Epitope mapping using combinatorial phage-display libraries: a graph-based algorithm

A phage-display library of random peptides is a combinatorial experimental technique that can be harnessed for studying antibody–antigen interactions. In this technique, a phage peptide library is scanned against an antibody molecule to obtain a set of peptides that are bound by the antibody with high affinity. This set of peptides is regarded as mimicking the genuine epitope of the antibody's interacting antigen and can be used to define it. Here we present PepSurf, an algorithm for mapping a set of affinity-selected peptides onto the solved structure of the antigen. The problem of epitope mapping is converted into the task of aligning a set of query peptides to a graph representing the surface of the antigen. The best match of each peptide is found by aligning it against virtually all possible paths in the graph. Following a clustering step, which combines the most significant matches, a predicted epitope is inferred. We show that PepSurf accurately predicts the epitope in four cases for which the epitope is known from a solved antibody–antigen co-crystal complex. We further examine the capabilities of PepSurf for predicting other types of protein–protein interfaces. The performance of PepSurf is compared to other available epitope mapping programs

Characterizing Complex Polysera Produced by Antigen-Specific Immunization through the Use of Affinity-Selected Mimotopes

BACKGROUND: Antigen-based (as opposed to whole organism) vaccines are actively being pursued for numerous indications. Even though different formulations may produce similar levels of total antigen-specific antibody, the composition of the antibody response can be quite distinct resulting in different levels of therapeutic activity. METHODOLOGY/PRINCIPAL FINDINGS: Using plasmid-based immunization against the proto-oncogene HER-2 as a model, we have demonstrated that affinity-selected epitope mimetics (mimotopes) can provide a defined signature of a polyclonal antibody response. Further, using novel computer algorithms that we have developed, these mimotopes can be used to predict epitope targets. CONCLUSIONS/SIGNIFICANCE: By combining our novel strategy with existing methods of epitope prediction based on physical properties of an individual protein, we believe that this method offers a robust method for characterizing the breadth of epitope-specificity within a specific polyserum. This strategy is useful as a tool for monitoring immunity following vaccination and can also be used to define relevant epitopes for the creation of novel vaccines

Site directed biotinylation of filamentous phage structural proteins

Filamentous bacteriophages have been used in numerous applications for the display of antibodies and random peptide libraries. Here we describe the introduction of a 13 amino acid sequence LASIFEAQKIEWR (designated BT, which is biotinylated in vivo by E. coli) into the N termini of four of the five structural proteins of the filamentous bacteriophage fd (Proteins 3, 7, 8 and 9). The in vivo and in vitro biotinylation of the various phages were compared. The production of multifunctional phages and their application as affinity reagents are demonstrated

Mammalian Otolin: A Multimeric Glycoprotein Specific to the Inner Ear that Interacts with Otoconial Matrix Protein Otoconin-90 and Cerebellin-1

The mammalian otoconial membrane is a dense extracellular matrix containing bio-mineralized otoconia. This structure provides the mechanical stimulus necessary for hair cells of the vestibular maculae to respond to linear accelerations and gravity. In teleosts, Otolin is required for the proper anchoring of otolith crystals to the sensory maculae. Otoconia detachment and subsequent entrapment in the semicircular canals can result in benign paroxysmal positional vertigo (BPPV), a common form of vertigo for which the molecular basis is unknown. Several cDNAs encoding protein components of the mammalian otoconia and otoconial membrane have recently been identified, and mutations in these genes result in abnormal otoconia formation and balance deficits.Here we describe the cloning and characterization of mammalian Otolin, a protein constituent of otoconia and the otoconial membrane. Otolin is a secreted glycoprotein of ∼70 kDa, with a C-terminal globular domain that is homologous to the immune complement C1q, and contains extensive posttranslational modifications including hydroxylated prolines and glycosylated lysines. Like all C1q/TNF family members, Otolin multimerizes into higher order oligomeric complexes. The expression of otolin mRNA is restricted to the inner ear, and immunohistochemical analysis identified Otolin protein in support cells of the vestibular maculae and semi-circular canal cristae. Additionally, Otolin forms protein complexes with Cerebellin-1 and Otoconin-90, two protein constituents of the otoconia, when expressed in vitro. Otolin was also found in subsets of support cells and non-sensory cells of the cochlea, suggesting that Otolin is also a component of the tectorial membrane.Given the importance of Otolin in lower organisms, the molecular cloning and biochemical characterization of the mammalian Otolin protein may lead to a better understanding of otoconial development and vestibular dysfunction

B-cell restriction – an alternative piece to the puzzle

Effective vaccination is based on three critical aspects of the B-cell response towards infectious agents: (i) that B-cells can generate specific antibodies towards a vast molecular diversity of antigens; proteins, sugars, DNA and lipids. There seems to be no limit to the ability to raise antibodies to everything. (ii) once stimulated, B-cells can perfect their antibodies through affinity maturation to complement every nook and cranny of the epitope and (iii) that the pathogen remains genetically stable and does not change to any great extent. Thus, antibodies produced against the vaccine and subsequent boosts recognize the viral virulent field isolates in future encounters and effectively knock them out. However, some vaccine targets, such as flu virus and HIV, are extremely genetically dynamic. The rapid genetic drift of these viruses renders them moving targets which assist in their ability to evade immune surveillance. Here we postulate that in the case of hyper-variable pathogens the B-cell response actually might be “too good”. We propose that restricting B-cell activities may prove effective in counteracting the genetic diversity of variant viruses such as flu and HIV. We suggest two levels of “B-cell restriction”: (i) to focus the B-cell response exclusively towards neutralizing epitopes by creating epitope-based immunogens; (ii) to restrict affinity maturation of B-cells to prevent the production of overly optimized exquisitely specific antibodies. Together, these “B-cell restrictions” provide a new modality for vaccine design