John E. Adamou

Use of a Whole Genome Approach To Identify Vaccine Molecules Affording Protection against Streptococcus pneumoniae Infection

ABSTRACT Microbial targets for protective humoral immunity are typically surface-localized proteins and contain common sequence motifs related to their secretion or surface binding. Exploiting the whole genome sequence of the human bacterial pathogen Streptococcus pneumoniae, we identified 130 open reading frames encoding proteins with secretion motifs or similarity to predicted virulence factors. Mice were immunized with 108 of these proteins, and 6 conferred protection against disseminated S. pneumoniaeinfection. Flow cytometry confirmed the surface localization of several of these targets. Each of the six protective antigens showed broad strain distribution and immunogenicity during human infection. Our results validate the use of a genomic approach for the identification of novel microbial targets that elicit a protective immune response. These new antigens may play a role in the development of improved vaccines against S. pneumoniae.

Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.

Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin‐2, MPIF‐2, or CKβ‐6, is a human CC chemokine with low amino acid sequence identity to other chemo‐ kines. Eotaxin‐2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross‐desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP‐4, but not RANTES, MIP‐lα, or MCP‐3, can completely cross‐desensitize the calcium response to eotaxin‐2 on these cells, indicating that eotaxin‐2 shares the same receptor used by eotaxin and MCP‐4. Eotaxin‐2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin‐2 also displaced 125I‐eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO‐CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP‐4. l25I‐Eotaxin‐2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin‐2 displace the ligand with equal affinity. Eotaxin and eotaxin‐2 promote a Ca2+ transient in RBL‐2H3 cells stably transfected with CCR3 (RBL‐2H3‐CCR3) and both ligands cross‐desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin‐2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor. J. Leukoc. Biol. 62: 667–675; 1997.

Molecular Characterization of a Novel Human Endothelin Receptor Splice Variant

Endothelin receptors are widely distributed throughout a number of tissues. A novel ETB receptor splice variant (ETB-SVR) was identified from a human placental cDNA library. Sequence analysis indicated that the ETB-SVR is 436 amino acids long and shares 91% identity to the human ETB-R. Northern blot analysis indicated an mRNA species of 2.7 kilobases, which is expressed in the lung, placenta, kidney, and skeletal muscle. Ligand binding studies of the cloned ETB-SVR and ETB-R receptors expressed in COS cells showed that ET peptides exhibited similar potency in displacing 125I-ET-1 binding. Functional studies showed that ET-1, ET-3, and sarafotoxin 6c displayed similar potencies for inositol phosphates accumulation in ETB-R-transfected COS cells, whereas no increase in inositol phosphate accumulation was observed in ETB-SVR-transfected cells. In addition, exposure of ETB-R-transfected cells to ET-1 caused an increase in the intracellular acidification rate whereas ETB-SVR-transfected cells did not respond to ET-1. These data suggest that the ETB-SVR and ETB-R are functionally distinct and the difference in the amino acid sequences between the two receptors may determine functional coupling. Availability of cDNA clones for endothelin receptors can facilitate our understanding of the role of ET in the pathophysiology of various diseases.