Identification of Schistosoma mansoni glycolipids that share immunogenic carbohydrate epitopes with glycoproteins

The immunoreactivity of sera of infected hosts against glycolipids derived from Schistosoma mansoni eggs, adult male worms, and cercariae was analyzed by immunostaining of glycolipids resolved by high-performance thin-layer chromatography. Eggs contained the greatest number of immunogenic glycolipids and bound the largest proportion of serum antibodies. Virtually all of the immunogenic egg glycolipids were neutrally charged and contained oligosaccharide chains larger in size than five sugar residues. The glycolipids of each developmental stage were shown by use of five monoclonal antibodies to share schistosome-specific carbohydrate epitopes that were also present on glycoproteins. Several of the carbohydrate epitopes were expressed throughout the life cycle, yet the overall structures of the glycolipids were not conserved. Quantitative analyses by solid-phase binding assays indicated that the carbohydrate epitopes were differentially expressed between the glycolipids and glycoproteins of developmental stages. Sera from infected humans and mice both contained very high levels of anti-carbohydrate antibodies that were reactive with the glycolipids, irrespective of the stage or intensity of disease. Mice harboring unisexual infections of either male or female worms also recognized the egg glycolipids in a pattern indistinguishable from that of patently infected mice. A greater proportion of the humoral response against egg antigens in infected humans was directed against protein determinants, as compared with infected mice.     

Methods and protocols for prediction of immunogenic epitopes

T-cell recognition of peptide/major histocompatibility complex (MHC) is a prerequisite for cellular immunity. Recently, there has been an influx of bioinformatics tools to facilitate the identification of T-cell epitopes to specific MHC alleles. This article examines existing computational strategies for the study of peptide/MHC interactions. The most important bioinformatics tools and methods with relevance to the study of peptide/MHC interactions have been reviewed. We have also provided guidelines for predicting antigenic peptides based on the availability of existing experimental data.     

Germline antibody recognition of distinct carbohydrate epitopes

High-resolution structures reveal how a germline antibody can recognize a range of clinically relevant carbohydrate epitopes. The germline response to a carbohydrate immunogen can be critical to survivability, with selection for antibody gene segments that both confer protection against common pathogens and retain the flexibility to adapt to new disease organisms. We show here that antibody S25-2 binds several distinct inner-core epitopes of bacterial lipopolysaccharides (LPSs) by linking an inherited monosaccharide residue binding site with a subset of complementarity-determining regions (CDRs) of limited flexibility positioned to recognize the remainder of an array of different epitopes. This strategy allows germline antibodies to adapt to different epitopes while minimizing entropic penalties associated with the immobilization of labile CDRs upon binding of antigen, and provides insight into the link between the genetic origin of individual CDRs and their respective roles in antigen recognition.     

Lactoseries carbohydrate epitopes in the vertebrate retina

The distribution of N-acetyl-lactosamine (NALA), a cell-surface carbohydrate epitope of the lactoseries, has been studied in the retina of representative species of all vertebrate classes by light microscope immunohistochemistry. In only some species of different classes (fish, amphibia and mammals) was NALA expression detected, and in these animals the distribution showed profound interspecies variability. In fishes and amphibia in which NALA was present, patterns ranged from single immunopositive cells to homogeneous labelling of cell layers. In mammals, NALA was found only in retinas that are cone dominated (tree squirrel and primates). In the tree squirrel, there was a dense cellular staining of the photoreceptor cell layer; whereas in primates, the carbohydrate epitope occur red only on some photoreceptor cells. From these receptor cells, positi ve axons could be traced to the inner plexiform layer. In spite of the profound interspecies differences, NALA is not randomly expressed, as its exclusive expression in mammals with cone- dominated vision indicates. The suggestion of a functional relevance for NALA glycosylation of retinal cells is supported by the labelling pattern for HNK-1 in these species, which was different from the pattern found in rod-dominated mammalian retinas. This revised version was published online in November 2006 with corrections to the Cover Date.     

In silico prediction of immunogenic T cell epitopes for HLA-DQ8

Background HLA-DQ alleles are involved in the pathogenesis of hypersensitivity reactions, with HLA-DQ8 associated with several human autoimmune disorders. Limited success has been achieved using sequence-based computational techniques for predicting HLA-DQ8-restricted T cell epitopes while accuracy and efficiency of recently developed structure-based models need to be improved. Results We describe a combined structure-based prediction approach for DQ8-restricted T cell epitope prediction using a recently developed fast and accurate docking protocol, pDOCK, and molecular surface electrostatic potential (MSEP)-based clustering of pMHC binding interfaces. The prediction model was rigorously trained, tested and validated using experimentally verified DQ8 binding and non-binding peptides. High prediction accuracy (average area under the ROC curve, average AROC>0.94) is validated against experimental data. Our model also predicts all binding registers correctly and known T cell activators with 77% accuracy. We also studied the patterns of DQ8-binding peptides and reassure the existence of epitopes not conforming to binding motifs. Conclusions We have developed a model that can be successfully applied as a generic protocol for easy in silico identification of potential immunogenic T cell epitopes. The current model is therefore applicable for screening vaccine candidates irrespective of sequence motifs. We have also illustrated efficient discrimination of different categories of binders from non-binders as well as different categories of pMHC agonists from non-agonists, while accurately predicting the binding registers of DQ8-restricted peptides. This combined approach provides a set of sensitive and specific computational tools to facilitate high-throughput screening of peptides for immunotherapeutic applications such as controlling allergic and autoimmune responses.     

In silico characterization of immunogenic epitopes presented by HLA-Cw*0401

Background

Arenaviruses are a family of rodent-borne viruses that cause several hemorrhagic fevers. These diseases can be devastating and are often lethal. Herein, to aid in the design and development of diagnostics, treatments and vaccines for arenavirus infections, we have developed a database containing protein sequences from the seven pathogenic arenaviruses (Junin, Guanarito, Sabia, Machupo, Whitewater Arroyo, Lassa and LCMV).

Results

The database currently contains a non-redundant set of 333 protein sequences which were manually annotated. All entries were linked to NCBI and cited PubMed references. The database has a convenient query interface including BLAST search. Sequence variability analyses were also performed and the results are hosted in the database.

Conclusion

The database is available at http://epitope.liai.org:8080/projects/arena and can be used to aid in studies that require proteomic information from pathogenic arenaviruses.     

Engineering immunogenic consensus T helper epitopes for a cross-clade HIV vaccine

Developing a vaccine that will stimulate broad HIV-specific T cell responses is difficult because of the variability in HIV T cell epitope sequences, which is in turn due to the high mutation rate and consequent strain diversity of HIV-1. We used a new Class II version of the EpiMatrix T cell epitope-mapping tool and Conservatrix to select highly conserved and promiscuous Class II HLA-restricted T cell epitopes from a database of 18,313 HIV-1 env sequences. Criteria for selection were: (1) number of HIV-1 strains represented as measured by Conservatrix; (2) EpiMatrix score; and (3) promiscuity (number of unique MHC motifs contained in the peptide). Using another vaccine design tool called the EpiAssembler, a new set of overlapping, conserved and immunogenic HIV-1 peptides were engineered creating extended "immunogenic consensus" sequences. Each overlapping 9-mer of the 20-23 amino acid long immunogenic consensus peptides was conserved in a large number (range 893-2254) of individual HIV-1 strains, although the novel peptides were not representative of any single strain of HIV. We synthesized nine representative peptides. T helper cell responses to the peptides were evaluated by ELISpot (gamma-interferon) assay, using peripheral blood monocytes (PBMC) obtained from 34 healthy long term non-progressor (LT) or moderate-progressor (MP) donors (median years infected = 8.88, median CD4 T cells = 595, median VL = 1044). Nine peptides were tested, of which eight were confirmed in ELISpot assays using PBMC from the LT/MP subjects. These epitopes were ranked by Conservation and EpiMatrix score 1, 2, 3, 5, 7, 11, and 14 out of the set of 9 original peptides. Five of these peptides were selected for inclusion in an epitope-driven cross-clade HIV-1 vaccine (the GAIA vaccine). These data confirm the utility of bioinformatics tools to select and construct novel "immunogenic consensus sequence" T cell epitopes for a globally relevant vaccine against HIV.     

Localization of defined carbohydrate epitopes in bovine polysialylated NCAM

Polysialylated neural cell adhesion molecule (NCAM) was immunoaffinity-purified from the brains of newborn calves. A degree of polymerization of up to 40 was chromatographically determined for released polysialic acid (PSA) chains. For characterization of N-glycan structures and attachment sites, PSA-NCAM was digested with trypsin, and the generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specific for PSA or the HNK1 epitope, i.e., HSO(3)-3GlcA(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-, yielding PSA-glycopeptides, HNK-glycopeptides and non-PSA/HNK1-(glyco) peptides. Using a combination of enzymatic deglycosylation, peptide fractionation, mass spectrometry and Edman degradation, HNK1-N-glycans could be assigned to glycosylation sites 2, 4, 5 and 6. Non-PSA/HNK1-glycans were assigned to glycosylation site 2, whereas PSA-N-glycans of bovine NCAM had been already previously shown to be restricted to glycosylation sites 5 and 6 (Glycobiology 12 (2002) 47). Respective oligosaccharides were enzymatically released, labeled with 2-aminopyridine and characterized by linkage analysis and mass spectrometry. Carbohydrate chains bearing PSA or the HNK1 epitope comprised mainly fucosylated, partially sulfated diantennary, triantennary or tetraantennary glycans without bisecting GlcNAc or fucosylated diantennary and triantennary species carrying, in part, bisecting GlcNAc residues, respectively. Some N-glycans simultaneously contained both the HNK1-epitope and PSA. Non-PSA/HNK1-glycans exhibited a heterogeneous pattern of partially truncated, mostly diantennary structures with one to three fucose residues, bisecting GlcNAc and/or sulfate residues. In addition, they were demonstrated to carry, to some extent, the Lewis X epitope. When compared with previous data on murine NCAM glycosylation, our results indicate a conservation of structural features and attachment sites for the different types of NCAM N-glycans.     

Definition of TCR epitopes for CTL-mediated attack of cutaneous T cell lymphoma

Therapeutic vaccination against cutaneous T cell lymphoma (CTCL) requires the characterization of cancer cell-specific CTL epitopes. Despite reported evidence for tumor-reactive cytotoxicity in CTCL patients, the nature of the recognized determinants remains elusive. The clonotypic TCR of CTCL cells is a promising candidate tumor-specific Ag. In this study, we report that the clonotypic and framework regions of the TCRs expressed in the malignant T cell clones of six CTCL patients contain multiple peptides with anchor residues fitting the patients' MHC class I molecules. We demonstrate that TCR peptide-specific T cells from the blood of healthy donors and patients can be induced to become cytotoxic effectors after repeated stimulation with 6 of 11 selected peptides with experimentally proven affinity for HLA-A*0201. Importantly, 4 of these 6 CTL lines reproducibly recognize and lyse autologous primary CTCL cells in MHC class I/CD8-dependent fashion. These tumoricidal CTL lines are directed against epitopes from V, hypervariable, and C regions of TCRalpha. We therefore conclude that recombined as well as V framework regions of the tumor cell TCRs contain predictable epitopes for CTL-mediated attack of CTCL cells. Our data further suggest that such peptides represent valuable tools for future anti-CTCL vaccination approaches.     

Investigation of pre-existing reactivity to biotherapeutics can uncover potential immunogenic epitopes and predict immunogenicity risk

ABSTRACT

Despite recent advances in the development of tools to predict immunogenicity risk of biotherapeutic molecules, the ability of a protein to elicit the formation of anti-drug antibodies (ADA) remains one of the most common causes for termination of clinical development programs. In this study, we use ADA assays to detect and measure pre-existing reactivity or the ability of a molecule to produce an ADA-like response in serum from treatment-naïve, healthy donors. We report herein that the magnitude of pre-existing reactivity evaluated pre-clinically and expressed as the 90th percentile of Tier 2 inhibition correlates with the subsequent rate of ADA emergence in the clinic. Furthermore, a multi-domain biotherapeutic (IgG-scFv bispecific antibody) showed the highest pre-existing reactivity and incidence of treatment-emergent ADA (TE-ADA) (57% and 93%, respectively). Using the components of the multidomain molecule in the Tier 2 step of the ADA assay, we were able to identify the scFv as the target of the serum pre-existing reactivity. Most importantly, the domain specificity of pre-existing ADA was the same as that of the TE-ADA from patients treated with the molecule. Based on these data, we propose the evaluation of the magnitude and of the domain specificity of pre-existing reactivity as a powerful tool to understand the immunogenic potential of novel biotherapeutics.     

Selection of HIV-specific immunogenic epitopes by screening random peptide libraries with HIV-1-positive sera

Efforts to develop a protective HIV-1 vaccine have been hindered by difficulties in identifying epitopes capable of inducing broad neutralizing Ab responses. In fact, the high mutation rate occurring in HIV-1 envelope proteins and the complex structure of gp120 as an oligomer associated with gp41 result in a high degree of antigenic polymorphism. To overcome these obstacles, we screened random peptide libraries using sera from HIV-infected subjects to identify antigenic and immunogenic mimics of HIV-1 epitopes. After extensive counterscreening with HIV-negative sera, we isolated peptides specifically recognized by Abs from HIV-1-infected individuals. These peptides behaved as antigenic mimics of linear or conformational HIV-1 epitopes generated in vivo in infected subjects. Consistent with these findings, sera of simian HIV-infected monkeys also recognized the HIV-specific epitopes. The selected peptides were immunogenic in mice, where they elicited HIV-specific Abs that effectively neutralized HIV-1 isolates. These results demonstrate that pools of HIV-1 mimotopes can be selected from combinatorial peptide libraries by taking advantage of the HIV-specific Ab repertoire induced by the natural infection.     

Immobilization of carbohydrate epitopes for surface plasmon resonance using the Staudinger ligation

The detection of carbohydrate-protein interactions is often performed using techniques that require surface immobilization of the lectin or the glycan. A commonly used assay for lectin binding is surface plasmon resonance (SPR). We describe an implementation of the Staudinger ligation as a method to immobilize carbohydrate epitopes to a biosensor surface. This was accomplished by first introducing an azide functionality to a carboxymethyldextran surface, followed by reaction with a phosphane-modified carbohydrate ligand. The chemistry employed is extremely mild and was easily adapted to a commercial biosensor system. Using this approach, we investigated the binding of jacalin and wheat germ agglutinin (WGA) to galactose, lactose, and N-acetyl-lactosamine. We observed that WGA binding shows evidence of multivalent interaction with the surface. Additionally, we found that jacalin binding was influenced by the presence of a flexible and hydrophobic galactosyl aglycone.     

The opportunistic fungal pathogen Scedosporium prolificans: carbohydrate epitopes of its glycoproteins

Isolated from the mycelium of Scedosporium prolificans were complex glycoproteins (RMP-Sp), with three structurally related components (HPSEC). RMP-Sp contained 35% protein and 62% carbohydrate with Rha, Ara, Man, Gal, Glc, and GlcNH(2) in a 18:1:24:8:6:5 molar ratio. Methylation analysis showed mainly nonreducing end- of Galp (13%), nonreducing end- (9%), 2-O- (13%), and 3-O-subst. Rhap (7%), nonreducing end- (11%), 2-O- (10%), 3-O- (14%), and 2,6-di-O-subst. Manp units (13%). Mild reductive beta-elimination of RMP-Sp gave alpha-l-Rhap-(1-->2)-alpha-l-Rhap-(1-->3)-alpha-l-Rhap-(1-->3)-alpha-d-Manp-(1-->2)-d-Man-ol, with Man-ol substituted at O-6 with beta-d-Galp units, a related pentasaccharide lacking beta-d-Galp units, and beta-d-Galp-(1-->6)-[alpha-d-Manp-(1-->2)]-d-Man-ol in a 16:3:1w/w ratio. Traces of Man-ol and Rha-ol were detected. ESI-MS showed HexHex-ol and Hex(3-6)Hex-ol components. Three rhamnosyl units were peeled off successively from the penta- and hexasaccharide by ESI-MS-MS. The carbohydrate epitopes of RMP-Sp differ from those of the glycoprotein of Pseudallescheria boydii, a related opportunistic pathogen.     

N-acetylgalactosaminyl disaccharide terminals contained in presumed carbohydrate epitopes specific to sea-squirt allergy

Using the methods of methylation/GLC, MS, and 1H-NMR analyses, a disaccharide structure of GalNAc alpha 1----2Fuc1---- was identified as one of the major structural units at non-reducing terminals of a highly branched and N-glycosidically linked carbohydrate on an allergenically active glycopeptide, Gp-2 (Mr ca. 8,000). Gp-2 was one of the major fractions in the Pronase digest of a sea-squirt antigen, Gi-rep, and is capable of eliciting the skin reaction specific to patients with sea-squirt allergy similarly to Gi-rep. An additional disaccharide structure of GalNAc1----(3/4)HexNAc1---- was also expected as the other major non-reducing terminal unit, though the anomeric configuration of the terminal GalNAc could not be identified. As Gp-2 carbohydrate was found to contain 3 mol each of the two types of terminal GalNAc-containing units, both were nominated as possible components of the IO4- oxidation-sensitive epitopes which are responsible for the allergenic activity in Gp-2 and its mother antigen, Gi-rep. A few moles of Fuc and Gal were also detected as minor non-reducing terminals in addition to the 6 mol of the GalNAc-containing units.     

Broadly neutralizing antibodies targeted to mucin-type carbohydrate epitopes of human immunodeficiency virus.

The cancer-related mucin-type carbohydrate neoantigen Tn was found on gp160 and gp120 of human immunodeficiency virus (HIV). Immunoglobulin G (IgG) and IgM monoclonal antibodies (MAbs) against Tn neutralized infection with cell-free virus and blocked fusion between HIV-infected and uninfected cells. This inhibition was found in infection of both lymphocytic cells and monocytoid cells. Viruses tested included six HIV-1 and five HIV-2 isolates propagated in different cells, as well as infectious plasma from AIDS patients. The antiviral effect of anti-Tn MAbs occurred by specific binding of the MAb to the virus; this binding was inhibitable by pure Tn antigen, and indications were found that this inhibition occurred at a pre-entry step. Boosting the naturally occurring low-titer anti-Tn activity may be of prophylactic value, as suggested by the in vitro neutralization found in this study.     

Blood-group-related carbohydrate antigens are expressed on human milk galactosyltransferase and are immunogenic in rabbits.

Immunochemical evidence is presented for the presence of blood-group-related carbohydrate structures on human milk galactosyltransferase and for the occurrence of the corresponding specificities among rabbit antibodies to this enzyme. Although these carbohydrate specificities constitute minor populations among antisera and affinity-purified antibodies to galactosyltransferase, their presence is important in the immunohistochemical approach to enzyme localization, since they give rise to strong reactivities with epithelial cells of the gastrointestinal tract.     

Definition of extracellular localized epitopes of Hsp70 involved in an NK immune response

In order to define extracellular localized epitopes of Hsp70 on human tumor cells which are accessible to the immune system, six commercially available Hsp70-specific monoclonal antibodies (mAb) with different recognition sites were examined by immunological approaches. The recognition pattern of these antibodies was analyzed on purified recombinant Hsp70 proteins (rHsp70, Hsc70, DnaK), on lysates of Hsp70-expressing colon carcinoma cells (CX+) and on lysates of M21 rat-1 cells that overexpress human Hsp70 or Hsp70 fragments: ΔBgl (del 120–428) consisting of the C-terminal part and ΔSma (del 438–618) consisting of the N-terminal part of human Hsp70. All antibodies reacted equally well with rHsp70 and cytoplasmic Hsp70 derived from human tumor cells or M21 rat-1 cells. Only one antibody (MA3–007; Hsp70, Hsc70) detects a region localized within the ATPase domain of Hsp70 (amino acid 122–264) and reacts positively with the C-terminal deletion mutant ΔSma. All other antibodies, including RPN1197 are directed against the C-terminal peptide binding domain of Hsp70 and react positively with the N-terminal deletion mutant ΔBgl. Although all six antibodies detect full-length Hsp70 protein, derived from plasma membrane fractions of CX+ tumor cells, cell surface expressed Hsp70 on viable CX+ tumor cells, as determined by flowcytometry, is only recognized with the antibodies MA3–006 (Hsp70, Hsc70; 504–617), MA3–009 (Hsp70; 504–617) and RPN1197 (Hsp70). An estimation of the ratio of membrane-bound to cytoplasmic Hsp70 molecules revealed that 15–20% of total Hsp70 molecules are expressed on the plasma membrane. This tumor-selective cell surface expression of Hsp70 correlates with an increased sensitivity to lysis mediated by non-MHC restricted natural killer (NK) cells. We demonstrate that only antibodies directed against membrane-bound Hsp70 (MA3–006, MA3–009, RPN1197) inhibit NK-killing activity against Hsp70-expressing tumor cells. Taken together our data indicate that at least the C-terminal region 504–617, that contains at least one single α-helix (amino acid 512–536), has to be localized extracellularly and might be of importance for an NK-mediated anti-tumor immune response.