Quorum sensing (QS) is the process through which bacteria communicate utilizing small diffusible molecules termed autoinducers. It has been demonstrated that QS controls a plethora of microbial processes including the expression of virulence factors. Here we report an immunopharmacotherapeutic approach for the attenuation of QS in the Gram-positive human pathogen Staphylococcus aureus. An anti-autoinducer monoclonal antibody, AP4-24H11, was elicited against a rationally designed hapten, and efficiently inhibited QS in vitro through the sequestration of the autoinducing peptide (AIP)-4 produced by S. aureus RN4850. Importantly, AP4-24H11 suppressed S. aureus pathogenicity in an abscess formation mouse model in vivo and provided complete protection against a lethal S. aureus challenge. These findings provide a strong foundation for further investigations of immunopharmacotherapy for the treatment of bacterial infections in which QS controls the expression of virulence factors.

We describe a system for direct selection of antibodies that are receptor agonists. Combinatorial antibody libraries in lentiviruses are used to infect eukaryotic cells that contain a fluorescent reporter system coupled to the receptor for which receptor agonist antibodies are sought. In this embodiment of the method, very large numbers of candidate antibodies expressing lentivirus and eukaryotic reporter cells are packaged together in a format where each is capable of replication, thereby forging a direct link between genotype and phenotype. Following infection, cells that fluoresce are sorted and the integrated genes encoding the agonist antibodies recovered. We validated the system by illustrating its ability to generate rapidly potent antibody agonists that are complete thrombopoietin phenocopies. The system should be generalizable to any pathway where its activation can be linked to production of a selectable phenotype.

B cells and their immunoglobulin products participate in allograft rejection of transplanted human kidneys in which an interesting feature is the presence of a germinal center like B-cell clusters in the allograft. We report here that the immunoglobulin repertoires of these infiltrating B cells are highly restricted and the B cells within a cluster are clonal. Antibody libraries made from the infiltrating B cells of individual patients unexpectedly revealed that each patient utilizes a particular set of dominant germ line genes as well as dominant complementarity determining region 3. Comparison of kidney and peripheral blood from the same patient showed that the immunoglobulin genes from both compartments had dominant clones, but they differed. The lymphocytes that infiltrate the kidneys express the immunoglobulin gene somatic recombination machinery usually restricted to highly activated lymphocytes in germinal centers and lymphomas. An analogy can be made between the inescapable antigenic drive in chronic infection versus that in an allograft, both of which may lead to emergence of dominant B-cell clones and even lymphoid malignancy.

Use of large combinatorial antibody libraries and next-generation sequencing of nucleic acids are two of the most powerful methods in modern molecular biology. The libraries are screened using the principles of evolutionary selection, albeit in real time, to enrich for members with a particular phenotype. This selective process necessarily results in the loss of information about less-fit molecules. On the other hand, sequencing of the library, by itself, gives information that is mostly unrelated to phenotype. If the two methods could be combined, the full potential of very large molecular libraries could be realized. Here we report the implementation of a phenotype-information-phenotype cycle that integrates information and gene recovery. After selection for phage-encoded antibodies that bind to targets expressed on the surface of Escherichia coli, the information content of the selected pool is obtained by pyrosequencing. Sequences that encode specific antibodies are identified by a bioinformatic analysis and recovered by a stringent affinity method that is uniquely suited for gene isolation from a highly degenerate collection of nucleic acids. This approach can be generalized for selection of antibodies against targets that are present as minor components of complex systems.A n important goal of modern molecular biology is to link the genotypic information that comes from nucleic acid sequencing to the phenotype of normal and diseased organisms. Here, the decoded nucleic acid information is directly linked to phenotype because, in evolutionary terms, this is an endpoint analysis and one assumes a high degree of fitness for any deduced protein.In other words, the information obtained from nucleic acid sequencing already relates to the highly selected phenotype of a functioning organism. Although such studies are proceeding rapidly for many genomes, deep sequencing has not been systematically used for the analyses of very large libraries of molecules that are related by sequence such as antibodies. This task is very different than the study of the genome of an organism because, for a given phenotype such as binding energy, one has a very large collection of related molecules that represent a spectrum of affinities. Furthermore, it is this very property of relatedness that makes the process of selective recovery of rare sequences from the larger collective so difficult (1). These problems become more acute when one wishes to select antibodies against targets that are components of complex mixtures such as cell surfaces.Although phage systems have the potential to link phenotype to genotype, their analysis is based on the process of selection which, when successful, necessarily results in the loss of information (2-7). For example, if molecules that bind less tightly but are otherwise related are discarded, one loses information concerning the evolutionary trajectory toward fitness. By contrast, nucleic acid sequencing of the libraries only gives information that without selection is, in the main, unrelated to p...

Obesity is a chronic, costly, and globally prevalent condition, with excess caloric intake a suspected etiologic factor. Nonsurgical treatments are modestly efficacious, and weight loss maintenance is hampered by anti-famine homeostatic mechanisms. Ghrelin, a gastric hormone linked to meal initiation, energy expenditure, and fuel partitioning, is hypothesized to facilitate weight gain and impede weight loss. Unique among known animal peptides, the serine-3 residue of ghrelin is posttranslationally acylated with an n-octanoic acid, a modification important for the peptide's active blood-brain transport and growth hormone secretagogue receptor-1 agonist activity. Pharmacological degradation of ghrelin would be hypothesized to reduce ghrelin's biological effects. To study endogenous ghrelin's role in appetite and energy expenditure, we generated antibodies that hydrolyze the octanoyl moiety of ghrelin to form des-acyl ghrelin. The most proficient antibody catalyst, GHR-11E11, was found to display a second-order rate constant of 18 M ؊ 1⅐s ؊1 for the hydrolysis of ghrelin to des-acyl ghrelin. I.v. administration of GHR-11E11 (50 mg/kg) maintained a greater metabolic rate in fasting C57BL/6J mice as compared with mice receiving a control antibody and suppressed 6-h refeeding after 24 h of food deprivation. Indirect respiratory measures of metabolism after refeeding and relative fuel substrate utilization were unaffected. The results support the hypothesis that acylated ghrelin stimulates appetite and curbs energy expenditure during deficient energy intake, whereas des-acyl ghrelin does not potently share these functions. Catalytic anti-ghrelin antibodies might thereby adjunctively aid consolidation of caloric restrictioninduced weight loss and might also be therapeutically relevant to Prader-Willi syndrome, characterized after infancy by hyperghrelinemia, hyperphagia, and obesity.hormone inactivation ͉ obesity ͉ neuropeptides A pproximately 1 billion people worldwide are overweight or obese (body mass index ϭ 25-29.9 or Ͼ30 kg/m 2 , respectively) (1), conditions associated with significant morbidity and mortality and for which new treatments are needed (2). Among recently characterized molecules implicated in energy homeostasis, ghrelin was identified in 1999 during a search for the endogenous ligand for the GH secretagogue receptor (GHSR) (ghrelin receptor), previously localized to peripheral tissues and to several CNS sites, including the arcuate nucleus and ventromedial hypothalamic nucleus (3). Human ghrelin is a 28-amino acid acylated peptide (GSS(n-octanoyl)FLSPEHQRVQQRKESKKPPAKLQPR) released mainly from endocrine cells of the stomach and upper gastrointestinal tract but also expressed in testes, kidney, pituitary, pancreas, lymphocytes, and brain (3-7). Several findings establish gastric ghrelin as an indicator of energy insufficiency and anabolic modulator of energy homeostasis (3). Ghrelin secretion is thought to stimulate food intake, slow metabolic rate, and spare lipid oxidation via central (8) and, possi...