Broadly Neutralizing Immune Responses against Hepatitis C Virus Induced by Vectored Measles Viruses and a Recombinant Envelope Protein Booster

Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination.     

Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells

Glycosylation is one of the most common posttranslational modifications of proteins. It has important roles for protein structure, stability and functions. In vivo the glycostructures influence pharmacokinetics and immunogenecity. It is well known that significant differences in glycosylation and glycostructures exist between recombinant proteins expressed in mammalian, yeast and insect cells. However, differences in protein glycosylation between different mammalian cell lines are much less well known. In order to examine differences in glycosylation in mammalian cells we have expressed 12 proteins in the two commonly used cell lines HEK and CHO. The cells were transiently transfected, and the expressed proteins were purified. To identify differences in glycosylation the proteins were analyzed on SDS-PAGE, isoelectric focusing (IEF), mass spectrometry and released glycans on capillary gel electrophoresis (CGE-LIF). For all proteins significant differences in the glycosylation were detected. The proteins migrated differently on SDS-PAGE, had different isoform patterns on IEF, showed different mass peak distributions on mass spectrometry and showed differences in the glycostructures detected in CGE. In order to verify that differences detected were attributed to glycosylation the proteins were treated with deglycosylating enzymes. Although, culture conditions induced minor changes in the glycosylation the major differences were between the two cell lines.     

Contextual specificity in peptide-mediated protein interactions

Most biological processes are regulated through complex networks of transient protein interactions where a globular domain in one protein recognizes a linear peptide from another, creating a relatively small contact interface. Although sufficient to ensure binding, these linear motifs alone are usually too short to achieve the high specificity observed, and additional contacts are often encoded in the residues surrounding the motif (i.e. the context). Here, we systematically identified all instances of peptide-mediated protein interactions of known three-dimensional structure and used them to investigate the individual contribution of motif and context to the global binding energy. We found that, on average, the context is responsible for roughly 20% of the binding and plays a crucial role in determining interaction specificity, by either improving the affinity with the native partner or impeding non-native interactions. We also studied and quantified the topological and energetic variability of interaction interfaces, finding a much higher heterogeneity in the context residues than in the consensus binding motifs. Our analysis partially reveals the molecular mechanisms responsible for the dynamic nature of peptide-mediated interactions, and suggests a global evolutionary mechanism to maximise the binding specificity. Finally, we investigated the viability of non-native interactions and highlight cases of potential cross-reaction that might compensate for individual protein failure and establish backup circuits to increase the robustness of cell networks.     

Pelagic larvae of New England intertidal gastropods II. Anachis avara

Summary The egg case, pelagic larvae, and young juvenile of Anachis avara Say, a species of prosobranch gastropod belonging to the family Columbellidae, are described and figured from specimes reared in the laboratory.

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Zusammenfassung Die Arbeit beschreibt die Eierkapseln, die pelagischen Larven und die Jugendformen von Anachis avara Say, einer prosobranchen Gastropoden-Spezies, die zur Familie der Columbelliden gehört. Abbildungen von Exemplaren, die im Laboratorium aufgezogen wurden, werden wiedergegeben.

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Contribution number 1341, Woods Hole Oceanographic Institution. This research was supported by N.S.F. grant no. 17883.