A distinct strategy to generate high-affinity peptide binders to receptor tyrosine kinases.

We describe a novel and general way of generating high affinity peptide (HAP) binders to receptor tyrosine kinases (RTKs), using a multi-step process comprising phage-display selection, identification of peptide pairs suitable for hetero-dimerization (non-competitive and synergistic) and chemical synthesis of heterodimers. Using this strategy, we generated HAPs with K(D)s below 1 nM for VEGF receptor-2 (VEGFR-2) and c-Met. VEGFR-2 HAPs bound significantly better (6- to 500-fold) than either of the individual peptides that were used for heterodimer synthesis. Most significantly, HAPs were much better (150- to 800-fold) competitors than monomers of the natural ligand (VEGF) in various competitive binding and functional assays. In addition, we also found the binding of HAPs to be less sensitive to serum than their component peptides. We believe that this method may be applied to any protein for generating high affinity peptide (HAP) binders.

Development of a cell line system susceptible to infection with vaccine strains of MDV.

Despite reliance on the need to continually prepare fresh cultures of chick embryo fibroblasts (CEFs) to make Marek's disease (MD) vaccines, MD vaccines are the most widely used vaccines in the poultry industry. Preparation of CEF's accounts for approximately 40% of the costs associated with producing MD vaccines. A significant reduction in MD vaccine production costs could be realized if a continuous cell lines were available for MD vaccine production. Recently, we reported development and characterization of a cell line system (OCL) that supports growth and replication of oncogenic serotype 1 Marek's disease virus (MDV). Here we report development of three cell line systems for production of MD vaccine. These cell lines support the growth and replication of attenuated serotype 1 MDV (CVI-OCL), serotype 2 MDV (SB1-OCL) and serotype 3 MDV (HVT-OCL). MDV is maintained in a stable state in the OCL cells and the infected cells can be continuously grown. The vaccines made from these cell lines are safe and protect White Leghorn chickens against challenge with very virulent serotype 1 MDV, similar to traditional vaccines made from CEF cells. These cell line systems can significantly reduce the costs associated with MD vaccine production. Furthermore, the increased stability of MDV and the potential for positive selection of recombinant MDV suggest that OCL will be ideal for production of more effective MDV vaccines using recombinant DNA technology.

Evidence that Marek's disease virus exists in a latent state in a sustainable fibroblast cell line.

Previously, we reported the development of two fibroblastic cell lines (MDV OU2.1 and OU2.2) infected with Marek's disease virus (MDV). The two cell lines, in nonconfluent continuous cultures, displayed characteristics consistent with MDV existing in a latent state. However, presence of distinct plaques in confluent cell monolayers and the ability to transfer cytolytic infection to susceptible birds and primary chick embryo fibroblasts, suggest that, if latent, the virus is easily reactivated from MDV OU2 cell lines. In this report, we present evidence which supports the hypothesis that MDV genomes in MDV OU2 cells are latent. PCR analyses and in vivo experiments demonstrate that CHCC-OU2 cells stabilize MDV so that serial in vitro passage does not attenuate the virus. Following two years of active culture, MDV genomes in MDV OU2 cells are still oncogenic, similar to that seen in MDV-lymphoblastoid cell lines. Expansion of the 132-bp repeat within MDV BamHI fragments H and D, typical of highly passaged serotype 1 MDV, has not been observed beyond two copies in MDV OU2 cells. Indirect immunofluorescence assays clearly demonstrate that MDV OU2 cells do not express glycoproteins B and I when subconfluent. However, upon reaching confluence these proteins are expressed in readily detectable amounts. Using RT-PCR we demonstrate that glycoproteins E and D are highly expressed in confluent MDV OU2 cells but absent from subconfluent cells, and MDV latency-associated transcripts (LATs), which are antisense to ICP4 transcripts and have been associated with latent MDV infection, are expressed in subconfluent MDV OU2 cells. Coincident with an increase in ICP4 expression, MDV LAT expression is down regulated when MDV OU2 cells become confluent.

Development of a sustainable chick cell line infected with Marek's disease virus.

Marek's disease virus (MDV) is a highly infectious and cell-associated avian herpesvirus. Fully productive infections with MDV are restricted to feather follicle epithelium of afflicted birds. In culture, MDV infection of primary chick (CEF) and duck embryo fibroblast cells is semiproductive. Passage of MDV and production of MDV vaccines is limited to these primary cell-associated systems. The finite life span of primary avian cell cultures has hampered efforts to use positive selection in generation of recombinant MDV and complicates studies of temporal gene regulation. In this report, we describe continuous chick fibroblast cell lines (MDV OU2.2 and MDV OU2.1) which support MDV replication. Southern blot and PCR analyses demonstrate that these cell lines harbor MDV DNA. Western blot analyses indicate that MDV OU2.2 cells express at least a limited set of viral proteins, pp38 and pp14, similar to that seen in MDV-lymphoblastoid cells. Presence of distinct plaques in confluent MDV OU2.2 cell monolayers is consistent with cytolytic semiproductive infection, similar to that observed in primary CEF. MDV OU2.2 cells are capable of transferring MDV infection to primary CEF cultures and inducing clinical signs of Marek's disease in susceptible birds. MDV OU2.2 cells have maintained a MDV-positive phenotype for over 16 months of active culture. Southern blot hybridization of MDV OU2.2 cell DNA reveals a distinct expansion of the MDV BamHI H fragment in a subset of viral genomes following long-term cultivation.