Dense Bodies of a gH/gL/UL128/UL130/UL131 Pentamer-Repaired Towne Strain of Human Cytomegalovirus Induce an Enhanced Neutralizing Antibody Response.

The development of a vaccine against human cytomegalovirus infection (HCMV) is a high-priority medical goal. The viral pentameric protein complex consisting of glycoprotein H (gH)/gL/UL128-131A (PC) is considered to be an important vaccine component. Its relevance to the induction of a protective antibody response is, however, still a matter of debate. We addressed this issue by using subviral dense bodies (DBs) of HCMV. DBs are exceptionally immunogenic. Laboratory HCMV strain DBs harbor important neutralizing antibody targets, like the glycoproteins B, H, L, M, and N, but they are devoid of the PC. To be able to directly compare the impact of the PC on the levels of neutralizing antibody (NT-abs) responses, a PC-positive variant of the HCMV laboratory strain Towne was established by bacterial artificial chromosome (BAC) mutagenesis (Towne-UL130rep). This strain synthesized PC-positive DBs upon infection of fibroblasts. These DBs were used in side-by-side immunizations with PC-negative Towne DBs. Mouse and rabbit sera were tested to address the impact of the PC on DB immunogenicity. The neutralizing antibody response to PC-positive DBs was superior to that of PC-negative DBs, as tested on fibroblasts, epithelial cells, and endothelial cells and for both animal species used. The experiments revealed the potential of the PC to enhance the antibody response against HCMV. Of particular interest was the finding that PC-positive DBs induced an antibody response that blocked the infection of fibroblasts by a PC-positive viral strain more efficiently than sera following immunizations with PC-negative particles.IMPORTANCE Infections with the human cytomegalovirus (HCMV) may cause severe and even life-threatening disease manifestations in newborns and immunosuppressed individuals. Several strategies for the development of a vaccine against this virus are currently pursued. A critical question in this respect refers to the antigenic composition of a successful vaccine. Using a subviral particle vaccine candidate, we show here that one protein complex of HCMV, termed the pentameric complex (PC), enhances the neutralizing antibody response against viral infection of different cell types. We further show for the first time that this not only relates to the infection of epithelial or endothelial cells; the presence of the PC in the particles also enhanced the neutralizing antibody response against the infection of fibroblasts by HCMV. Together, these findings argue in favor of including the PC in strategies for HCMV vaccine development.

Evaluation of life cycle defective adenovirus mutants for production of adeno-associated virus vectors.

BACKGROUND: Adeno-associated virus-based vectors are efficient and safe drug candidates for different in vivo gene therapy applications. With increasing numbers of clinical studies based on AAV2 vectors that include not only rare, but also common diseases as a therapeutic target, there is an increased demand for the development of improved production technologies. METHODS: In the present study, we compared two life cycle defective adenovirus mutants as helper viruses for AAV2 vector production. They had deletions either in the gene coding for the preterminal protein (pTP) that is expressed early in the viral life cycle and is essential for genome replication or in the gene coding for the 100K protein, a protein with many functions, one of which is involved in virus assembly. AAV2 vector production efficiencies were evaluated by analyzing genome-containing particles using a real-time polymerase chain reaction and functional units were investigated by transduction assays. RESULTS: Somewhat contrary to our expectations, the ∆100K mutant virus showed only a moderate efficiency as a helper virus for AAV2 vector production, whereas the replication-deficient ∆pTP mutant supported AAV2 production almost as efficiently as adenovirus wild-type. We also showed that a temperature shift to 32°C together with extended incubation times improved AAV2 vector productivity. CONCLUSIONS: The present study indicates the advantages of using a ∆pTP mutant adenovirus rather than adenovirus wild-type as a helper virus for AAV2 production and also indicates that temperature shifts to lower temperatures may improve AAV2 vector production rates.

Global grass (Poaceae) success underpinned by traits facilitating colonization, persistence and habitat transformation.

Poaceae (the grasses) is arguably the most successful plant family, in terms of its global occurrence in (almost) all ecosystems with angiosperms, its ecological dominance in many ecosystems, and high species richness. We suggest that the success of grasses is best understood in context of their capacity to colonize, persist, and transform environments (the "Viking syndrome"). This results from combining effective long-distance dispersal, efficacious establishment biology, ecological flexibility, resilience to disturbance and the capacity to modify environments by changing the nature of fire and mammalian herbivory. We identify a diverse set of functional traits linked to dispersal, establishment and competitive abilities. Enhanced long-distance dispersal is determined by anemochory, epizoochory and endozoochory and is facilitated via the spikelet (and especially the awned lemma) which functions as the dispersal unit. Establishment success could be a consequence of the precocious embryo and large starch reserves, which may underpin the extremely short generation times in grasses. Post-establishment genetic bottlenecks may be mitigated by wind pollination and the widespread occurrence of polyploidy, in combination with gametic self-incompatibility. The ecological competitiveness of grasses is corroborated by their dominance across the range of environmental extremes tolerated by angiosperms, facilitated by both C3 and C4 photosynthesis, well-developed frost tolerance in several clades, and a sympodial growth form that enabled the evolution of both annual and long-lived life forms. Finally, absence of investment in wood (except in bamboos), and the presence of persistent buds at or below ground level, provides tolerance of repeated defoliation (whether by fire, frost, drought or herbivores). Biotic modification of environments via feedbacks with herbivory or fire reinforce grass dominance leading to open ecosystems. Grasses can be both palatable and productive, fostering high biomass and diversity of mammalian herbivores. Many grasses have a suite of architectural and functional traits that facilitate frequent fire, including a tufted growth form, and tannin-like substances in leaves which slow decomposition. We mapped these traits over the phylogeny of the Poales, spanning the grasses and their relatives, and demonstrated the accumulation of traits since monocots originated in the mid-Cretaceous. Although the sympodial growth form is a monocot trait, tillering resulting in the tufted growth form most likely evolved within the grasses. Similarly, although an ovary apparently constructed of a single carpel evolved in the most recent grass ancestor, spikelets and the awned lemma dispersal units evolved within the grasses. Frost tolerance and C4 photosynthesis evolved relatively late (late Palaeogene), and the last significant trait to evolve was probably the production of tannins, associated with pyrophytic savannas. This fits palaeobotanical data, suggesting several phases in the grass success story: from a late Cretaceous origin, to occasional tropical grassland patches in the later Palaeogene, to extensive C3 grassy woodlands in the early-middle Miocene, to the dramatic expansion of the tropical C4 grass savannas and grasslands in the Pliocene, and the C3 steppe grasslands during the Pleistocene glacial periods. Modern grasslands depend heavily on strongly seasonal climates, making them sensitive to climate change.

Disturbance of firefly luciferase-based bioassays by different aluminum species.

Luciferase-dependent assays, important for biochemical analyses of cytotoxicity and reporter genes, may be perturbed by compounds interfering with the luciferase reaction. We analyzed the impact of different aluminum (Al) species on a luciferase-based assay for determination of cellular adenosine triphosphate. Al(0) nanoparticles (Al(0)-NPs) but not Al2O3-NPs decreased luminescence, correlated to high absorbance of Al(0)-NPs. By contrast, Al ions increased the luminescent signal. Data demonstrate that luciferase-dependent assays can be reciprocally disturbed by Al-NPs or Al ions in a specific manner, depending on the particular Al species. Careful interpretation of data from such experiments is essential in order to obtain conclusive results.

Identification of SLURP-1 as an epidermal neuromodulator explains the clinical phenotype of Mal de Meleda.

Mal de Meleda is an autosomal recessive inflammatory and keratotic palmoplantar skin disorder due to mutations in the ARS B gene, encoding for SLURP-1 (secreted mammalian Ly-6/uPAR-related protein 1). SLURP-1 belongs to the Ly-6/uPAR superfamily of receptor and secreted proteins, which participate in signal transduction, immune cell activation or cellular adhesion. The high degree of structural similarity between SLURP-1 and the three fingers motif of snake neurotoxins and Lynx1 suggests that this protein interacts with the neuronal acetylcholine receptors. We found that SLURP-1 potentiates the human alpha 7 nicotinic acetylcholine receptors that are present in keratinocytes. These results identify SLURP-1 as a secreted epidermal neuromodulator which is likely to be essential for both epidermal homeostasis and inhibition of TNF-alpha release by macrophages during wound healing. This explains both the hyperproliferative as well as the inflammatory clinical phenotype of Mal de Meleda.