A Novel Virus-Like Particle Based Vaccine Platform Displaying the Placental Malaria Antigen VAR2CSA

Placental malaria caused by Plasmodium falciparum is a major cause of mortality and severe morbidity. Clinical testing of a soluble protein-based vaccine containing the parasite ligand, VAR2CSA, has been initiated. VAR2CSA binds to the human receptor chondroitin sulphate A (CSA) and is responsible for sequestration of Plasmodium falciparum infected erythrocytes in the placenta. It is imperative that a vaccine against malaria in pregnancy, if administered to women before they become pregnant, can induce a strong and long lasting immune response. While most soluble protein-based vaccines have failed during clinical testing, virus-like particle (VLP) based vaccines (e.g., the licensed human papillomavirus vaccines) have demonstrated high efficacy, suggesting that the spatial assembly of the vaccine antigen is a critical parameter for inducing an optimal long-lasting protective immune response. We have developed a VLP vaccine display platform by identifying regions of the HPV16 L1 coat protein where a biotin acceptor site (AviTag^TM) can be inserted without compromising VLP-assembly. Subsequent biotinylation of Avi-L1 VLPs allow us to anchor monovalent streptavidin (mSA)-fused proteins to the biotin, thereby obtaining a dense and repetitive VLP-display of the vaccine antigen. The mSA-VAR2CSA antigen was delivered on the Avi-L1 VLP platform and tested in C57BL/6 mice in comparison to two soluble protein-based vaccines consisting of naked VAR2CSA and mSA-VAR2CSA. The mSA-VAR2CSA Avi-L1 VLP and soluble mSA-VAR2CSA vaccines induced higher antibody titers than the soluble naked VAR2CSA vaccine after three immunizations. The VAR2CSA Avi-L1 VLP vaccine induced statistically significantly higher endpoint titres compared to the soluble mSA-VAR2CSA vaccine, after 1^st and 2^nd immunization; however, this difference was not statistically significant after 3^rd immunization. Importantly, the VLP-VAR2CSA induced antibodies were functional in inhibiting the binding of parasites to CSA. This study demonstrates that the described Avi-L1 VLP-platform may serve as a versatile system for facilitating optimal VLP-display of large and complex vaccine antigens.     

Involvement of a natural fusion of a cytochrome P450 and a hydrolase in mycophenolic acid biosynthesis

Mycophenolic acid (MPA) is a fungal secondary metabolite and the active component in several immunosuppressive pharmaceuticals. The gene cluster coding for the MPA biosynthetic pathway has recently been discovered in Penicillium brevicompactum, demonstrating that the first step is catalyzed by MpaC, a polyketide synthase producing 5-methylorsellinic acid (5-MOA). However, the biochemical role of the enzymes encoded by the remaining genes in the MPA gene cluster is still unknown. Based on bioinformatic analysis of the MPA gene cluster, we hypothesized that the step following 5-MOA production in the pathway is carried out by a natural fusion enzyme MpaDE, consisting of a cytochrome P450 (MpaD) in the N-terminal region and a hydrolase (MpaE) in the C-terminal region. We verified that the fusion gene is indeed expressed in P. brevicompactum by obtaining full-length sequence of the mpaDE cDNA prepared from the extracted RNA. Heterologous coexpression of mpaC and the fusion gene mpaDE in the MPA-nonproducer Aspergillus nidulans resulted in the production of 5,7-dihydroxy-4-methylphthalide (DHMP), the second intermediate in MPA biosynthesis. Analysis of the strain coexpressing mpaC and the mpaD part of mpaDE shows that the P450 catalyzes hydroxylation of 5-MOA to 4,6-dihydroxy-2-(hydroxymethyl)-3-methylbenzoic acid (DHMB). DHMB is then converted to DHMP, and our results suggest that the hydrolase domain aids this second step by acting as a lactone synthase that catalyzes the ring closure. Overall, the chimeric enzyme MpaDE provides insight into the genetic organization of the MPA biosynthesis pathway.     

Dynamics in the microbiology of maize silage during whole‐season storage

Aims: To monitor seasonal variations in the microbiology of maize silage and to determine whether the risk of fungal spoilage varies during whole‐year storage. Methods and Results: A continuous survey of 20 maize silage stacks was conducted over a period from three to 11 months after ensiling. Filamentous fungi, yeasts and lactic acid bacteria (LAB) were enumerated at five time‐points, and cultivable species of filamentous fungi were identified. Significant differences in the numbers of filamentous fungi, yeast and LAB were detected. The highest numbers of fungi were five to seven and the lowest 11 months after ensiling, while the LAB decreased in numbers during the study. Filamentous fungi were isolated from all stacks at all time‐points. The most abundant toxigenic mould species were Penicillium roqueforti, Penicillium paneum and Aspergillus fumigatus. Conclusions: There are significant variations in the microbiology of maize silage over a whole storage season. The risk of fungal spoilage was highest 5–7 months after ensiling and lowest after 11 months. Significance and Impact of the Study: This information is valuable in the assessment of health risks connected with spoiled maize silage and may be useful in the management of maize silage stacks, when whole‐season storage is applied.     

Rebound effects of price differences

Goal, Scope and Background  Traditionally, comparative life cycle assessments (LCA) have not considered rebound effects, for instance in case of significant price differences among the compared products. No justifications have been made for this delimitation in scope. This article shows that price differences and the consequent effects of marginal consumer expenditure may influence the conclusions of comparative LCA significantly. We also show that considerations about rebound effects of price differences can be included in LCAs. Methods  The direct rebound effect of a price difference is marginal consumption. Based on statistical data on private consumption in different income groups (Statistics Denmark 2005a, 2005b), the present article provides an estimate of how an average Danish household will spend an additional 1 DKK for further consumer goods, when the household has gained money from choosing a cheaper product alternative. The approach is to use marginal income changes and the following changes in consumption patterns as an expression for marginal consumption. Secondly, the environmental impact potentials related to this marginal consumption are estimated by the use of environmental impact intensity data from an IO-LCA database (Weidema et al. 2005). Finally, it is discussed whether, and in which ways the conclusions of comparative LCAs can be affected by including the price difference between product alternatives. This is elucidated in a case study of a comparative LCA screening of two different kinds of Danish cheese products (Fricke et al. 2004). Results  Car purchase and driving, use and maintenance of dwelling, clothing purchase and insurance constitutes the largest percentages of the marginal consumption. In a case study of two cheeses, the including the impact potentials related to the price difference results in significant changes in the total impact potentials. Considering the relatively small price difference of the two products, it is likely also to have a significant influence on the results of comparative LCAs more generally. Discussion  The influence of marginal consumption in comparative LCAs is relevant to consider in situations with large differences in the price of the product alternatives being compared, and in situations with minor differences in the impact potentials related to the alternatives. However, different uncertainties are linked to determining the pattern for marginal consumption and the environmental impact potential related to this. These are first of all related to the method used, but also include inaccurate data of consumption in households, aggregation and weighting of income groups, aggregation of product groups, estimation and size of the price difference, and the general applicability of the results. Conclusion  Incorporating marginal consumption in consequential LCAs is possible in practice. In the case study used, including the rebound effects of the price difference has a significant influence on the result of the comparative LCA, as the result for the impact categories acidification and nutrient enrichment changes in favour of the expensive product. Recommendations and Perspectives  It is recommended that the rebound effects of price differences should be included more frequently in LCAs. In order to ensure this, further research in marginal consumption and investment patterns and IO data for different countries or regions is required. Furthermore, this study does not consider the economic distributional consequences of buying an expensive product instead of a cheaper product (e.g. related to how the profit is spent by those who provided the product). It should also be noted, that more expensive products not necessarily result in less consumption, as those who provided the product also will spend the money they have earned from the sale. Ideally, these consequences should also be further investigated. Likewise, the development of databases to include marginal consumption in PC-tools is needed. In general, considerations of marginal consumption would favour expensive product alternatives, depending, however, on the type of consumer. ESS-Submission Editor: Dr. David Hunkeler (david.hunkeler@aquaplustech.ch)     

Identification of Trichoderma strains from building materials by ITS1 ribotyping, UP-PCR fingerprinting and UP-PCR cross hybridization

To study the role of Trichoderma in sick building syndrome, it is essential to be able to accurately identify species. Forty-four strains of Trichoderma spp. isolated from Danish buildings damaged by water leaks were identified using ITS1 ribotyping and universally primed PCR, UP-PCR. Ribotyping allowed the assignment of the strains into three distinct groups. High similarity of UP-PCR banding profiles of the strains allowed species designation for almost all strains (43 out of 44) when compared with the UP-PCR banding profiles obtained from reference strains of T. atroviride, T. citrinoviride, T. harzianum, T. longibrachiatum and T. viride. However, cross hybridization of UP-PCR products showed that the latter strain had high DNA homology to the ex-type strain of T. hamatum. The combined approach is a convenient way for reliable identification of Trichoderma strains.     

The glucose repressor genecre1 ofTrichoderma: Isolation and expression of a full-length and a truncated mutant form

Thecre1 genes of the filamentous fungiTrichoderma reesei andT. harzianum were isolated and characterized. The deduced CREI proteins are 46% identical to the product of the glucose repressor genecreA ofAspergillus nidulans, encoding a DNA-binding protein with zinc fingers of the C₂H₂ type. Thecre1 promoters contain several sequence elements that are identical to the previously identified binding sites forA. nidulans CREA. Steady-state mRNA levels forcre1 of theT. reesei strain QM9414 varied depending on the carbon source, being low on glucose-containing media. These observations suggest thatcre1 expression may be autoregulated. TheT. reesei strain Rut-C30, a hyperproducer of cellulolytic enzymes, was found to express a truncated form of thecre1 gene (cre1-1) with an ORF corresponding to a protein of 95 amino acids with only one zinc finger. Unlike QM9414 the strain Rut-C30 produced cellulase mRNAs on glucose-containing medium and transformation of the full-lengthcre1 gene into this strain caused glucose repression ofcbh1 expression, demonstrating thatcre1 regulates cellulase expression.     

The glucose repressor genecre1 ofTrichoderma: Isolation and expression of a full-length and a truncated mutant form

Thecre1 genes of the filamentous fungiTrichoderma reesei andT. harzianum were isolated and characterized. The deduced CREI proteins are 46% identical to the product of the glucose repressor genecreA ofAspergillus nidulans, encoding a DNA-binding protein with zinc fingers of the C₂H₂ type. Thecre1 promoters contain several sequence elements that are identical to the previously identified binding sites forA. nidulans CREA. Steady-state mRNA levels forcre1 of theT. reesei strain QM9414 varied depending on the carbon source, being low on glucose-containing media. These observations suggest thatcre1 expression may be autoregulated. TheT. reesei strain Rut-C30, a hyperproducer of cellulolytic enzymes, was found to express a truncated form of thecre1 gene (cre1-1) with an ORF corresponding to a protein of 95 amino acids with only one zinc finger. Unlike QM9414 the strain Rut-C30 produced cellulase mRNAs on glucose-containing medium and transformation of the full-lengthcre1 gene into this strain caused glucose repression ofcbh1 expression, demonstrating thatcre1 regulates cellulase expression.