Vaccine therapy for P. acnes-associated diseases.

Recent studies have afforded abundant evidences showing that Propionibacterium acnes (P. acnes) is involved not only in acne vulgaris, but also in many diseases, including endocarditis, endophthalmitis, osteomyelitis, joint, nervous system, cranial neurosurgery infections, and implanted biomaterial contamination. In spite of a range of P. acnes pathogenicity, its vaccine therapies have been studied much less intensively than antibiotic therapies which have been mainstay of treatment for P. acnes-associated diseases. Therefore, we have recently developed effective vaccines for P. acnes-associated inflammatory acne, consisting of a cell wall-anchored sialidase of P. acnes or killed-whole organism of P. acnes. Our data strongly show that immunization of ICR mice with the vaccines provides in vivo protective immunity against P. acnes challenge and decreases P. acnes-induced elevation of cytokine production. This review highlights the potential functions of killed P. acnes- and sialidase-based vaccines as novel treatments for P. acnes-associated diseases.

Inactivation and purification of cowpea mosaic virus-like particles displaying peptide antigens from Bacillus anthracis.

Chimeric cowpea mosaic virus (CPMV) particles displaying foreign peptide antigens on the particle surface are suitable for development of peptide-based vaccines. However, commonly used PEG precipitation-based purification methods are not sufficient for production of high quality vaccine candidates because they do not allow for separation of chimeric particles from cleaved contaminating species. Moreover, the purified particles remain infectious to plants. To advance the CPMV technology further, it is necessary to develop efficient and scalable purification strategies and preferably eliminate the infectivity of chimeric viruses. CPMV was engineered to display a 25 amino acid peptide derived from the Bacillus anthracis protective antigen on the surface loop of the large coat protein subunit. The engineered virus was propagated in cowpea plants and assembled into chimeric virus particles displaying 60 copies of the peptide on the surface. An effective inactivation method was developed to produce non-infectious chimeric CPMV virus-like particles (VLPs). Uncleaved VLPs were separated from the contaminating cleaved forms by anion exchange chromatography. The yield of purified chimeric VLPs was 0.3 g kg(-1) of leaf tissue. The results demonstrate the ability to generate multi-gram quantities of non-infectious, chimeric CPMV VLPs in plants for use in the development of peptide-based vaccines.

Expression and self-assembly of cowpea chlorotic mottle virus-like particles in Pseudomonas fluorescens.

Coat protein of the cowpea chlorotic mottle virus (CCMV), a plant bromovirus, has been expressed in a soluble form in a prokaryote, Pseudomonas fluorescens, and assembled into virus-like particles (VLPs) in vivo that were structurally similar to the native CCMV particles derived from plants. The CCMV VLPs were purified by PEG precipitation followed by separation on a sucrose density gradient and analyzed by size exclusion chromatography, UV spectrometry, and transmission electron microscopy. DNA microarray experiments revealed that the VLPs encapsulated very large numbers of different host RNAs in a non-specific manner. The development of a P. fluorescens expression system now enables production of CCMV VLPs by bacterial fermentation for use in pharmaceutical or nanotechnology applications.

Development of a plant viral-vector-based gene expression assay for the screening of yeast cytochrome p450 monooxygenases.

Development of a gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant virus gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity. Yarrowia lipolytica CYP52 gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant virus gene expression vector, and used as a model system for determining heterologous expression. Plants infected with virus vectors expressing the yeast CYP52 genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative control constructs. YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods. The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant virus expression library. A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants. All YlALK gene constructs scored positive in the randomized screen. Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis. This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of gene function.