Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential

The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro, the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)₂. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.     

Patterns of MADS-box gene expression mark flower-type development in Gerbera hybrida (Asteraceae)

Background  

The inflorescence of the cut-flower crop Gerbera hybrida (Asteraceae) consists of two principal flower types, ray and disc, which form a tightly packed head, or capitulum. Despite great interest in plant morphological evolution and the tractability of the gerbera system, very little is known regarding genetic mechanisms involved in flower type specification. Here, we provide comparative staging of ray and disc flower development and microarray screening for differentially expressed genes, accomplished via microdissection of hundreds of coordinately developing flower primordia.     

Mandipropamid targets the cellulose synthase-like PiCesA3 to inhibit cell wall biosynthesis in the oomycete plant pathogen, Phytophthora infestans

Oomycete plant pathogens cause a wide variety of economically and environmentally important plant diseases. Mandipropamid (MPD) is a carboxylic acid amide (CAA) effective against downy mildews, such as Plasmopara viticola on grapes and potato late blight caused by Phytophthora infestans . Historically, the identification of the mode of action of oomycete-specific control agents has been problematic. Here, we describe how a combination of biochemical and genetic techniques has been utilized to identify the molecular target of MPD in P. infestans . Phytophthora infestans germinating cysts treated with MPD produced swelling symptoms typical of cell wall synthesis inhibitors, and these effects were reversible after washing with H₂O. Uptake studies with ¹⁴C-labelled MPD showed that this oomycete control agent acts on the cell wall and does not enter the cell. Furthermore, ¹⁴C glucose incorporation into cellulose was perturbed in the presence of MPD which, taken together, suggests that the inhibition of cellulose synthesis is the primary effect of MPD. Laboratory mutants, insensitive to MPD, were raised by ethyl methane sulphonate (EMS) mutagenesis, and gene sequence analysis of cellulose synthase genes in these mutants revealed two point mutations in the PiCesA3 gene, known to be involved in cellulose synthesis. Both mutations in the PiCesA3 gene result in a change to the same amino acid (glycine-1105) in the protein. The transformation and expression of a mutated PiCesA3 allele was carried out in a sensitive wild-type isolate to demonstrate that the mutations in PiCesA3 were responsible for the MPD insensitivity phenotype.