Epitope Clusters Research Articles

Identification of a Conserved Epitope Cluster in the N Protein of Porcine Reproductive and Respiratory Syndrome Virus

In this study, 4 overlapping fragments and 12 overlapping peptides of the nucleocapsid (N) protein from porcine reproductive and respiratory syndrome virus (PRRSV) were expressed as glutathione S-transferase (GST) fusion proteins and used to probe a panel of 16 anti-N protein monoclonal antibodies (mAbs) by ELISA. The minimal epitope sequence of the following seven mAbs was determined by sequential deletion of terminal amino acid residues from each peptide: N2H7 corresponded to H54FPLA58; N2F7 corresponded to K52PHFPLA58; and N1A2, N1E3, N1G4, and N2E5 were reactive against E51KPHFP56. Furthermore, a polypeptide containing this epitope cluster was recognized by PRRSV-immune pig serum by Western blot, suggesting that residues 51-58 represent an immunodominant region of the N protein. Sequence alignment revealed that these epitopes are well conserved among North American and European genotypes of PRRSV. These findings enhance our knowledge of the antigenic structure of N protein and may facilitate the development of better diagnostic methods for PRRSV.

Mapping of B cell epitopes in measles virus nucleocapsid protein

The B-cell response against measles nucleoprotein (MeN) plays an important role in the control of measles infection. However, the data on B cell epitopes of MeN are still limited. The objective of this study was to identify B cell epitopes in MeN using monoclonal and polyclonal antibodies raised against recombinant yeast-expressed MeN (rMeN) as well as human sera from measles-positive individuals. After immunization of mice, 15 monoclonal antibodies (mAbs) against rMeN were generated. The B cell epitopes were localized using recombinant overlapping MeN fragments, PepScan analysis, and competitive ELISA. The epitopes of 14 mAbs were mapped within the C-terminus of MeN between amino acids (aa) 419 and 525. Four mAbs recognized a linear epitope located within a sequence of aa 440-448. Competitive ELISA revealed a cluster of conformational mAb epitopes. Cross-inhibition studies with human sera demonstrated similar localization of B cell epitopes recognized by serum antibodies from naturally infected individuals. Thus, the majority of B cell epitopes are located at the C-terminal domain of MeN. These findings provide new data on the antigenic structure of MeN and are in agreement with recent experimental evidence indicating that the C-terminal domain of MeN is well accessible on the surface of nucleocapsid-like structures.

Induction of Human Immunodeficiency Virus Type-1-Specific Immunity with a Novel Gene Transport Unit (GTU)-MultiHIV DNA Vaccine

A multiHIV fusion gene expressing an antigenic fusion protein composed of regulatory HIV-1 proteins Rev, Nef, and Tat, as well as Gag p17/p24 and a stretch of 11 cytotoxic T lymphocyte (CTL) epitope clusters from Pol and Env, was cloned into a novel DNA vector named the Gene Transport Unit (GTU). A mouse H-2(d)-restricted HIV-1 gp120 epitope (RGPGRAFVTI) was cloned into the fusion gene as well. In addition to the HIV- 1 genes the GTU codes for a nuclear anchoring protein (bovine papilloma virus E2), ensuring the long maintenance of the vector and a high expression level of the selected immunogens. BALB/c mice were immunized with the GTU-MultiHIV DNA construct by different routes and regimens of immunization to assess the immunogenicity of the DNA vaccine in vivo. Mice developed strong CD8(+) CTL responses to HIV-1 Env and Gag measured by an ELISPOT-IFN-gamma assay and chromium release assay. In addition, T cell responses to regulatory proteins Rev, Nef, and Tat were induced. Antibody responses were detected to each of the HIV antigens encoded by the DNA construct. Minimal doses of the GTU-MultiHIV DNA delivered by gene gun were potent in inducing significant HIV-specific CTL responses. The equivalent doses of the conventional plasmid expressing MultiHIV DNA delivered by gene gun failed to do so. An ideal DNA vaccine should yield high expression of the viral antigens for a prolonged period of time, and expression of the multiple viral antigens is probably required for the induction of a broad and protective immune response. The GTU-MultiHIV DNA vaccine described is a good vaccine candidate that meets the above criteria.

Spatial clustering of the IgE epitopes on the major timothy grass pollen allergen Phl p 1: Importance for allergenic activity

The major timothy grass pollen allergen Phl p 1 is one of the most potent and frequently recognized environmental allergens. We sought to study at a molecular and structural level the IgE recognition of Phl p 1 and its relation to allergenic activity. Monoclonal human IgE antibody fragments specific for Phl p 1 and group 1 allergens from various grasses were isolated from a combinatorial library made of lymphocytes from patients with grass pollen allergy. Recombinant Phl p 1 fragments and the 3-dimensional structure of Phl p 1 were used to localize the major binding site for the IgE antibodies. A rPhl p 1 fragment containing this binding site was expressed in Escherichia coli, purified, and tested for IgE reactivity and allergenic activity with sera and basophils from patients with grass pollen allergy. Monoclonal antibodies, as well as polyclonal serum IgE, from patients with grass pollen allergy defined a C-terminal fragment of Phl p 1 that represents a sterically oriented portion on the Phl p 1 structure. This Phl p 1 portion bound most of the allergen-specific IgE antibodies and contained the majority of the allergenic activity of Phl p 1. IgE recognition of spatially clustered epitopes on allergens might be a general factor determining their allergenic activity. Geographic distribution of IgE epitopes on an allergen might influence its allergenic activity and hence explain discrepancies between diagnostic test results based on IgE serology and provocation testing. It might also form a basis for the development of low allergenic vaccines.

Lessons Learned from Clustering of Fluorinated Glycolipids on Selectin Ligand Function in Cell Rolling,

Selectin-induced leukocyte rolling along the endothelium is an essential step in the cellular immune response. Since clustering of binding epitopes is thought to be crucial for selectin-ligand interaction, we focused on requirements of ligand clusters in a flow chamber study. Neoglycolipids bearing the binding epitope sialyl Lewis X (sLeX) were used as artificial ligands in model membranes. sLeX ligands or matrix lipids or both were applied with partially fluorinated alkyl chains to increase the ligand cluster separation tendency. Cluster size, their inner structure, and separation distance were evaluated with high resolution by scanning force microscopy (SFM) and correlated with binding or rolling of E-selectin-expressing cells. Fluorination of only one component, sLeX ligand or matrix lipid, leads to a very high separation tendency and impeded cell rolling, although firm cell adhesion could be observed down to 0.005 mol % ligand concentration. As a sign of total immiscibility, cluster size increased with ligand concentration, and resulting excessive ligand densities within the clusters prevent cell rolling. Fluorination of both sLeX ligands and matrix created small clusters which could serve as rolling patches. Our results confirmed that cluster size and separation distance controlled by a certain miscibility of ligand and matrix as well as a sufficiently diluted ligand concentration within the clusters are necessary for cell rolling. Within this work, selectin ligand clustering and its ability to mediate cell rolling are presented as a balance between multivalency of binding and sufficient flexibility of the single epitopes. This might be helpful for better understanding the function of the natural selectin ligands.

SARS coronavirus nucleocapsid immunodominant T-cell epitope cluster is common to both exogenous recombinant and endogenous DNA-encoded immunogens

Correspondence between the T-cell epitope responses of vaccine immunogens and those of pathogen antigens is critical to vaccine efficacy. In the present study, we analyzed the spectrum of immune responses of mice to three different forms of the SARS coronavirus nucleocapsid (N): (1) exogenous recombinant protein (N-GST) with Freund's adjuvant; (2) DNA encoding unmodified N as an endogenous cytoplasmic protein (pN); and (3) DNA encoding N as a LAMP-1 chimera targeted to the lysosomal MHC II compartment (p-LAMP-N). Lysosomal trafficking of the LAMP/N chimera in transfected cells was documented by both confocal and immunoelectron microscopy. The responses of the immunized mice differed markedly. The strongest T-cell IFN-γ and CTL responses were to the LAMP-N chimera followed by the pN immunogen. In contrast, N-GST elicited strong T cell IL-4 but minimal IFN-γ responses and a much greater antibody response. Despite these differences, however, the immunodominant T-cell ELISpot responses to each of the three immunogens were elicited by the same N peptides, with the greatest responses being generated by a cluster of five overlapping peptides, N 76–114, each of which contained nonameric H2 d binding domains with high binding scores for both class I and, except for N 76–93, class II alleles. These results demonstrate that processing and presentation of N, whether exogenously or endogenously derived, resulted in common immunodominant epitopes, supporting the usefulness of modified antigen delivery and trafficking forms and, in particular, LAMP chimeras as vaccine candidates. Nevertheless, the profiles of T-cell responses were distinctly different. The pronounced Th-2 and humoral response to N protein plus adjuvant are in contrast to the balanced IFN-γ and IL-4 responses and strong memory CTL responses to the LAMP-N chimera.

Cross-Clade Protection Induced by Human Immunodeficiency Virus-1 DNA Immunogens Expressing Consensus Sequences of Multiple Genes and Epitopes From Subtypes A, B, C, and FGH

The correlate of protection in human immunodeficiency virus (HIV) infection is not known, but preclinical and clinical studies support the involvement of both antibodies and cellular immunity. In addition, the existence of multiple HIV clades makes HIV vaccine design especially challenging. We have constructed a vaccine platform with an HIV-1 subtype B DNA immunogen expressing full length consensus sequences from HIV-1 rev, nef, tat, and gag with additional cellular epitope clusters from the env and pol regions. Furthermore, this platform has been extended to three additional plasmids expressing the same immunogens but originating from subtypes A or C consensus or FGH ancestral sequences. Immunogenicity in BALB/c mice, by gene gun or intramuscular delivery, revealed strong IFN-gamma production in response to in vitro re-stimulation with a H-2d restricted gag peptide (AMQMLKETI) or even stronger toward an env epitope (RGPGRAFVTI). Weak humoral immunity was detected. Gene gun immunization with a cocktail of all four plasmids induced pre-challenge cellular immunity in C57Bl6/A2.01 mice and subsequently a robust frequency of protection (11/12 animals) after experimental challenge with subtype A or B HIV-1/Murine Leukemia Virus (HIV-1/MuLV). The cross-clade protection observed in this challenge experiment demonstrates that these multigene/multiepitope HIV DNA immunogens are likely to be potent immunogens also against the HIV-infection of human beings.

Antigenic structures recognized by anti-β2-glycoprotein I auto-antibodies

Beta2-glycoprotein I (beta2-GPI) is a major antigen for anti-cardiolipin antibodies and their epitopes are cryptic. Conformation of each domain of beta2-GPI was optimized from its crystal structure by energy minimization and by molecular dynamics simulation. Three electrostatic interactions, i.e. D193-K246, D222-K317 and E228-K308, were observed between domains IV and V in the optimized structure that was constructed based on the consensus sequences obtained by the phage-displayed random peptide library. Antigenic structures determined by the epitope mapping mainly consisted of hydrophobic amino acids located on two discontinuous sequences in domain IV. These amino acid clusters, as an epitope, were covered by domain V and were of a hidden nature. A similar but incomplete counterpart to the epitopic clusters was found in domain I but was not in domains II or III. Binding of anti-beta2-GPI auto-antibodies to solid-phase beta2-GPI was significantly reduced either by L replacement for W235, a common amino acid component for the epitopes, or by V replacement for all of D193, D222 and E228. Structural analysis indicated a hypothesis that these electrostatic interactions between domains IV and V retained exposure to W235 and that epitope spreading occurred in the region surrounding W235. Thus, epitopic structures recognized by anti-beta2-GPI auto-antibodies are cryptic and inter-domain electrostatic interactions are involved in their in exposure.

Evaluation of high functional avidity CTL to Gag epitope clusters in EIAV carrier horses

Cytotoxic T lymphocytes (CTL) are critical for lentivirus control including EIAV. Since CTL from most EIAV carrier horses recognize Gag epitope clusters (EC), the hypothesis that carrier horses would have high functional avidity CTL to optimal epitopes in Gag EC was tested. Twenty-two optimal EC epitopes were identified; two in EC1, six in EC2, and seven each in EC3 and 4. However, only five of nine horses had high functional avidity CTL (≤11 nM) recognizing six epitopes in EC; four in relatively conserved EC3; and one each in EC1 and 2. Horses with high functional avidity CTL had significantly more days since the last clinical episode than horses with low avidity CTL, and this was not explained by analyzing duration of infection. Furthermore, there was a significant inverse correlation between the CTL functional avidity of the nine horses and the days since the last clinical episode. Gag CTL epitope escape variants were found in three horses, but only one of these was recognized by high functional avidity CTL. Thus, not all carrier horses had high functional avidity CTL to Gag EC, but those that did had longer periods without disease episodes.

Mapping the functional domains of human transcobalamin using monoclonal antibodies

Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA...TAAM(103-198) or the C-terminal peptide LEPA...LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS...QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA...HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment KRSN...RTVR(207-227), which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor.

Anaplasma marginale Major Surface Protein 2 CD4 + -T-Cell Epitopes Are Evenly Distributed in Conserved and Hypervariable Regions (HVR), Whereas Linear B-Cell Epitopes Are Predominantly Located in the HVR

Organisms in the genus Anaplasma express an immunodominant major surface protein 2 (MSP2), composed of a central hypervariable region (HVR) flanked by highly conserved regions. Throughout Anaplasma marginale infection, recombination results in the sequential appearance of novel MSP2 variants and subsequent control of rickettsemia by the immune response, leading to persistent infection. To determine whether immune evasion and selection for variant organisms is associated with a predominant response against HVR epitopes, T-cell and linear B-cell epitopes were localized by measuring peripheral blood gamma interferon-secreting cells, proliferation, and antibody binding to 27 overlapping peptides spanning MSP2 in 16 cattle. Similar numbers of MSP2-specific CD4(+) T-cell epitopes eliciting responses of similar magnitude were found in conserved and hypervariable regions. T-cell epitope clusters recognized by the majority of animals were identified in the HVR (amino acids [aa] 171 to 229) and conserved regions (aa 101 to 170 and 272 to 361). In contrast, linear B-cell epitopes were concentrated in the HVR, residing within hydrophilic sequences. The pattern of recognition of epitope clusters by T cells and of HVR epitopes by B cells is consistent with the influence of protein structure on epitope recognition.

Glycomic profiling of developmental changes in bovine testis by lectin histochemistry and further analysis of the most prominent alteration on the level of the glycoproteome by lectin blotting and lectin affinity chromatography.

The emerging concept of the sugar code attributes functional significance to oligosaccharides of cellular glycoconjugates by protein (lectin)-carbohydrate interactions. Hence it follows that monitoring of glycan expression (glycomic profiling) is not only valuable to delineate characteristic (phenomenological) changes in the cell's glycosylation but will also come up with the localization of epitopes with potential in biorecognition. It is for this purpose that we have set up a panel of 16 markers (plant lectins and a carbohydrate-specific antibody). The selection met two criteria: a) to be able to detect the common constituents of natural glycans; and b) to place emphasis on detection of neutral carbohydrate units at the spatially accessible branch ends of glycan chains, which are known to be active as ligands for endogenous lectins in situ. Next, we incorporated recent insights into the importance of epitope clustering to turn less abundant oligosaccharides into potent ligands into our study design. To be able to focus on such high-affinity sites, we performed systematic titration studies aimed at defining the probe concentration at which carbohydrate-independent background staining is minimal while still yielding a clear signal. These requirements were met by marker concentrations of 1.25-2.5 microg/ml. Under these conditions, we defined cell-type- and differentiation-dependent changes in bovine testis. Sertoli cells lacked reactivity, whereas gonocytes were differentially reactive with the tested markers. The extent of staining intensity was subject to developmental changes, preferentially for Gal/GalNAc presentation and in this group most prominently with the galactoside-specific lectin from Viscum album L. (mistletoe). Of interest in this context, this lectin is known as a potent mitogen and signal inductor as well as haemagglutinin. The Gal/GalNAc-dependent signals decreased markedly in the course of development and staining was completely lost in the case of mistletoe lectin 12 weeks after gestation. Spermatids of adult testis presented respective glycan epitopes. In contrast to this developmental course of staining, endothelial cells either maintained a constant signal intensity or revealed a signal increase during development for Gal/GalNAc-specific lectins. Their binding of concanavalin A and the two phyto-haemagglutinins (PHA-E/L), which were not or only weakly reactive for gonocytes, served as inherent activity control. Based on lectin blot analysis with the mistletoe lectin as the marker which detected the most prominent change, the glycoprotein patterns from fetal and adult tissue specimens were qualitatively different, rendering changes in expression of the protein part of glycoproteins more likely than remodeling a glycoprotein's glycan chains. Methodologically, results of this procedure were compared to data obtained with lectin affinity chromatography and the combination of the two procedures. Differences in the profiles were discovered that can be assigned to the disparate ways to process the detergent extracts. When access to sample quantity is limited, as is possible in the case of fetal tissue, direct lectin blotting is recommended.

CTL from EIAV carrier horses with diverse MHC class I alleles recognize epitope clusters in Gag matrix and capsid proteins

Cytotoxic T lymphocytes (CTL) are important for controlling equine infectious anemia virus (EIAV). Because Gag matrix (MA) and capsid (CA) are the most frequently recognized proteins, the hypothesis that CTL from EIAV-infected horses with diverse MHC class I alleles recognize epitope clusters (EC) in these proteins was tested. Four EC were identified by CTL from 15 horses and 8 of these horses had diverse MHC class I alleles. Two of the eight had CTL to EC1, six to EC2, five to EC3, and four to EC4. Because EC2–4 were recognized by CTL from >50% of horses with diverse alleles, the hypothesis was accepted. EC1 and EC3 were the most conserved EC and these more conserved broadly recognized EC may be most useful for CTL induction, helping overcome MHC class I polymorphism and antigenic variation.

Differences in the expressed HLA class I alleles effect the differential clustering of HIV type 1-specific T cell responses in infected Chinese and caucasians.

China is a region of the world with a rapidly spreading HIV-1 epidemic. Studies providing insights into HIV-1 pathogenesis in infected Chinese are urgently needed to support the design and testing of an effective HIV-1 vaccine for this population. HIV-1-specific T cell responses were characterized in 32 HIV-1-infected individuals of Chinese origin and compared to 34 infected caucasians using 410 overlapping peptides spanning the entire HIV-1 clade B consensus sequence in an IFN-gamma ELISpot assay. All HIV-1 proteins were targeted with similar frequency in both populations and all study subjects recognized at least one overlapping peptide. HIV-1-specific T cell responses clustered in seven different regions of the HIV-1 genome in the Chinese cohort and in nine different regions in the caucasian cohort. The dominant HLA class I alleles expressed in the two populations differed significantly, and differences in epitope clustering pattern were shown to be influenced by differences in class I alleles that restrict immunodominant epitopes. These studies demonstrate that the clustering of HIV-1-specific T cell responses is influenced by the genetic HLA class I background in the study populations. The design and testing of candidate vaccines to fight the rapidly growing HIV-1 epidemic must therefore take the HLA genetics of the population into account as specific regions of the virus can be expected to be differentially targeted in ethnically diverse populations.

Comprehensive analysis of human immunodeficiency virus type 1-specific CD4 responses reveals marked immunodominance of gag and nef and the presence of broadly recognized peptides.

Increasing evidence suggests that human immunodeficiency virus type 1 (HIV-1)-specific CD4 T-cell responses contribute to effective immune control of HIV-1 infection. However, the breadths and specificities of these responses have not been defined. We screened fresh CD8-depleted peripheral blood mononuclear cells (PBMC) from 36 subjects at different stages of HIV-1 infection for virus-specific CD4 responses by gamma interferon enzyme-linked immunospot assay, using 410 overlapping peptides spanning all HIV-1 proteins (based on the clade B consensus sequence). HIV-1-specific CD4 responses were identified in 30 of the 36 individuals studied, with the strongest and broadest responses detected in persons treated in acute infection who underwent treatment interruption. In individuals with identified responses, the total number of recognized HIV-1 peptides ranged from 1 to 36 (median, 7) and the total magnitude of responses ranged from 80 to >14,600 (median, 990) spot-forming cells/10(6) CD8-depleted PBMC. Neither the total magnitude nor the number of responses correlated with viremia. The most frequent and robust responses were directed against epitopes within the Gag and Nef proteins. Peptides targeted by >/=25% of individuals were then tested for binding to a panel of common HLA-DR molecules. All bound broadly to at least four of the eight alleles tested, and two bound to all of the HLA-DR molecules studied. Fine mapping and HLA restriction of the responses against four of these peptides showed a combination of clustering of epitopes and promiscuous presentation of the same epitopes by different HLA class II alleles. These findings have implications for the design of immunotherapeutic strategies and for testing candidate HIV vaccines.

Antibodies to GAD65 epitopes at diagnosis and over the first 10 years of clinical type 1 diabetes mellitus.

Antibodies to glutamate decarboxylase (GAD65Ab) may persist, and their titres even increase after the clinical onset of type 1 diabetes. To characterize this phenomenon in detail, we analysed sequentially antibodies to GAD65 epitope clusters in a radio-binding assay in patients with type 1 diabetes. Serum samples were taken at diagnosis and 2, 5 and 10 years later from 50 young patients who had tested positive for GAD65Ab at least once during observation. The levels of GAD65Ab peaked in 21 patients after diagnosis. Antibodies to the middle region of GAD65 (GAD65-M-Ab, 88%) were more common at diagnosis than antibodies to the C-terminal (GAD65-C-Ab, 68%, P < 0.05) or N-terminal region (4%, P < 0.001). Antibodies to middle and especially to C-terminal epitopes decreased in those with decreasing levels of GAD65Ab (P < 0.001), whereas the frequencies of GAD65-M-Ab and GAD65-C-Ab remained quite stable among the subjects with increasing levels. Lower exogenous insulin dose and HbA(1) levels and stronger humoral immune response to islet cells were observed in those with increasing levels of GAD65-M-Ab than in those with decreasing levels (P < 0.05). The present observation supports the view that the middle region of GAD65 comprises immunodominant epitopes. An enhanced humoral immune response to GAD65 after diagnosis is related to persistent immune reactivity to the middle and C-terminal regions.

Reverse transcriptase-based DNA vaccines against drug-resistant HIV-1 tested in a mouse model

Drug resistance is becoming a problem in the treatment of the human immunodeficiency virus type one (HIV-1). To obtain therapeutic DNA vaccines that would target multiple drug-resistance (DR) mutations, we cloned genes for DR HIV-1 reverse transcriptase (RT) and codon-optimized synthetic genes encoding clusters of human CTL epitopes located at the sites of DR-mutations (RT minigenes) and antibody and CTL-epitope tags. Expression of RT genes/minigenes in eukaryotic cells was confirmed by Western blotting and immunofluoresence staining with RT- or tag-specific antibodies. Immunization of mice with DR–RT gene induced no RT-specific antibodies. Immunization of HLA-A ∗0201-transgenic mice with RT minigenes induced RT-specific cellular responses detected by interferon-γ secretion. This documents first steps in creating therapeutic vaccine against drug-resistant HIV strains.

Clustering of Th cell epitopes on exposed regions of HIV envelope despite defects in antibody activity.

A long-standing question in the field of immunology concerns the factors that contribute to Th cell epitope immunodominance. For a number of viral membrane proteins, Th cell epitopes are localized to exposed protein surfaces, often overlapping with Ab binding sites. It has therefore been proposed that Abs on B cell surfaces selectively bind and protect exposed protein fragments during Ag processing, and that this interaction helps to shape the Th cell repertoire. While attractive in concept, this hypothesis has not been thoroughly tested. To test this hypothesis, we have compared Th cell peptide immunodominance in normal C57BL/6 mice with that in C57BL/6( micro MT/ micro MT) mice (lacking normal B cell activity). Animals were first vaccinated with DNA constructs expressing one of three different HIV envelope proteins, after which the CD4(+) T cell response profiles were characterized toward overlapping peptides using an IFN-gamma ELISPOT assay. We found a striking similarity between the peptide response profiles in the two mouse strains. Profiles also matched those of previous experiments in which different envelope vaccination regimens were used. Our results clearly demonstrate that normal Ab activity is not required for the establishment or maintenance of Th peptide immunodominance in the HIV envelope response. To explain the clustering of Th cell epitopes, we propose that localization of peptide on exposed envelope surfaces facilitates proteolytic activity and preferential peptide shuttling through the Ag processing pathway.

Differential proteasomal processing of hydrophobic and hydrophilic protein regions: contribution to cytotoxic T lymphocyte epitope clustering in HIV-1-Nef.

HIV proteins contain a multitude of naturally processed cytotoxic T lymphocyte (CTL) epitopes that concentrate in clusters. The molecular basis of epitope clustering is of interest for understanding HIV immunogenicity and for vaccine design. We show that the CTL epitope clusters of HIV proteins predominantly coincide with hydrophobic regions, whereas the noncluster regions are predominantly hydrophilic. Analysis of the proteasomal degradation products of full-length HIV-Nef revealed a differential sensitivity of cluster and noncluster regions to proteasomal processing. Compared with the epitope-scarce noncluster regions, cluster regions are digested by proteasomes more intensively and with greater preference for hydrophobic P1 residues, resulting in substantially greater numbers of fragments with the sizes and COOH termini typical of epitopes and their precursors. Indeed, many of these fragments correspond to endogenously processed Nef epitopes and/or their potential precursors. The results suggest that differential proteasomal processing contributes importantly to the clustering of CTL epitopes in hydrophobic regions.

New Approaches for the Detection and Characterization of Alpha-Fetoprotein Epitope Variants

Alpha-fetoprotein (AFP) is an embryo-specific protein of mammalian sera that consists of a single glycosylated polypeptide chain (∼70 kD) with 5–6 epitope clusters located on the native molecule. We have elaborated three approaches for the separation of AFP into fractions (variants) that are different in the distinct epitope expression on the native molecule. For this, we have used three technologies largely based on immunoaffinity electrochromatography and electrophoresis/immunoblotting. The separation of these variants was facilitated using monoclonal antibodies previously characterized to the 5 epitope clusters and two individual epitopes on AFP in the ISOBM international workshop. We have shown that these approaches facilitate the identification of cryptic epitopes in AFP, and the relevance of these findings is discussed.