Fighting smart diseases requires smart vaccines. Novel ways to present protective immunogenic peptide epitopes to human immune systems are needed. Herein, we focus on Self Assembling Protein ...Nanoparticles (SAPNs) as scaffolds/platforms for vaccine delivery that produce strong immune responses against Toxoplasma gondii in HLA supermotif, transgenic mice. Herein, we present a useful platform to present peptides that elicit CD4
, CD8
T and B cell immune responses in a core architecture, formed by flagellin, administered in combination with TLR4 ligand-emulsion (GLA-SE) adjuvant. We demonstrate protection of HLA-A*11:01, HLA-A*02:01, and HLA-B*07:02 mice against toxoplasmosis by (i) this novel chimeric polypeptide, containing epitopes that elicit CD8
T cells, CD4
T helper cells, and IgG2b antibodies, and (ii) adjuvant activation of innate immune TLR4 and TLR5 pathways. HLA-A*11:01, HLA-A*02:01, and HLA-B*07:02q11 transgenic mouse splenocytes with peptides demonstrated predicted genetic restrictions. This creates a new paradigm-shifting vaccine approach to prevent toxoplasmosis, extendable to other diseases.
The parasitic disease malaria remains a major global public health concern and no truly effective vaccine exists. One approach to the development of a malaria vaccine is to target the asexual blood ...stage that results in clinical symptoms. Most attempts have failed. New antigens such as P27A and P27 have emerged as potential new vaccine candidates. Multiple studies have demonstrated that antigens are more immunogenic and are better correlated with protection when presented on particulate delivery systems. One such particulate delivery system is the self-assembling protein nanoparticle (SAPN) that relies on coiled-coil domains of proteins to form stable nanoparticles. In the past we have used de novo designed amino acid domains to drive the formation of the coiled-coil scaffolds which present the antigenic epitopes on the particle surface.
Here we use naturally occurring domains found in the tex1 protein to form the coiled-coil scaffolding of the nanoparticle. Thus, by engineering P27A and a new extended form of the coiled-coil domain P27 onto the N and C terminus of the SAPN protein monomer we have developed a particulate delivery system that effectively displays both antigens on a single particle that uses malaria tex1 sequences to form the nanoparticle scaffold. These particles are immunogenic in a murine model and induce immune responses similar to the ones observed in seropositive individuals in malaria endemic regions.
We demonstrate that our P27/P27A-SAPNs induce an immune response akin to the one in seropositive individuals in Burkina Faso. Since P27 is highly conserved among different Plasmodium species, these novel SAPNs may even provide cross-protection between Plasmodium falciparum and Plasmodium vivax the two major human malaria pathogens. As the SAPNs are also easy to manufacture and store they can be delivered to the population in need without complication thus providing a low cost malaria vaccine.
Abstract Current influenza vaccines should be improved by the addition of universal influenza vaccine antigens in order protect against multiple virus strains. We used our self-assembling protein ...nanoparticles (SAPNs) to display the two conserved influenza antigens M2e and Helix C in their native oligomerization states. To further improve the immunogenicity of the SAPNs we designed and incorporated the TLR5 agonist flagellin into the SAPNs to generate self-adjuvanted SAPNs. We demonstrate that addition of flagellin does not affect the ability of SAPNs to self-assemble and that they are able to stimulate TLR5 in a dose dependent manner. Chickens vaccinated with the self-adjuvanted-SAPNs induce significantly higher levels of antibodies than unadjuvanted-SAPNs and show higher cross-neutralizing activity compared to a commercial inactivated virus vaccine. Upon immunization with self-adjuvanted-SAPNs mice were completely protected against a lethal challenge. Thus, we have generated a self-adjuvanted-SAPN with a great potential as a universal influenza vaccine.
Nuclear pore complexes (NPCs) facilitate macromolecular exchange between the nucleus and cytoplasm of eukaryotic cells. The vertebrate NPC is composed of ∼30 different proteins (nucleoporins), of ...which around one third contain phenylalanine-glycine (FG)-repeat domains that are thought to mediate the main interaction between the NPC and soluble transport receptors. We have recently shown that the FG-repeat domain of Nup153 is flexible within the NPC, although this nucleoporin is anchored to the nuclear side of the NPC. By using domain-specific antibodies, we have now mapped the domain topology of Nup214 in
Xenopus oocytes and in human somatic cells by immuno-EM. We have found that whereas Nup214 is anchored to the cytoplasmic side of the NPC
via its N-terminal and central domain, its FG-repeat domain appears flexible, residing on both sides of the NPC. Moreover, the spatial distribution of the FG-repeat domains of both Nup153 and Nup214 shifts in a transport-dependent manner, suggesting that the location of FG-repeat domains within the NPC correlates with cargo/receptor interactions and that they concomitantly move with cargo through the central pore of the NPC.
Nucleoporins represent the molecular building blocks of nuclear pore complexes (NPCs), which mediate facilitated macromolecular trafficking between the cytoplasm and nucleus of eukaryotic cells. ...Phenylalanine-glycine (FG) repeat motifs are found in about one-third of the nucleoporins, and they provide major binding or docking sites for soluble transport receptors. We have shown recently that localization of the FG-repeat domains of vertebrate nucleoporins Nup153 and Nup214 within the NPC is influenced by its transport state. To test whether chemical effectors, such as calcium and ATP, influence the localization of the FG-repeat domains of Nup153 and Nup214 within the NPC, we performed immuno-electron microscopy of
Xenopus oocyte nuclei using domain-specific antibodies against Nup153 and Nup214, respectively. Ca
2+ and ATP are known to induce conformational changes in the NPC architecture, especially at the cytoplasmic face, but also at the nuclear basket of the NPC. We have found concentrations of calcium in the micromolar range or 1 mM ATP in the surrounding buffer leaves the spatial distribution of the FG-repeat of Nup153 and Nup214 largely unchanged. In contrast, ATP depletion, calcium store depletion by EGTA or thapsigargin, and high concentrations of divalent cation (i.e. 2 mM Ca
2+ and 2 mM Mg
2+) constrain the distribution of the FG-repeats of Nup153 and Nup214. Our data suggest that the location of the FG-repeat domains of Nup153 and Nup214 is sensitive to chemical changes within the near-field environment of the NPC.
Three actinomycete strains isolated from soil treated with 2,4-D were able to degrade the herbicide Diuron
in vitro. Strain CCT 4916 was the most efficient, degrading up to 37% of applied Diuron (100 ...mg Kg
−1 soil) in 7 days, as measured by HPLC and UV/VIS spectroscopy. All strains showed protease and urease activity; intracellular activity of metapyrocatechase and pyrocatechase were not found. Actinomycete strain CCT 4916 produced manganese peroxidase, which could be potentially related to degradation of Diuron.
We investigate the interaction of hepatitis B virus capsids lacking a nuclear localization signal with nuclear pore complexes (NPCs) in permeabilized HeLa cells. Confocal and wide-field optical ...images of the nuclear envelope show well-spaced individual NPCs. Specific interactions of capsids with single NPCs are characterized by extended residence times of capsids in the focal volume which are characterized by fluorescence correlation spectroscopy. In addition, single-capsid-tracking experiments using fast wide-field fluorescence microscopy at 50 frames/s allow us to directly observe specific binding via a dual-color colocalization of capsids and NPCs. We find that binding occurs with high probability on the nuclear-pore ring moiety, at 44±9nm radial distance from the central axis.
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