A Phase I Double Blind,Placebo-Controlled,Randomized Study of the Safety and Immunogenicity of an Adjuvanted HIV-1 Gag-Pol-Nef Fusion Protein and Adenovirus 35 Gag-RT-Int-Nef Vaccine in Healthy HIV-Uninfected African Adults

Background

Sequential prime-boost or co-administration of HIV vaccine candidates based on an adjuvanted clade B p24, RT, Nef, p17 fusion protein (F4/AS01) plus a non-replicating adenovirus 35 expressing clade A Gag, RT, Int and Nef (Ad35-GRIN) may lead to a unique immune profile, inducing both strong T-cell and antibody responses.

Methods

In a phase 1, double-blind, placebo-controlled trial, 146 healthy adult volunteers were randomized to one of four regimens: heterologous prime-boost with two doses of F4/AS01_E or F4/AS01_B followed by Ad35-GRIN; Ad35-GRIN followed by two doses of F4/AS01_B; or three co-administrations of Ad35-GRIN and F4/AS01_B. T cell and antibody responses were measured.

Results

The vaccines were generally well-tolerated, and did not cause serious adverse events. The response rate, by IFN-γ ELISPOT, was greater when Ad35-GRIN was the priming vaccine and in the co-administration groups. F4/AS01 induced CD4+ T-cells expressing primarily CD40L and IL2 +/- TNF-α, while Ad35-GRIN induced predominantly CD8+ T-cells expressing IFN-γ +/- IL2 or TNF-α. Viral inhibition was induced after Ad35-GRIN vaccination, regardless of the regimen. Strong F4-specific antibody responses were induced. Immune responses persisted at least a year after the last vaccination. The complementary response profiles, characteristic of each vaccine, were both expressed after co-administration.

Conclusion

Co-administration of an adjuvanted protein and an adenovirus vector showed an acceptable safety and reactogenicity profile and resulted in strong, multifunctional and complementary HIV-specific immune responses.

Trial Registration

ClinicalTrials.gov NCT01264445     

Legionella pneumophila Subversion of Host Vesicular Transport by SidC Effector Proteins

Tethering proteins play a key role in vesicular transport, ensuring that cargo arrives at a specific destination. The bacterial effector protein SidC and its paralog SdcA have been described as tethering factors encoded by the intracellular pathogen Legionella pneumophila. Here, we demonstrate that SidC proteins are important for early events unique to maturation of vacuoles containing Legionella and discover monoubiquitination of Rab1 as a new SidC‐dependent activity. The crystal structure of the SidC N‐terminus revealed a novel fold that is important for function and could be involved in Legionella adaptations to evolutionarily divergent host cells it encounters in natural environments.      

Dissociation by Chelating Agents and Substructure of the Thermophilic Bacteriophage TP84

The thermophilic bacteriophage TP84 is dissociated into its head, tail, and released deoxyribonucleic acid (DNA) by chelating agents such as ethylenediaminetetraacetic acid (EDTA) and phosphate. The phage is more sensitive to EDTA than to phosphate, and dialysis against either agent causes more effective dissociation than standing in their presence. The tail possesses a knobbed structure which is inserted into the head of the intact phage and to which the DNA appears to be attached. The method of dissociating TP84 described in this paper provides a source of undamaged structural components and intact strands of DNA for subsequent investigations. A possible mechanism of chelate inactivation is discussed.     

Brassica carinata CIL1 mediates extracellular ROS production during auxin- and ABA-regulated lateral root development

In the plant cytoplasm signals from multiple sources interact to control development. Lateral root proliferation is controlled by the antagonistic action of the hormones auxin and abscisic acid (ABA). Auxin stimulates the separation of pericycle initials and cell expansion. ABA is required for lateral root initiation and elongation and acts as an antagonist through auxin-dependent pathways. These hormones also mediate reactive oxygen species (ROS) accumulation in growing roots and promote cell expansion. We describe the isolation and characterization of a copper- and auxin-induced gene, COPPER INDUCED in LEAVES (CIL1), from Brassica carinata. Transgenic B. carinata seedlings expressing antisense CIL1 were used to determine the biological function of this gene. Lines with reduced CIL1 expression showed a decrease in lateral root development, as well as reduced sensitivity to auxin and ABA. Steady-state analysis of redox components showed a decrease in NADPH oxidase, superoxide dismutase, and catalase activity, accompanied by an increase in hydrogen peroxide concentration. GFP-tagged CIL1 accumulated at the plasma membrane and in the apoplast indicating CIL1 is likely extracellular. From these data we propose that CIL1 is an extracellular protein involved in ROS cycling mediating auxin and ABA signals.     

Presence and isolation of 2 lipoproteins immunologically related to apolipoprotein A I in human serum

Very high density lipoproteins d : 1.23--1.25 g/ml (VHDL2) have been isolated from human serum by preparative ultracentrifugation. They contain 80 per cent proteins and 20 per cent lipids. Lipids are mainly phospholipids (80 per cent). The proportion of lysolecithin (50 per cent) is higher than that of lecithin (40 per cent). The quantity of cholesterol is low, the free cholesterol: total cholesterol ratio is 0.35. VHDL2 consisted principally in lipoprotein D and two lipoproteins immunologically apparented to apolipoprotein A I, called LP A I1 and LP A I2. The LP A I1 has a molecular weight slightly higher and a hydrated density lower than that of LP AI2. Our experiments suggest that LP A I1 exists in the serum before ultracentrifugation while LP A I 2 comes from HDL degradation during ultracentrifugation. The immunological heterogeneity of apo A I forming different protein-lipid complexes is discussed.     

Phospholipase A2 action on planar lipid bilayers generates a small, transitory current that is voltage independent.

Addition of either bee venom or Trimeresurus flavoviridis phospholipase A2 (PLA2) to the solution bathing the front side of a voltage-clamped, planar lipid bilayer consistently produced a transitory current lasting approximately 100 s. This current is consistent with anions moving through the membrane to the rear side. The peak current is independent of holding potential. PLA2 activity on phospholipid membranes not only produced a current but also led to membrane rupture within 300 s. The current depends on Ca2+ and lipid type. Addition of PLA2 in the absence of Ca2+ or to membranes made of nonsubstrate lipids (e.g., glycerol monooleate or lysophosphatidylcholine) produced no current and did not break the bilayer. Peak current height, signal decay time, and time to membrane rupture all depended on PLA2 dose, whereas total charge produced was constant. This current does not flow through ion channels because there are no channels present and the current is not voltage dependent. The evidence is consistent with the hypothesis that the current is generated by the movement of ionized fatty acid produced by PLA2 action. These results demonstrate a simple method to measure enzyme activity in the presence of different substrates and varied environmental conditions.     

Synthesis and characterisation of sulfated amphiphilic alpha-, beta- and gamma-cyclodextrins: application to the complexation of acyclovir

The synthesis of sulfated amphiphilic alpha-, beta- and gamma-cyclodextrins was achieved according to the standard protection-deprotection procedure. The formation of inclusion complexes between the amphiphilic alpha-, beta- and gamma-cyclodextrins and an antiviral molecule, acyclovir (ACV) was investigated by UV-visible spectroscopy (UV-Vis) and electrospray ionisation mass spectrometry (ESIMS). UV-Vis spectroscopy allowed determination of the stoichiometry and stability constants of complexes, whereas ESIMS, a soft ionisation technique, allowed the detection of the inclusion complexes. The results showed that the non-sulfated amphiphilic cyclodextrins exhibit a 1:2 stoichiometry with acyclovir, while sulfated amphiphilic cyclodextrins, except gamma-cyclodextrin, exhibit a 1:1 stoichiometry indicating the loss of one interaction site. Non-covalent interactions between acyclovir and non-sulfated amphiphilic cyclodextrins appear to take place both in the cavity of the cyclodextrin and inside the hydrophobic zone generated by alkanoyl chains. In contrast, in the case of sulfated amphiphilic cyclodextrins, the interactions appear to involve only the hydrophobic region of the alkanoyl chains.