HuD antibodies are associated with small cell lung cancer. To identify relevant epitopes of HuD antibodies, patient sera and a monoclonal antibody were analyzed for their reactivity to linear 20mer peptides spanning the human HuD protein. The HuD monoclonal antibody recognized a single fragment located in the first RNA recognition motif. Thorough analysis identified VRDKITQGSL as the actual epitope. Screening of anti-HuD positive patients and healthy controls identified eight peptides as potential subdominant epitopes. The majority of these peptides were located in the N-terminal end as well as in the linker region between the second and third RNA recognition motifs.

Neutralizing antibodies (Nab) are a principal component of an effective human immune response to many pathogens, yet their role in HIV-1 infection is unclear. To gain a better understanding of this role, we examined plasma from patients with acute HIV infection. Here we report the detection of autologous Nab as early as 52 days after detection of HIV-specific antibodies. The viral inhibitory activity of Nab resulted in complete replacement of neutralization-sensitive virus by successive populations of resistant virus. Escape virus contained mutations in the env gene that were unexpectedly sparse, did not map generally to known neutralization epitopes, and involved primarily changes in N-linked glycosylation. This pattern of escape, and the exceptional density of HIV-1 envelope glycosylation generally, led us to postulate an evolving 'glycan shield' mechanism of neutralization escape whereby selected changes in glycan packing prevent Nab binding but not receptor binding. Direct support for this model was obtained by mutational substitution showing that Nab-selected alterations in glycosylation conferred escape from both autologous antibody and epitope-specific monoclonal antibodies. The evolving glycan shield thus represents a new mechanism contributing to HIV-1 persistence in the face of an evolving antibody repertoire.

Using an approach for isolating and characterizing peptide fractions that are intracellularly associated with major histocompatibility complex class I molecules, the major peptide recognized by cytotoxic T cells specific for the vesicular stomatitis virus has been isolated from the H-2Kb molecule of infected cells. This endogenously processed octapeptide is allele-specific as it does not bind to H-2Db molecules, and contains the core sequence of the epitope of the nucleocapsid protein of the vesicular stomatitis virus identified by testing with exogenous synthetic peptides.

We have produced a monoclonal antibody that reacts with all classes of intermediate filaments in immunofluorescence assays, including glial filaments in astrocytes, neurofilaments in axons, tonofilaments in epithelial PtK2 cells and intermediate filaments in fibroblasts. It also binds to Z lines in skeletal muscle. In SDS-polyacrylamide gels, the antibody binds to most and perhaps all of the major intermediate filament proteins that have been previously defined, including glial fibrillary acidic protein, the three vertebrate neurofilament proteins (the "neurofilament triplet"), vimentin, desmin, several cytokeratins and the neurofilament proteins of squid and the marine worm Myxicola. In addition, the antibody binds to a protein with an approximate molecular weight of 66,000 that may be a component of all intermediate filaments. These findings suggest that all vertebrate and invertebrate intermediate filament proteins share a common antigenic determinant and raise the possibility that all intermediate filaments contain a 66,000 molecular weight protein.

The action of tissue Transglutaminase (TGase) on specific protein-bound glutamine residues plays a critical role in numerous biological processes. Here we provide evidence for a new role of this enzyme in the common, HLA-DQ2 (and DQ8) associated enteropathy, celiac disease (CD). The intestinal inflammation in CD is precipitated by exposure to wheat gliadin in the diet and is associated with increased mucosal activity of TGase. This enzyme has also been identified as the main target for CD-associated anti-endomysium autoantibodies, and is known to accept gliadin as one of its few substrates. We have examined the possibility that TGase could be involved in modulating the reactivity of gliadin specific T cells. This could establish a link between previous reports of the role of TGase in CD and the prevailing view of CD as a T-cell mediated disorder. We found a specific effect of TGase on T-cell recognition of gliadin. This effect was limited to gliadin-specific T cells isolated from intestinal CD lesions. We demonstrate that TGase mediates its effect through an ordered and specific deamidation of gliadins. This deamidation creates an epitope that binds efficiently to DQ2 and is recognized by gut-derived T cells. Generation of epitopes by enzymatic modification is a new mechanism that may be relevant for breaking of tolerance and initiation of autoimmune disease.

Cytotoxic T lymphocytes (CTLs) are thought to play a crucial role in the termination of the acute primary HIV-1 syndrome, but clear evidence for this presumption has been lacking. Here we demonstrate positive selection of HIV-1 proviral sequences encoding variants within a CTL epitope in Nef, a gene product critical for viral pathogenicity, during and after seroconversion. These positively selected HIV-1 variants carried epitope sequence changes that either diminished or escaped CTL recognition. Other proviruses had mutations that abolished the Nef epitope altogether. These results provide clear evidence that CTLs exert selection pressure on the viral population in acute HIV-1 infection.

Human melanoma MZ2-MEL expresses several distinct antigens that are recognized by autologous cytolytic T lymphocytes (CTL). Some of these antigens are encoded by genes MAGE-1, MAGE-3, and BAGE, which are expressed in a large fraction of tumors of various histological types but are silent in normal adult tissues with the exception of testis. We report here the identification of the gene coding for MZ2-F, another antigen recognized by autologous CTL on MZ2-MEL cells. This gene, which was named GAGE-1, is not related to any presently known gene. It belongs to a family of genes that are expressed in a variety of tumors but not in normal tissues, except for the testis. Antigenic peptide YRPRPRRY, which is encoded by GAGE-1, is recognized by anti-MZ2-F CTL on class I molecule HLA-Cw6. The two genes of the GAGE family that code for this peptide, namely GAGE-1 and GAGE-2, are expressed in a significant proportion of melanomas (24%), sarcomas (25%), non-small cell lung cancers (19%), head and neck tumors (19%), and bladder tumors (12%). About 50% of melanoma patients carry on their tumor at least one of the presently defined antigens encoded by the MAGE, BAGE, and GAGE genes.

EBV is a gammaherpesvirus that can establish both nonproductive (latent) and productive (lytic) infections within the cells of its host. Although T cell responses to EBV latent proteins have been well characterized, little is known about the importance of responses to lytic proteins in long term virus carriers. Here we have compared the frequencies of CD8+ T cells specific for EBV latent and lytic Ags in healthy virus carriers, using three techniques: limiting dilution analysis, enzyme-linked immunospot assay, and FACS staining with tetrameric MHC-peptide complexes. T cells specific for EBV lytic protein epitopes were readily detectable in all donors and were usually more abundant than those specific for latent epitopes. We infer that direct T cell control of viral replicative lesions is maintained in long term carriers of EBV and is an important component of the immune response to this virus. Estimates of CD8+ T cell frequencies varied considerably according to methodology; values obtained from MHC-peptide tetramer staining were, on the average, 4.4-fold higher than those obtained from enzyme-linked immunospot assays, which were, in turn, on the average, 5.3-fold higher than those obtained from limiting dilution analysis. Tetramer staining showed that as many as 5.5% circulating CD8+ T cells in a virus carrier were specific for a single EBV lytic protein epitope. Such values are much greater than previously imagined and illustrate how antigenic challenge from a persistent herpesvirus can influence the composition of the host's CD8+ T cell pool.

During the past year, many new antibody structures have been determined, increasing our understanding of these immunologically important molecules. Of special interest are new catalytic antibodies, antibody-peptide and antibody-virus complexes, NMR structures, and structures illustrating conformational changes and antibody cross-reactivity.

A comprehensive analysis of the energetic importance of the 31 side-chains buried at the interface between human growth hormone (hGH) and the extracellular binding domain of its receptor (hGHbp) has been carried out to assess the roles of contact side-chains in modulating the affinity and kinetics of binding. Each side-chain in hGH was converted to alanine, and the kinetics and affinity were measured using a biosensor device. This detects binding of the mutated hormones to the immobilized hGHbp by changes induced in refractive index. The data generated on the biosensor match affinities obtained by radio-immune assay in solution. The study shows that only one-quarter of the side-chains buried at the interface can account for the majority of the binding energy. These residues cluster near the center of the structural epitope. The role of these side-chains is predominantly to slow dissociation because most of the effect of the alanine substitutions is to increase the off-rate, not to slow the on-rate. The hormone associates about 10,000 times slower than expected from random diffusion but 1000 times faster than may be expected if one imposes strict orientation restraints for a productive collision. Electrostatic interactions partly modulate association because mutations at Arg residues most affect association and together contribute a factor of about 20 to the on-rate. The data suggest that the hormone and receptor associate by diffusion and electrostatics to form an ensemble of weak collisional complexes. From these a bound complex is produced that is stabilized by only a small proportion of the contacts. We suggest that solvation energies and/or side-chains interactions within the free hormone or receptor may be so favorable that little energy is gained at most side-chains upon binding. The fact that the functional binding epitope is much smaller than the structural epitope suggests it may be possible to design smaller hormone mimics.

Synthetic peptide analogues of sequences in the HER-2 protooncogene (HER-2) were selected based on the presence of HLA-A2.1 anchor motifs to identify the epitopes on HER-2 recognized by ovarian tumor-reactive CTL. 19 synthetic peptides were evaluated for recognition by four HLA-A2 ovarian-specific cytotoxic T lymphocyte (CTL) lines obtained from leukocytes associated with ovarian tumors. The nonapeptide E75 (HER-2, 369-377:KIFGSLAFL) was efficient in sensitizing T2 cells for lysis by all four CTL lines. This peptide was specifically recognized by cloned CD8+ CTL isolated from one of the ovarian-specific CTL lines. E75-pulsed T2 cells inhibited lysis by the same CTL clone of both an HLA-A2+ HER-2high ovarian tumor and a HER-2high cloned ovarian tumor line transfected with HLA-A2, suggesting that this or a structurally similar epitope may be specifically recognized by these CTL on ovarian tumors. Several other HER-2 peptides were recognized preferentially by one or two CTL lines, suggesting that both common and private HER-2 epitopes may be immunogenic in patients with ovarian tumors. Since HER-2 is a self-antigen, these peptides may be useful for understanding mechanisms of tumor recognition by T cells, immunological tolerance to tumor, and structural characterization of tumor antigens.