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1.
Vaccine ; 29(37): 6352-7, 2011 Aug 26.
Article in English | MEDLINE | ID: mdl-21596075

ABSTRACT

West Nile Virus (WNV) is an emerging pathogenic flavivirus with increasing distribution worldwide. Birds are the natural host of the virus, but also mammals, including humans, can be infected. In some cases, a WNV infection can be associated with severe neurological symptoms. All currently available WNV vaccines are in the veterinary sector, and there is a need to develop safe and effective immunization technologies, which can also be used in humans. An alternative to current vaccination methods is DNA immunization. Most current DNA vaccine candidates against flaviviruses simultaneously express the viral envelope (E) and membrane (prM) proteins, which leads to the formation of virus-like particles. Here we generated a DNA plasmid, which expresses only the E-protein ectodomain. Vaccination of mice stimulated anti-WNV T-cell responses and neutralizing antibodies that were higher than those obtained after immunizing with a recombinant protein previously shown to be a protective WNV vaccine. A single dose of the plasmid was sufficient to protect animals from a lethal challenge with the virus. Moreover, immunogenicity could be boosted when DNA injection was followed by immunization with recombinant domain DIII of the E-protein. This resulted in significantly enhanced neutralizing antibody titers and a more prominent cellular immune response. The results suggest that the WNV E-protein is sufficient as a protective antigen in DNA vaccines and that protection can be significantly improved by adding a recombinant protein boost to the DNA prime.


Subject(s)
Plasmids , Vaccines, DNA/immunology , Viral Envelope Proteins/immunology , West Nile Virus Vaccines , Adjuvants, Immunologic , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Chlorocebus aethiops , Female , HeLa Cells , Humans , Immunization, Secondary , Interferon-gamma/biosynthesis , Mice , Mice, Inbred BALB C , Plasmids/administration & dosage , Plasmids/genetics , Plasmids/immunology , Recombinant Proteins/immunology , T-Lymphocytes/immunology , Vaccination , Vaccines, DNA/genetics , Vero Cells , West Nile Fever/immunology , West Nile Fever/prevention & control , West Nile Virus Vaccines/administration & dosage , West Nile Virus Vaccines/genetics , West Nile Virus Vaccines/immunology , West Nile virus/genetics , West Nile virus/immunology
2.
J Biol Phys ; 37(1): 117-31, 2011 Jan.
Article in English | MEDLINE | ID: mdl-22210966

ABSTRACT

The compaction of DNA by the HU protein from Thermotoga maritima (TmHU) is analysed on a single-molecule level by the usage of an optical tweezers-assisted force clamp. The condensation reaction is investigated at forces between 2 and 40 pN applied to the ends of the DNA as well as in dependence on the TmHU concentration. At 2 and 5 pN, the DNA compaction down to 30% of the initial end-to-end distance takes place in two regimes. Increasing the force changes the progression of the reaction until almost nothing is observed at 40 pN. Based on the results of steered molecular dynamics simulations, the first regime of the length reduction is assigned to a primary level of DNA compaction by TmHU. The second one is supposed to correspond to the formation of higher levels of structural organisation. These findings are supported by results obtained by atomic force microscopy.

3.
Eur Biophys J ; 37(6): 927-34, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18379774

ABSTRACT

Optical tweezers (OT) are ideally suited to study the interaction of single receptor-ligand bonds. Here we introduce a newly developed assay using OT to investigate the interactions between Protein A from Staphylococcus aureus and Immunoglobulin G from rabbit serum (RIgG). We demonstrate that the rupture forces depend on the loading rate and on the sodium chloride concentration. The measured loading rate effect is well known in the literature and the data we obtained were found to be in good agreement with an already published theoretical model. The dependence of the rupture forces on the salt concentration demonstrates the influence of hydrophobic interactions on the bond strength. Our experimental setup can probe the interaction between a single receptor and its specific ligand under changing conditions and hence offers manifold applications in single molecule biotechnology.


Subject(s)
Immunoglobulin G/chemistry , Models, Chemical , Models, Molecular , Optical Tweezers , Staphylococcal Protein A/chemistry , Computer Simulation , Elasticity , Protein Binding , Stress, Mechanical
4.
Microsc Res Tech ; 70(11): 938-43, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17661366

ABSTRACT

The kinetics of binding for the histone-like protein TmHU (from Thermotoga maritima) to DNA is analyzed on a single molecule level by use of optical tweezers. For the reaction rate a pronounced concentration-dependence is found with an "all or nothing"-limit which suggests the cooperative nature of the binding-reaction. By analyzing the statistics of mechanically induced dissociation-events of TmHU from DNA multiple reaction sites are observed to become more likely with increasing TmHU concentration. This is interpreted as a hint for a secondary organizational level of the TmHU/DNA complex. The reaction rate of TmHU binding to DNA is remarkably higher than that of the HU protein from Escherichia coli which will be discussed.


Subject(s)
Bacterial Proteins/metabolism , DNA/metabolism , Optical Tweezers , Thermotoga maritima/chemistry , DNA-Binding Proteins/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Protein Binding
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