIDS vaccine candidate MVA-B administered as a single immunogen in humans triggers robust, polyfunctional, and selective effector memory T cell responses to HIV-1 antigens.

Attenuated poxvirus vectors expressing human immunodeficiency virus type 1 (HIV-1) antigens are considered promising HIV/AIDS vaccine candidates. Here, we describe the nature of T cell immune responses induced in healthy volunteers participating in a phase I clinical trial in Spain after intramuscular administration of three doses of the recombinant MVA-B-expressing monomeric gp120 and the fused Gag-Pol-Nef (GPN) polyprotein of clade B. The majority (92.3%) of the volunteers immunized had a positive specific T cell response at any time postvaccination as detected by gamma interferon (IFN-γ) intracellular cytokine staining (ICS) assay. The CD4(+) T cell responses were predominantly Env directed, whereas the CD8(+) T cell responses were similarly distributed against Env, Gag, and GPN. The proportion of responders after two doses of MVA-B was similar to that obtained after the third dose of MVA-B vaccination, and the responses were sustained (84.6% at week 48). Vaccine-induced CD8(+) T cells to HIV-1 antigens after 1 year were polyfunctional and distributed mainly within the effector memory (TEM) and terminally differentiated effector memory (TEMRA) T cell populations. Antivector T cell responses were mostly induced by CD8(+) T cells, highly polyfunctional, and of TEMRA phenotype. These findings demonstrate that the poxvirus MVA-B vaccine candidate given alone is highly immunogenic, inducing broad, polyfunctional, and long-lasting CD4 and CD8 T cell responses to HIV-1 antigens, with preference for TEM. Thus, on the basis of the immune profile of MVA-B in humans, this immunogen can be considered a promising HIV/AIDS vaccine candidate.