A novel neutralizing human monoclonal antibody broadly abrogates hepatitis C virus infection in vitro and in vivo

Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the effi cacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization levels observed for mAb 2A5 were generally high and mostly superior to those obtained with AP33, a well-characterized HCV-neutralizing monoclonal antibody. Using humanized mice, complete protection was observed after genotype 1a and 4a HCV challenge, while only partial protection was achieved using gt1b and 6a isolates. Epitope mapping revealed that mAb 2A5 binding is conformation-dependent and identified the E2-region spanning amino acids 434 to 446 (epitope II) as the predominant contact domain. Conclusion : mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence represent a valuable candidate to prevent HCV recurrence in LT-patients. In addition, the detailed identification of the neutralizing epitope can be applied for the design of prophylactic HCV vaccines

Vectors for expression of proteins with single or combinatorial fluorescent protein and tandem affinity purification tags in Dictyostelium

We constructed a series of expression vectors for purification of native proteins and protein complexes in Dictyostelium. Protein purification is achieved by either a C-terminal or N-terminal fusion of the protein of choice to the tandem affinity purification (TAP) tag. The TAP tag consists of a protein A tag and a calmodulin binding peptide (CBP) and has been successfully used for purification of native protein complexes from yeast and animal cells. Protein expression is driven by the constitutive actin 15 promoter and the vectors optionally carry additional green- or yellow fluorescent protein (GFP or YFP) tags for fusion at either a C- or N-terminal location. Tandem affinity purification of native Dictyostelium protein complexes was tested by using pArc-34, one of the members of the well characterized Dictyostelium Arp2/3 complex, as bait. After denaturation and SDS–PAGE separation of the pArc-34 associated proteins all members of the Arp2/3 complex could be identified

Characterization of a cAMP-stimulated cAMP phosphodiesterase in Dictyostelium discoideum

A cyclic nucleotide phosphodiesterase, PdeE, that harbors two cyclic nucleotide binding motifs and a binuclear Zn2+-binding domain was characterized in Dictyostelium. In other eukaryotes, the Dictyostelium domain shows greatest homology to the 73-kDa subunit of the pre-mRNA cleavage and polyadenylation specificity factor. The Dictyostelium PdeE gene is expressed at its highest levels during aggregation, and its disruption causes the loss of a cAMP-phosphodiesterase activity. The pdeE null mutants show a normal cAMP-induced cGMP response and a 1.5-fold increase of cAMP-induced cAMP relay. Overexpression of a PdeE-yellow fluorescent protein (YFP) fusion construct causes inhibition of aggregation and loss of the cAMP relay response, but the cells can aggregate in synergy with wild-type cells. The PdeE-YFP fusion protein was partially purified by immunoprecipitation and biochemically characterized. PdeE and its Dictyostelium ortholog, PdeD, are both maximally active at pH 7.0. Both enzymes require bivalent cations for activity. The common cofactors Zn2+ and Mg2+ activated PdeE and PdeD maximally at 10 mm, whereas Mn2+ activated the enzymes to 4-fold higher levels, with half-maximal activation between 10 and 100 gm. PdeE is an allosteric enzyme, which is similar to4-fold activated by cAMP, with half-maximal activation occurring at about 10 muM and an apparent K-m of similar to1 mm. cGMP is degraded at a 6-fold lower rate than cAMP. Neither cGMP nor 8-Br-cAMP are efficient activators of PdeE activity

Vaccine-induced monoclonal antibodies targeting circumsporozoite protein prevent Plasmodium falciparum infection.

Contains fulltext : 137167.pdf (publisher's version ) (Open Access

Microorganism Response to Stressed Terrestrial Environments: A Raman Spectroscopic Perspective of Extremophilic Life Strategies

Raman spectroscopy is a valuable analytical technique for the identification of biomolecules and minerals in natural samples, which involves little or minimal sample manipulation. In this paper, we evaluate the advantages and disadvantages of this technique applied to the study of extremophiles. Furthermore, we provide a review of the results published, up to the present point in time, of the bio- and geo-strategies adopted by different types of extremophile colonies of microorganisms. We also show the characteristic Raman signatures for the identification of pigments and minerals, which appear in those complex samples

TARP is an immunotherapeutic target in acute myeloid leukemia expressed in the leukemic stem cell compartment

Immunotherapeutic strategies targeting the rare leukemic stem cell compartment might provide salvage to the high relapse rates currently observed in acute myeloid leukemia (AML). We applied gene expression profiling for comparison of leukemic blasts and leukemic stem cells with their normal counterparts. Here, we show that the T-cell receptor ? chain alternate reading frame protein (TARP) is over-expressed in de novo pediatric (n=13) and adult (n=17) AML sorted leukemic stem cells and blasts compared to hematopoietic stem cells and normal myeloblasts (15 healthy controls). Moreover, TARP expression was significantly associated with a Emslike tyrosine kinase receptor-3 internal tandem duplication in pediatric AML. TARP overexpression was confirmed in AML cell lines (n=9), and was found to be absent in B-cell acute lymphocytic leukemia (n=5) and chronic myeloid leukemia (n=1). Sequencing revealed that both a classical TARP transcript, as described in breast and prostate adenocarcinoma, and an AML-specific alternative TARP transcript, were present. Protein expression levels mostly matched transcript levels. TARP was shown to reside in the cytoplasmic compartment and showed sporadic endoplasmic reticulum co-localization. TARP-T-cell receptor engineered cytotoxic T-cells in vitro killed AML cell lines and patient leukemic cells co-expressing TARP and HLA-A*0201. In conclusion, TARP qualifies as a relevant target for immunotherapeutic T-cell therapy in AML