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Search Results: 1 - 10 of 4583 matches for " Klaus überla "
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HIV Vaccine Development in the Aftermath of the STEP Study: Re-Focus on Occult HIV Infection?
Klaus überla
PLOS Pathogens , 2008, DOI: 10.1371/journal.ppat.1000114
Developing an HIV Vaccine: The Role of Efficacy Studies in Nonhuman Primates
Klaus überla
PLOS Medicine , 2005, DOI: 10.1371/journal.pmed.0020119
Characterization of cationic lipid DNA transfection complexes differing in susceptability to serum inhibition
Godwin Nchinda, Klaus überla, Olaf Zsch?rnig
BMC Biotechnology , 2002, DOI: 10.1186/1472-6750-2-12
Abstract: Increasing the ratio of DOTAP or DC to DNA led to a dose dependent enhancement of transfection efficiency in the presence of high serum concentrations up to a ratio of approximately 128 nmol lipid/μg DNA. Transfection efficiency could be further increased for all ratios of DOTAP and DC to DNA by addition of the DNA condensing agent protamine sulfate (PS). For DOTAP DNA complexes with ratios of ≤ 32 nmol/μg DNA, peak transfection efficiencies were obtained with 4 μg PS/μg DNA. In contrast, increasing the amount of PS of DC complexes above 0.5 μg PS /μg DNA did not lead to significant further increases in transfection efficiency in the presence of high serum concentrations. Four complexes, which had a similar high transfection efficiency in cell culture in the presence of low serum concentrations but which differed largely in the lipid to DNA ratio and the amount of PS were selected for further analysis. Intravenous injection of the selected complexes led to 22-fold differences in transduction efficiency, which correlated with transfection efficiency in the presence of high serum concentrations. The complex with the highest transfection efficiency in vivo consisted of 64 nmol DC/ 16 μg PS/ μg DNA. Physical analysis revealed a predicted size of 440 nm and the highest zeta potential of the complexes analyzed.Optimization of cationic lipid DNA complexes for transfection efficiency in the presence of high concentrations of serum led to the identification of a DC complex with high transduction efficiency in mice. This complex differs from previously described ones by higher lipid to DNA and PS to DNA ratios. The stability of this complex in the presence of high concentrations of serum and its high transduction efficiency in mice suggests that it is a promising candidate vehicle for in vivo gene delivery.Cationic lipid DNA complexes have been used as a non-viral gene delivery system in cell culture [1-5] and in several animal models [6-11]. Apart from their relative ease of
Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps
Nicola Ternette, Daniela Stefanou, Seraphin Kuate, Klaus überla, Thomas Grunwald
Virology Journal , 2007, DOI: 10.1186/1743-422x-4-51
Abstract: Insertion of an intron flanked by exonic sequences 5'-terminal to the open reading frames (ORF) of VSV-G and RSV-F led to detectable cytoplasmic mRNA levels of both genes. While the exonic sequences were sufficient to stabilise the VSV-G mRNA, cytoplasmic mRNA levels of RSV-F were dependent on the presence of a functional intron. Cytoplasmic VSV-G mRNA levels led to readily detectable levels of VSV-G protein, whereas RSV-F protein expression remained undetectable. However, RSV-F expression was observed after mutating two of four consensus sites for polyadenylation present in the RSV-F ORF. Expression levels could be further enhanced by codon optimisation.Insufficient cytoplasmic mRNA levels and premature polyadenylation prevent expression of RSV-F by RNA polymerase II dependent expression plasmids. Since RSV replicates in the cytoplasm, the presence of premature polyadenylation sites and elements leading to nuclear instability should not interfere with RSV-F expression during virus replication. The molecular mechanisms responsible for the destabilisation of the RSV-F and VSV-G mRNAs and the different requirements for their rescue by insertion of an intron remain to be defined.Eukaryotic cells differ from prokaryotic cells by increased compartmentalisation of the intracellular environment to facilitate complex enzymatic reactions required for efficient protein expression and modification, cell metabolism and/or cell division. Adaptation to the host cell and particularly to its expression machinery is the key requirement for the replication of any virus. Several RNA viruses only replicate in the cytoplasm of their eukaryotic host cell. These viruses possess their own transcription machinery involving a viral RNA-dependent RNA polymerase which allows cytoplasmic mRNA synthesis from the viral genomic RNA. Therefore, these viruses are not adapted to the complex nuclear milieu of the eukaryotic host cell. Inefficient expression of genes from RNA viruses by RNA polymerase
The HIV-1 Rev Protein Enhances Encapsidation of Unspliced and Spliced, RRE-Containing Lentiviral Vector RNA
Bastian Grewe, Katrin Ehrhardt, Bianca Hoffmann, Maik Blissenbach, Sabine Brandt, Klaus überla
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0048688
Abstract: Background During the RNA encapsidation process of human immunodeficiency virus (HIV) viral genomic, unspliced RNA (gRNA) is preferentially incorporated into assembling virions. However, a certain amount of spliced viral transcripts can also be detected in viral particles. Recently, we observed that nuclear export of HIV and lentiviral vector gRNA by Rev is required for efficient encapsidation. Since singly-spliced HIV transcripts also contain the Rev-response element (RRE), we investigated if the encapsidation efficiency of RRE-containing spliced HIV-vector transcripts is also increased by the viral Rev protein. Findings Starting with a lentiviral vector imitating the splicing pattern of HIV, we constructed vectors that express an unspliced transcript either identical in sequence to the singly-spliced or the fully-spliced RNA of the parental construct. After transfection of the different lentiviral vectors cytoplasmic and virion-associated RNA levels and vector titers were determined in the presence and absence of Rev. Rev enhanced the infectious titer of vectors containing an RRE 6 to 37-fold. Furthermore, Rev strongly increased encapsidation efficiencies of all RRE-containing transcripts up to 200-fold. However, a good correlation between encapsidation efficiency and lentiviral vector titer could only be observed for the gRNA. The infectious titer of the vector encoding the fully-spliced RNA without RRE as well as the encapsidation efficiency of all transcripts lacking the RRE was not influenced by Rev. Interestingly, the splicing process itself did not seem to interfere with packaging, since the encapsidation efficiencies of the same RNA expressed either by splicing or as an unspliced transcript did not differ significantly. Conclusions Rev-mediated nuclear export enhances the encapsidation efficiency of RRE-containing lentiviral vector RNAs independently of whether they have been spliced or not.
Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8+ T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity
Matthias Tenbusch, Seraphin Kuate, Bettina Tippler, Nicole Gerlach, Simone Schimmer, Ulf Dittmer, Klaus überla
BMC Immunology , 2008, DOI: 10.1186/1471-2172-9-13
Abstract: In vitro, the GM-CSF ovalbumin fusion protein (GM-OVA) led to the better stimulation of OVA-specific CD8+ T cells by antigen-presenting cells than OVA and GM-CSF given as two separate proteins. However, prime-boost immunizations of mice with DNA and adenoviral vector vaccines encoding GM-OVA suppressed CD8+ T-cell responses to OVA. OVA-specific IgG2a antibody levels were also reduced, while the IgG1 antibody response was enhanced. Suppression of CD8+ T cell responses by GM-OVA vaccines was associated with the induction of neutralizing antibodies to GM-CSF. In contrast, the coexpression of GM-CSF and antigens in DNA prime adenoviral boost immunizations led to a striking expansion of polyfunctional OVA-specific CD8+ T cells without the induction of autoantibodies.The induction of autoantibodies suggests a general note of caution regarding the use of highly immunogenic viral vector vaccines encoding fusion proteins between antigens and host proteins. In contrast, the expansion of polyfunctional OVA-specific CD8+ T cells after immunizations with bicistronic vectors further support a potential application of GM-CSF as an adjuvant for heterologous prime-boost regimens with genetic vaccines. Since DNA prime adenoviral vector boost regimenes are presently considered as one of the most efficient ways to induce CD8+ T cell responses in mice, non-human primates and humans, further enhancement of this response by GM-CSF is a striking observation.The induction of strong CTL responses by prophylactic and therapeutic vaccines is considered necessary for the control of chronic viral infections and cancer [1-3]. Genetic vaccines seem to be promising tools, since the expression of antigen by the vaccinee leads to improved MHC-I restricted cellular immune responses. DNA vaccines have been shown to elicit CTL, T helper and antibody responses in a variety of animal models [4-8]. However, DNA vaccines alone stimulated only weak T-cell responses in monkeys [9] and humans [10]. To enhance
T cell independent secondary antibody responses to the envelope protein of simian immunodeficiency virus
Ghulam Nabi, Vladimir Temchura, Claudius Gro?mann, Seraphin Kuate, Matthias Tenbusch, Klaus überla
Retrovirology , 2012, DOI: 10.1186/1742-4690-9-42
Abstract: To explore differential regulation of Gag and Env antibody responses, immunocompetent BALB/c and T cell deficient nude mice were immunized with virus like particles (VLP) of simian immunodeficiency virus or adenoviral vectors expressing SIV Gag and Env. High levels of antibodies against Gag and Env could only be induced in immunocompetent mice, but not in the immunodeficient mice. Thus, neither cells expressing Env after adenoviral gene transfer nor VLPs induce a T cell independent primary anti-Env antibody response. However, secondary B cell responses to Env, but not to Gag, were observed in immunodeficient mice after transfer of primed B cells and boosting with VLPs or adenoviral vectors expressing Gag and Env. This T cell independent secondary antibody response to Env was reduced after stimulation with VLPs modified to contain monomeric membrane bound gp130 surface subunit of Env and undetectable after injection of soluble gp130.Membrane-bound trimeric Env seems to be responsible for the maintenance of high levels of anti-Env antibodies during progression to AIDS. This T cell independent secondary antibody response may prevent T cell-dependent affinity maturation and thus contribute to viral immune escape by favoring persistence of non-protective antibodies.
Rev Proteins of Human and Simian Immunodeficiency Virus Enhance RNA Encapsidation
Sabine Brandt equal contributor,Maik Bli?enbach equal contributor,Bastian Grewe,Rebecca Konietzny,Thomas Grunwald,Klaus überla
PLOS Pathogens , 2007, DOI: 10.1371/journal.ppat.0030054
Abstract: The main function attributed to the Rev proteins of immunodeficiency viruses is the shuttling of viral RNAs containing the Rev responsive element (RRE) via the CRM-1 export pathway from the nucleus to the cytoplasm. This restricts expression of structural proteins to the late phase of the lentiviral replication cycle. Using Rev-independent gag-pol expression plasmids of HIV-1 and simian immunodeficiency virus and lentiviral vector constructs, we have observed that HIV-1 and simian immunodeficiency virus Rev enhanced RNA encapsidation 20- to 70-fold, correlating well with the effect of Rev on vector titers. In contrast, cytoplasmic vector RNA levels were only marginally affected by Rev. Binding of Rev to the RRE or to a heterologous RNA element was required for Rev-mediated enhancement of RNA encapsidation. In addition to specific interactions of nucleocapsid with the packaging signal at the 5′ end of the genome, the Rev/RRE system provides a second mechanism contributing to preferential encapsidation of genomic lentiviral RNA.
CCL28 Induces Mucosal Homing of HIV-1-Specific IgA-Secreting Plasma Cells in Mice Immunized with HIV-1 Virus-Like Particles
Veronica Rainone, Gregor Dubois, Vladimir Temchura, Klaus überla, Alberto Clivio, Manuela Nebuloni, Eleonora Lauri, Daria Trabattoni, Francisco Veas, Mario Clerici
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0026979
Abstract: Mucosae-associated epithelial chemokine (MEC or CCL28) binds to CCR3 and CCR10 and recruits IgA-secreting plasma cells (IgA-ASCs) in the mucosal lamina propria. The ability of this chemokine to enhance migration of IgA-ASCs to mucosal sites was assessed in a mouse immunization model using HIV-1IIIB Virus-like particles (VLPs). Mice receiving either HIV-1IIIB VLPs alone, CCL28 alone, or the irrelevant CCL19 chemokine were used as controls. Results showed a significantly increased CCR3 and CCR10 expression on CD19+ splenocytes of HIV-1IIIB VPL-CCL28-treated mice. HIV-1 Env-specific IFN-γ, IL-4 and IL-5 production, total IgA, anti-Env IgA as well as gastro-intestinal mucosal IgA-secreting plasma cells were also significantly augmented in these mice. Notably, sera and vaginal secretions from HIV-1IIIB VLP-CCL28-treated mice exhibited an enhanced neutralizing activity against both a HIV-1/B-subtype laboratory strain and a heterologous HIV-1/C-subtype primary isolate. These data suggest that CCL28 could be useful in enhancing the IgA immune response that will likely play a pivotal role in prophylactic HIV vaccines.
Enhancement of the priming efficacy of DNA vaccines encoding dendritic cell-targeted antigens by synergistic toll-like receptor ligands
Claudius Grossmann, Matthias Tenbusch, Godwin Nchinda, Vladimir Temchura, Ghulam Nabi, Geoffrey W Stone, Richard S Kornbluth, Klaus überla
BMC Immunology , 2009, DOI: 10.1186/1471-2172-10-43
Abstract: Mice were primed with the adjuvanted DEC-205 targeted DNA vaccines and boosted with adenoviral vectors encoding the same antigens. CD8+ T cell responses were determined after the adenoviral booster immunization, to determine how well the different DNA immunization regimens prime for the adenoviral boost. In the absence of adjuvants, targeting of DNA-encoded ovalbumin to DCs suppressed CD8+ T-cell responses after the adenoviral booster immunization. CD8+ T-cell responses to the DEC205 targeted DNA vaccines increased only slightly by adding either the TLR-9 ligand CpG, the TLR-3 ligand Poly I:C, or CD40 ligand expression plasmids. However, the combination of both TLR-ligands led to a strong enhancement of CD8+ T-cell responses compared to a non-targeted DNA vaccine. This finding was confirmed using HIV Gag as antigen.Although DNA prime adenoviral vector boost immunizations belong to the strongest inducers of cytotoxic T cell responses in different animal models and humans, the CD8+ T cell responses can be further improved by targeting the DNA encoded antigen to DEC205 in the presence of synergistic TLR ligands CpG and Poly I:C.Depending on their state of maturation or activation dendritic cells (DC) can prime antigen-specific T cells with substantially different functional activities. While DC can mediate peripheral T cell tolerance in the steady-state, activation and maturation turns them into potent inducers of CD4+ and CD8+ T cell immunity (reviewed in [1]). Thus, targeting antigens to DCs is a promising strategy to improve prevention and treatment of autoimmunity and to raise the efficacy of vaccines against tumors and infectious diseases. For targeting of DC in vivo, most of the studies use recombinant protein antigens coupled or fused to an antibody specific for a DC surface marker. In addition to targeting the DCs their differentiation and activation status needs to be controlled in order to obtain the desired T cell response. A striking example of how DC activ
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