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Endogenous tissue inhibitors of metalloproteinases (TIMPs) have key roles in regulating physiological and pathological cellular processes (1-3). Imitating the inhibitory molecular mechanisms of TIMPs while increasing selectivity has been a challenging but desired approach for antibody-based therapy (4). TIMPs use hybrid protein-protein interactions to form an energetic bond with the catalytic metal ion, as well as with enzyme surface residues (5-7). We used an innovative immunization strategy that exploits aspects of molecular mimicry to produce inhibitory antibodies that show TIMP-like binding mechanisms toward the activated forms of gelatinases (matrix metalloproteinases 2 and 9). Specifically, we immunized mice with a synthetic molecule that mimics the conserved structure of the metalloenzyme catalytic zinc-histidine complex residing within the enzyme active site. This immunization procedure yielded selective function-blocking monoclonal antibodies directed against the catalytic zinc-protein complex and enzyme surface conformational epitopes of endogenous gelatinases. The therapeutic potential of these antibodies has been demonstrated with relevant mouse models of inflammatory bowel disease (8). Here we propose a general experimental strategy for generating inhibitory antibodies that effectively target the in vivo activity of dysregulated metalloproteinases by mimicking the mechanism employed by TIMPs., Mice were immunized using a small, synthetic, organic metal-ligand molecule--tris-imidazole zinc complex (Zn-tripod)--which was specifically designed to mimic structural and chemical motifs of the relatively exposed catalytic zinc-protein machinery in [...]

The recognition of virus infected or malignantly transformed cells by cytotoxic T lymphocytes critically depends on the transporter associated with antigen processing (TAP), which delivers proteasomal degradation products into the endoplasmic reticulum lumen for subsequent loading of major histocompatibility complex class I molecules. Here we have identified a single cysteinyl residue in the TAP complex that modulates peptide binding and translocation, thereby restricting the epitope repertoire. Cysteine 213 in human TAP2 was found to be part of a newly uncovered substrate-binding site crucial for peptide recognition. This residue contacts the peptide in the binding pocket in an orientated manner. The translocation complex can be reversibly inactivated by thiol modification of this cysteinyl residue. As part of an unexpected mechanism, this residue is crucial in complementing the binding pocket for a given subset of epitopes as well as in maintaining a substrate-receptive conformation of the translocation complex. ATP-binding cassette transporter | cysteine scanning mutagenesis | membrane proteins | molecular recognition | substrate specificity doi/ 10.1073/pnas.1001308107

Background: Our group has previously used HLA-II associated HIV viral polymorphisms to predict CD4+ T-cell escape in HIV infected individuals. In both acute and chronic HIV infection, adapted epitopes (AE) encompassing these polymorphisms elicited weaker CD4 immune responses than the corresponding non-adapted epitopes (NAE). A significant proportion (30% to 40%) of these HLA-II associated viral polymorphisms are encoded in HIV-1 vaccine immunogens. However, the impact of these HLA-II associated adaptations on vaccine immuno-genicity has not been previously studied. Methods: Vaccine recipient and placebo PBMC samples were obtained from three different HIV-1 vaccine trials. These included MRKAd5 or HVTN 502 (n = 24), DNA/rAd5 or HVTN 505 (n = 24), and DNA/MVA or HVTN 106 (n = 40). Vaccine-matched epitopes were then grouped together into adapted epitope and non-adapted epitope pools. Immunogenicity was then tested utilizing a CD8-depleted IFNg ELISpot assay (n = 88) and a more sensitive flow-based activation induced marker (AIM) assay (n = 58) to identify antigen-specific CD4+ T cells. Results: In the 88 samples, we identified 10 positive IFNg ELISpot responses to non-adapted epitopes, while we only identified 1 positive response to adapted epitopes (p = 0.013, Fisher's Exact). Out of the 58 samples tested by the AIM assay, we identified a trend towards more non-adapted epitope responses, with 28 responses to non-adapted epitopes and 18 responses to adapted epitopes (p = 0.087, Fisher's Exact). Overall, there was an increased magnitude of AIM marker expression in response to non-adapted epitopes (p < 0.0001, Wilcoxon). This shows that CD4+ T cells from vaccine recipients produce more IFNg and are more activated in response to vaccine-matched non-adapted epitopes. Finally, we also report a positive correlation between non-adapted epitope-specific CD4 response magnitude and Env-specific IgG antibody generation in DNA/rAd5 (p = 0.014, Spearman's), suggesting that these CD4 responses may play a role in eliciting HIV-specific antibodies. Ongoing studies are being performed to determine the phenotype and function of these CD4+ T-cell responses at the single-cell level. Conclusions: Our data demonstrate that non-adapted epitopes encoded within human HIV-1 vaccine immunogens are immunogenic. This suggests that modified vaccine inserts should be considered to enhance CD4 responses and that such optimization strategies may have implications for future HIV-1 vaccine design., https://doi.org/10.1002/jia2.25659 Please Note: Illustration(s) are not available due to copyright [...]

The entomopathogenic fungus Beauveria bassiana and its insect host target represent a model system with which to examine host-pathogen interactions. Carbohydrate epitopes on the surfaces of fungal cells play diverse roles in processes that include adhesion, non-self recognition and immune invasion with respect to invertebrate hosts. B. bassiana produces a number of distinct cell types that include aerial conidia, submerged conidia, blastospores and haemolymph-derived cells termed in vivo blastospores or hyphal bodies. In order to characterize variations in the surface carbohydrate epitopes among these cells, a series of fluorescently labelled lectins, combined with confocal microscopy and flow cytometry to quantify the response, was used. Aerial conidia displayed the most diverse lectin binding characteristics, showing reactivity against concanavalin A (ConA), Galanthus nivalis (GNL), Griffonia simpficifolia (GSII), Helix pomatia (HPA), Griffonia simplicifolia isolectin (GSI), peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEAI) and wheatgerm agglutinin (WGA), and weak reactivity against Ricinus communis I (RCA), Sambucus nigra (SNA), Limax flavus (LFA) and Sophora japonica (SJA) lectins. Lectin binding to submerged conidia was similar to that to aerial conidia, except that no reactivity against UEAI, HPA and SJA was noted, and WGA appeared to bind strongly at specific polar spots. In contrast, the majority of in vitro blastospores were not bound by ConA, GNL, GSII, GSI, SNA, UEAI, LFA or SJA, with PNA binding in large patches, and some polarity in WGA binding noted. Significant changes in lectin binding also occurred after aerial conidial germination and in cells grown on either lactose or trehalose. For germinated conidia, differential lectin binding was noted between the conidial base, the germ tube and the hyphal tip. Fungal cells isolated from the haemolymph of the infected insect hosts Manduca sexta and Heliothis virescens appeared to shed most carbohydrate epitopes, displaying binding only to the GNL, PNA and WGA lectins. Ultrastructural examination of the haemolymph-derived cells revealed the presence of a highly ordered outermost brush-like structure not present on any of the in vitro cells. Haemolymph-derived hyphal bodies placed into rich broth medium showed expression of several surface carbohydrate epitopes, most notably showing increased PNA binding and strong binding by the RCA lectin. These data indicate robust and diverse production of carbohydrate epitopes on different developmental stages of fungal cells and provide evidence that surface carbohydrates are elaborated in infection-specific patterns.

Human leukocyte antigen (HLA) class I molecules present a variety of posttranslationally modified epitopes at the cell surface, although the consequences of such presentation remain largely unclear. Phosphorylation plays a critical cellular role, and deregulation in phosphate metabolism is associated with disease, including autoimmunity and tumor immunity. We have solved the high-resolution structures of 3 HLA A2-restricted phosphopeptides associated with tumor immunity and compared them with the structures of their nonphosphorylated counterparts. Phosphorylation of the epitope was observed to affect the structure and mobility of the bound epitope. In addition, the phosphoamino acid stabilized the HLA peptide complex in an epitope-specific manner and was observed to exhibit discrete flexibility within the antigen-binding cleft. Collectively, our data suggest that phosphorylation generates neoepitopes that represent demanding targets for T-cell receptor ligation. These findings provide insights into the mode of phosphopeptide presentation by HLA as well as providing a platform for the rational design of a generation of posttranslationally modified tumor vaccines. antigen presentation | HLA | phosphopeptide | T cells | X-ray crystallography

Activation of dendritic cells (DCs) enhances their ability to prime naive T cells. How activation renders them immunogenic rather than tolerogenic is unclear. Here, we show, using temporally regulated expression of a transgene-encoded neoself antigen in DCs, that either prolonged antigen presentation or DC activation could elicit full expansion, effector cytokine production, and memory-cell differentiation. Microarray analysis of gene expression in T cells showed that all changes linked to DC activation through CD40 could be reproduced by persistent antigen delivery, suggesting that stabilization of antigen presentation is an important consequence of DC activation in vivo. In this system, DC activation by CD40 engagement indeed extended their ability to present antigen to [CD4.sup.+] T cells in vivo, although different results were obtained with antigen delivered to DCs by means of endocytosis from the cell surface. These results suggest that antigen persistence may be an important discriminator of immunogenic and tolerogenic antigen exposure. immune response | immune tolerance | regulated transgene | MHC class II

Cryptococcus neoformans is a major pathogen of humans throughout the world. Using commercial monoclonal antibodies to capsular epitopes, strains of C. neoformans manifest five serotypes: A, B, C, D and AD. Previous studies demonstrated significant divergence among serotypes A, B, C and D, which are typically haploid. In contrast, most strains of serotype AD are diploid or aneuploid and result from recent hybridization between strains of serotypes A and D. Whether serotypes A, B, C and D represent strictly asexual lineages is not known. Using comparative genealogical analyses of two genes, the authors investigated whether recombination occurred among strains within serotypes A and D. For each of 14 serotype AD strains, a portion (642 bp) of the orotidine monophosphate pyrophosphorylase (URA5) gene was cloned and sequenced. Each of these 14 strains contained two different alleles and sequences for both alleles were obtained. The URA5 gene genealogy was compared to that derived from the laccase (LAC) gene, which was reported recently for the same 14 strains. For both genes, each of the 14 serotype AD strains contained two phylogenetically distinct alleles: one allele was highly similar to those from serotype A strains and the other to alleles from serotype D strains. However, within both the serotype A allelic group and the serotype D allelic group, there was significant incongruence between genealogies derived from URA5 and LAC. The results suggest recombination in natural populations of both serotypes A and D.

Transgenic PDAPP mice, which express a disease-linked isoform of the human amyloid precursor protein, exhibit CNS pathology that is similar to Alzheimer's disease. In an age-dependent fashion, the mice develop plaques containing [beta]-amyloid peptide (A[beta]) and exhibit neuronal dystrophy and synaptic loss. It has been shown in previous studies that pathology can be prevented and even reversed by immunization of the mice with the A[beta] peptide. Similar protection could be achieved by passive administration of some but not all monoclonal antibodies against A[beta]. In the current studies we sought to define the optimal antibody response for reducing neuropathology. Immune sera with reactivity against different A[beta] epitopes and monoclonal antibodies with different isotypes were examined for efficacy both ex vivo and in vivo. The studies showed that: (i) of the purified or elicited antibodies tested, only anti-bodies against the N-terminal regions of A[beta] were able to invoke plaque clearance; (ii) plaque binding correlated with a clearance response and neuronal protection, whereas the ability of antibodies to capture soluble A[beta] was not necessarily correlated with efficacy; (iii) the isotype of the antibody dramatically influenced the degree of plaque clearance and neuronal protection; (iv) high affinity of the antibody for Fc receptors on microglial cells seemed more important than high affinity for A[beta] itself; and (v) complement activation was not required for plaque clearance. These results indicate that antibody Fc-mediated plaque clearance is a highly efficient and effective process for protection against neuropathology in an animal model of Alzheimer's disease.

The nuclear structures that contain symmetrical dimethylated arginine (sDMA)-modified proteins and the role of this posttranslational modification is unknown. Here we report that the Cajal body is a major epitope in HeLa cells for an sDMA-specific antibody and that coilin is an sDMA-containing protein as analyzed by using the sDMA-specific antibody and matrix-assisted laser desorption ionization time of flight mass spectrometry. The methylation inhibitor 5'-deoxy-5'-methylthioadenosine reduces the levels of coilin methylation and causes the appearance of SMN-positive gems. In cells devoid of Cajal bodies, such as primary fibroblasts, sDMA-containing proteins concentrated in speckles. Cells from a patient with spinal muscular atrophy, containing low levels of the methyl-binding protein SMN, localized sDMA-containing proteins in the nucleoplasm as a discrete granular pattern. Splicing reactions are efficiently inhibited by using the sDMA-specific antibody or by using hypomethylated nuclear extracts, showing that active spliceosomes contain sDMA polypeptides and suggesting that arginine methylation is important for efficient pre-mRNA splicing. Our findings support a model in which arginine methylation is important for the localization of coilin and SMN in Cajal bodies.

Research has been conducted on the protein-protein interactions. The functional consequences of these interactions have been investigated via the addition of the artificial protein binding interface carried out in modifying proteins and the results are reported.

Research has been conducted on the antibody-dependent cellular cytotoxicity in a host defence mechanism. The study of the murine monoclonal antibody which mediates cytotoxicity's activity against HIV-infected cells is presented.

Two recently identified immunodominant epitopes from [alpha]-gliadin account for most of the stimulatory activity of dietary gluten on intestinal and peripheral T lymphocytes in patients with celiac sprue. The proteolytic kinetics of peptides containing these epitopes were analyzed in vitro using soluble proteases from bovine and porcine pancreas and brush-border membrane vesicles from adult rat intestine. We showed that these proline-glutamine-rich epitopes are exceptionally resistant to enzymatic processing. Moreover, as estimated from the residual peptide structure and confirmed by exogeneous peptidase supplementation, dipeptidyl peptidase IV and dipeptidyl carboxypeptidase I were identified as the rate-limiting enzymes in the digestive breakdown of these peptides. A similar conclusion also emerged from analogous studies with brush-border membrane from a human intestinal biopsy. Supplementation of rat brush-border membrane with trace quantities of a bacterial prolyl endopeptidase led to the rapid destruction of the immunodominant epitopes in these peptides. These results suggest a possible enzyme therapy strategy for celiac sprue, for which the only current therapeutic option is strict exclusion of gluten-containing food. celiac sprue; brush-border membrane; peptidase; prolyl endopeptidase; kinetics

Results reveal that the alpha module of the self-recognizing outer membrane protein of Escherichia coli, antigen 43, can accommodate and display correctly folded proteins containing entire functional domains in gram-negativve baceria including Salmonella strains. Implications of the research for vaccine development is discussed.

Research has been conducted on the EF-hand calcium-binding protein, S100B. The effect of the S100B linker and C-terminal regions on the protein-protein interactions has been investigated via the use of the calcium-sensitive S100B-binding epitope TRTK-12 and the details are reported.

Research has been conducted on the small molecules with insulin mimetic effects and oral availability. The search for the compounds capable to mimick an epitope of the insulin molecule involved in the insulin receptor binding have been carried out and the results are presented.

Research has been conducted on the urokinase-type plasminogen activator and its glycolipid-anchored receptor. The interactions between these activator and receptor important for the pericellular plasminogen activation have been examined and discussed.

Binding of synthetic disaccharides representing epitopes of chlamydial lipopolysaccharide to antibodies has been studied using NMR. The nonreducing pyranose unit was found to be the major binding epitope. Individual differences depended on engagement of other parts of disaccharide ligands.

Results show that DNA sequences of donor silent pilin gene and recipient expressed pilin gene act differently during gonococcal pilin antigenic variation in Neisseria gonorrhoeae. Data further suggest that more than one recombination mechanism may be operating in this process.

To determine whether established CD8+ T cell memory to an epitope prominent during the replicative phase of a y. herpesvirus infection protects against subsequent challenge, mice were primed with a recombinant vaccinia virus expressing the p56 peptide and then boosted by intranasal exposure to an influenza A virus incorporating p56 in the neuraminidase protein. Clonally expanded populations of functional, p56-specific [CD8.sup.+] T cells were present at high frequency in both the lung and the lymphoid tissue 1 month later, immediately before respiratory challenge with [Gamma]HV-68. This prime-and-boost regime led to a massive reduction of productive [Gamma]HV-68 infection in the respiratory tract and, initially, to much lower levels of latency in both the regional lymph nodes and the spleen. The [CD8.sup.+] T cell response to another epitope (p79) was diminished, there was less evidence of B cell activation, and the onset of the [CD4.sup.+] T cell-dependent splenomegaly was delayed. Within 3-4 weeks of the [Gamma]HV-68 challenge, however, the extent of latent infection in the lymph nodes and spleen was equivalent, and both groups developed the prominent infectious mononucleosis-like syndrome that is characteristic of this infection. The reverse protocol (influenza then vaccinia) seemed to be slightly less effective. Even though immune [CD8.sup.+] T cells may be present at the time and site of virus challenge, establishing a high level of [CD8.sup.+] T cell memory to lytic-phase epitopes alone does not protect against the longer-term consequences of this [Gamma]HV infection.

Tetrameric HLA class I-minor histocompatibility antigen peptide complexes show minor histocompatibility antigen (mHag)-specific cytotoxic T lymphocytes (CTLs) in those with graft-versus-host disease (GvHD). The direct ex vivo visualization of mHag-specific CTLs during GvHD using tetrameric HLA-class and I-mHAg HA-1 and HY peptide complexes is shown. I-mHag peptide tetramers may be useful as clinical tools for diagnosis/monitoring of GvHD patients.

Endogenously synthesized antigenic determinants are generally presented on major histocompatibility complex (MHC) class I molecules, whereas exogenous determinants are presented by MHC class II molecules. Here, it is shown that exogenous antigens chaperoned by a heat shock protein can be channeled into the endogenous pathway, presented by MHC class I molecules, and recognized by (CD8.sup.+) T lymphocytes. This pathway is functional only in a subset of macrophages among the cell types tested. These observations provide a basis for the tumor-specific and virus-specific immunogenicity of cognate heat shock protein preparations and offer a mechanism for the classical phenomenon of cross-priming., Heat shock proteins (HSPs) isolated from cancer cells or virus-infected cells elicit protective immunity or cytotoxic T lymphocytes (CTLs) to the cognate tumor or viral antigen (1-3). In contrast, HSPs [...]

A study of the immunoreactivity of epitopes in the apoA-I sequence (apolipoprotein A-I) in lipid-free apoA-I and lipid bound apoA-I show that lipid binding influences apoA-I conformation. Experiments used reconstituted discoidal lipoproteins, LpA-I, characterized by the number of apoA-I per particle, to indicate that particle size can change apoA-I conformation. Four LpA-I were used in the study, two in the Lp2A-I class with diameters of 7.8 and 9.6 nm, and two in the Lp3A-I class with diameters of 10.8 and 13.4 nm.