Epitope Clusters Research Articles

Physical, Spatial, and Molecular Aspects of Extracellular Matrix of In Vivo Niches and Artificial Scaffolds Relevant to Stem Cells Research.

Extracellular matrix can influence stem cell choices, such as self-renewal, quiescence, migration, proliferation, phenotype maintenance, differentiation, or apoptosis. Three aspects of extracellular matrix were extensively studied during the last decade: physical properties, spatial presentation of adhesive epitopes, and molecular complexity. Over 15 different parameters have been shown to influence stem cell choices. Physical aspects include stiffness (or elasticity), viscoelasticity, pore size, porosity, amplitude and frequency of static and dynamic deformations applied to the matrix. Spatial aspects include scaffold dimensionality (2D or 3D) and thickness; cell polarity; area, shape, and microscale topography of cell adhesion surface; epitope concentration, epitope clustering characteristics (number of epitopes per cluster, spacing between epitopes within cluster, spacing between separate clusters, cluster patterns, and level of disorder in epitope arrangement), and nanotopography. Biochemical characteristics of natural extracellular matrix molecules regard diversity and structural complexity of matrix molecules, affinity and specificity of epitope interaction with cell receptors, role of non-affinity domains, complexity of supramolecular organization, and co-signaling by growth factors or matrix epitopes. Synergy between several matrix aspects enables stem cells to retain their function in vivo and may be a key to generation of long-term, robust, and effective in vitro stem cell culture systems.

Assessing Structure-Activity Relationships in FVIII By Integration of Structural, Biophysical, and Biochemical Data Obtained with Anti-FVIII Antibodies

IntroductionAnti-factor VIII (FVIII) antibodies are valuable probes for evaluating structure-activity relationships in FVIII. In this study, we structurally and functionally characterized a panel of anti-FVIII antibodies comprising 36 mouse monoclonal antibodies and 1 camelid nanobody with specificities that span all 5 structural domains of FVIII. Integration of these data into a comprehensive functional interaction map reveals regions that are essential for the function of FVIII and provides a useful guide for researchers engaged in the engineering, quantitation, and purification of FVIII and variants thereof.Materials and MethodsMonoclonal mouse IgGs were raised by conventional hybridoma methods or obtained from commercial sources (Green Mountain Antibodies, American Diagnostica). Recombinant B domain-deleted human FVIII (BDD-rFVIII), consisting of a mixture of proteolytically processed forms and dual-chain forms, was generated by transfection of HEK293 cells and purified by immuno-affinity chromatography to >95% purity. Human plasma-derived von Willebrand factor (VWF) was purchased from Haematologic Technologies.The chain and domain specificities of each antibody and competition with VWF for FVIII binding were evaluated by biolayer interferometry (BLI; ForteBio Octet). Pairwise epitope overlap analysis (binning) was performed by both BLI and multiplexed surface plasmon resonance (SPR; BioRad ProteOn). The affinity of each antibody for FVIII was determined by SPR (ProteOn, Biacore T200), and the inhibitory activity of each antibody was determined by the standard Bethesda assay using normal human plasma. The ability of antibodies to inhibit the thrombin-mediated cleavage of FVIII was determined by resolving samples from a digestion time-course on SDS-PAGE and subsequent quantitation by gel densitometry. The morphology of FVIII-Fab fragment complexes was determined by negative stain electron microscopy (EM) with images classified into 25 groups. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to localize antibody epitopes on FVIII by evaluating deuterium uptake in the presence and absence of selected antibodies. Differential deuterium uptake over four hours was evaluated for over 500 unique FVIII-derived peptides that resulted from cleavage with pepsin, with greater than 95% sequence coverage of BDD-FVIII.Results and ConclusionsAntibodies in this panel were characterized with respect to FVIII chain and domain specificity. Fifteen antibodies were mapped to the FVIII heavy chain (3 in the A1 domain, 12 in the A2 domain), and 22 antibodies were mapped to the FVIII light chain (8 in A3/C1 domains, 14 in C2 domain). Affinities for FVIII varied widely, with KD values ranging from ~10 nM to ~10 pM. Bethesda inhibitory activities also varied widely, from undetectable levels to greater than 40,000 Bethesda units (BU) per milligram. No correlation was observed between antibody affinity and inhibitory activity, but inhibitory activity was strongly associated with distinct clusters of antibodies having overlapping epitopes. Nine antibodies competed with VWF for binding to FVIII (2 in C1 domain, 7 in C2 domain), and these antibodies were distributed among 3 distinct epitope clusters, indicating that the VWF binding interface is broadly distributed across the C1 and C2 domains. Three C2 domain-specific antibodies that compete with VWF for binding to FVIII also interfered with the liberation of the acidic a3 peptide by thrombin in the absence of VWF. Thrombin-mediated cleavage at other sites in FVIII was not affected by these antibodies, suggesting the presence of a thrombin binding site within the C2 domain that is required specifically for cleavage by thrombin at the N-terminus of the FVIII light chain. The structures of FVIII in complex with 15 individual antibody Fab fragments were determined by negative stain EM for both inhibitory and non-inhibitory antibodies, and precise epitope mapping of selected antibodies was achieved by HDX-MS. Collectively, these data were used to generate an FVIII-antibody interaction map that integrates activity data with structural data spanning the atomic level to the gross morphological level. This integrated map can serve as a resource for the evaluation of structure-activity relationships in FVIII. DisclosuresLiu:Biogen Idec: Employment. Bou-Assaf:Biogen Idec: Employment. Zhang:Biogen Idec: Employment. Goodman:Biogen Idec: Employment. Walz:Biogen Idec: Honoraria, Research Funding. Peters:Biogen Idec: Employment. Kulman:Biogen Idec: Employment.

C3d adjuvant effects are mediated through the activation of C3d-specific autoreactive T cells.

Complement fragment C3d covalently attached to antigens enhances immune responses, particularly for antigens lacking T cell epitopes. Enhancement has been attributed to receptor cross-linking between complement receptor CR2 (CD21) and polysaccharide antigen to surface IgM on naïve B cells. Paradoxically, C3d has still been shown to increase immune responses in CD21 KO mice, suggesting that an auxiliary activation pathway exists. In prior studies, we demonstrated the CD21-independent C3d adjuvant effect might be due to T cell recognition of C3d T helper epitopes processed and presented by MHC class II on the B cell surface. C3d peptide sequences containing concentrated clusters of putative human C3 T cell epitopes were identified using the epitope-mapping algorithm, EpiMatrix. These peptide sequences were synthesized and shown in vitro to bind multiple HLA-DR alleles with high affinity, and induce IFNγ responses in healthy donor PBMCs. In the present studies, we establish further correlations between HLA binding and HLA-specific lymphocyte reactions with select epitope clusters. Additionally, we show that the T cell phenotype of C3d-specific reactive T cells is CD4+CD45RO+ memory T cells. Finally, mutation of a single T cell epitope residing within the P28 peptide segment of C3d resulted in significantly diminished adjuvant activity in BALB/c mice. Collectively, these studies support the hypothesis that the paradoxical enhancement of immune responses by C3d in the absence of CD21 is due to internalization and processing of C3d into peptides that activate autoreactive CD4+ T helper cells in the context of HLA class II.

T cell epitope clustering in the highly immunogenic BZLF1 antigen of Epstein-Barr virus.

Polymorphism in the human leukocyte antigen (HLA) loci ensures that the CD8(+) T cell response to viruses is directed against a diverse range of antigenic epitopes, thereby minimizing the impact of virus escape mutation across the population. The BZLF1 antigen of Epstein-Barr virus is an immunodominant target for CD8(+) T cells, but the response has been characterized only in the context of a limited number of HLA molecules due to incomplete epitope mapping. We have now greatly expanded the number of defined CD8(+) T cell epitopes from BZLF1, allowing the response to be evaluated in a much larger proportion of the population. Some regions of the antigen fail to be recognized by CD8(+) T cells, while others include clusters of overlapping epitopes presented by different HLA molecules. These highly immunogenic regions of BZLF1 include polymorphic sequences, such that up to four overlapping epitopes are impacted by a single amino acid variation common in different regions of the world. This focusing of the immune response to limited regions of the viral protein could be due to sequence similarity to human proteins creating "immune blind spots" through self-tolerance. This study significantly enhances the understanding of the immune response to BZLF1, and the precisely mapped T cell epitopes may be directly exploited in vaccine development and adoptive immunotherapy. Epstein-Barr virus (EBV) is an important human pathogen, associated with several malignancies, including nasopharyngeal carcinoma and Hodgkin lymphoma. T lymphocytes are critical for virus control, and clinical trials aimed at manipulating this arm of the immune system have demonstrated efficacy in treating these EBV-associated diseases. These trials have utilized information on the precise location of viral epitopes for T cell recognition, for either measuring or enhancing responses. In this study, we have characterized the T cell response to the highly immunogenic BZLF1 antigen of EBV by greatly expanding the number of defined T cell epitopes. An unusual clustering of epitopes was identified, highlighting a small region of BZLF1 that is targeted by the immune response of a high proportion of the world's population. This focusing of the immune response could be utilized in developing vaccines/therapies with wide coverage, or it could potentially be exploited by the virus to escape the immune response.

Multiple independent IgE epitopes on the highly allergenic grass pollen allergen Phl p 5.

BackgroundGroup 5 allergens are small proteins that consist of two domains. They belong to the most potent respiratory allergens.ObjectiveTo determine the binding sites and to study allergic patients' IgE recognition of the group 5 allergen (Phl p 5) from timothy grass pollen using human monoclonal IgE antibodies that have been isolated from grass pollen allergic patients.MethodsUsing recombinant isoallergens, fragments, mutants and synthetic peptides of Phl p 5, as well as peptide-specific antibodies, the interaction of recombinant human monoclonal IgE and Phl p 5 was studied using direct binding and blocking assays. Cross-reactivity of monoclonal IgE with group 5 allergens in several grasses was studied and inhibition experiments with patients' polyclonal IgE were performed.ResultsMonoclonal human IgE showed extensive cross-reactivity with group 5 allergens in several grasses. Despite its small size of 29 kDa, four independent epitope clusters on isoallergen Phl p 5.0101, two in each domain, were recognized by human IgE. Isoallergen Phl p 5.0201 carried two of these epitopes. Inhibition studies with allergic patients' polyclonal IgE suggest the presence of additional IgE epitopes on Phl p 5.Conclusions & Clinical RelevanceOur results reveal the presence of a large number of independent IgE epitopes on the Phl p 5 allergen explaining the high allergenic activity of this protein and its ability to induce severe allergic symptoms. High-density IgE recognition may be a general feature of many potent allergens and form a basis for the development of improved diagnostic and therapeutic procedures in allergic disease.

Development and characterization of three novel monoclonal antibodies against CA-125.

Cancer antigen 125 (CA-125) is the most widely used tumor marker for ovarian cancer. Thus, monoclonal antibodies (MAbs) against CA-125 are valuable reagents for the development of diagnostic tests and immunotherapy. We describe here the generation and characterization of three novel hybridoma cell lines producing MAbs against CA-125. CA-125 purified from culture supernatant of ovarian carcinoma cell line OVCAR-3 by affinity chromatography, was used for immunization of BALB/c mice. Three stable cell lines (3C8, 2B6, and 5A12) were selected for production of antibodies against CA-125 and were expanded in mass culture. All three antibodies were shown to recognize linear epitopes. Antibodies 2B6 and 5A12 were determined to recognize epitope cluster B (M 11-like); MAb 3C8 was classified as group A-epitope binders (OC 125-like). The antibodies produced may be used for the development and improvement of CA-125 immunoassays.

Human Leukocyte Antigen B-Cell Epitope Tertiary Structure and Surface Electrostatic Potential Reveals the Molecular Basis for Alloantibody Binding and Epitope Immunogenicity.

Introduction The potential of donor HLA B-cell epitopes to induce recipient humoral alloimmunity depends on their structural and physiochemical properties. We determined the three-dimensional structure and electrostatic potential of two widely-expressed HLA B-cell epitopes and examined the impact of amino acid mutations on HLA antibody reactivity. Methods The structure of 24 HLA-B alleles, expressing either the Bw4 or Bw6 epitope (defined by sequence motifs at positions 77-83), were predicted using homology modelling (MODELLER) based on the structure of crystallographically-resolved HLA molecules. Multiple conformers were sampled from a 1 ns molecular dynamics simulations using GROMACS. The electrostatic potential in three-dimensional space encompassing the Bw4/Bw6 epitope, for each HLA-B molecule, was computed by solving the Poisson-Boltzmann equation and quantitatively compared with one another to form dendrograms that cluster epitopes with similar electrostatics properties. Amino acid mutations within the 77-83 sequence motifs were also examined. Results Comparison of the electrostatic potential in the space surrounding residues 77-83 allowed tight clustering of HLA-B molecules according to Bw4 or Bw6 epitope expression, independent of epitope amino acid composition and variability in the structural context of epitope expression. This provides a molecular basis for known patterns of serological cross-reactivity. Critical amino acid mutations that abrogated antibody binding to Bw6-expressing HLA-B*07:02 (G83R, R79G, R82L) induced distinct electrostatic potential changes displacing the mutants from the Bw6 epitope cluster; in contrast, mutation N80T did not affect antibody binding and had negligible physiochemical effect. Conclusion This study suggests that HLA B-cell epitopes are characterized by distinct topographic patterns of electrostatic potential explaining HLA-specific antibody binding and enabling novel insights into HLA immunogenicity.

Multivalent dendrimers presenting spatially controlled clusters of binding epitopes in thermoresponsive hyaluronan hydrogels

The controlled presentation of biofunctionality is of key importance for hydrogel applications in cell-based regenerative medicine. Here, a versatile approach was demonstrated to present clustered binding epitopes in an injectable, thermoresponsive hydrogel. Well-defined multivalent dendrimers bearing four integrin binding sequences and an azido moiety were covalently grafted to propargylamine-derived hyaluronic acid (Hyal-pa) using copper-catalyzed alkyne–azide cycloaddition (CuAAC), and then combined with pN-modified hyaluronan (Hyal-pN). The dendrimers were prepared by synthesizing a bifunctional diethylenetriamine pentaacetic acid core with azido and NHBoc oligo(ethylene glycol) aminoethyl branches, then further conjugated with solid-phase synthesized RGDS and DGRS peptides. Azido terminated pN was synthesized by reversible addition-fragmentation chain transfer polymerization and reacted to Hyal-pa via CuAAC. Nuclear magnetic resonance (NMR), high performance liquid chromatography, size exclusion chromatography and mass spectroscopy proved that the dendrimers had well-defined size and were disubstituted. NMR and atomic absorption analysis confirmed the hyaluronan was affixed with dendrimers or pN. Rheological measurements demonstrated that dendrimers do not influence the elastic or viscous moduli of thermoresponsive hyaluronan compositions at a relevant biological concentration. Finally, human mesenchymal stromal cells were encapsulated in the biomaterial and cultured for 21days, demonstrating the faculty of this dendrimer-modified hydrogel as a molecular toolbox for tailoring the biofunctionality of thermoresponsive hyaluronan carriers for biomedical applications.

Conserved B-Cell Epitopes among Human Bocavirus Species Indicate Potential Diagnostic Targets

BackgroundHuman bocavirus species 1–4 (HBoV1–4) have been associated with respiratory and enteric infections in children. However, the immunological mechanisms in response to HBoV infections are not fully understood. Though previous studies have shown cross-reactivities between HBoV species, the epitopes responsible for this phenomenon remain unknown. In this study, we used genomic and immunologic approaches to identify the reactive epitopes conserved across multiple HBoV species and explored their potential as the basis of a novel diagnostic test for HBoVs.Methodology/Principal FindingsWe generated HBoV1–3 VP2 gene fragment phage display libraries (GFPDLs) and used these libraries to analyze mouse antisera against VP2 protein of HBoV1, 2, and 3, and human sera positive for HBoVs. Using this approach, we mapped four epitope clusters of HBoVs and identified two immunodominant peptides–P1 (1MSDTDIQDQQPDTVDAPQNT20), and P2 (162EHAYPNASHPWDEDVMPDL180)–that are conserved among HBoV1–4. To confirm epitope immunogenicity, we immunized mice with the immunodominant P1 and P2 peptides identified in our screen and found that they elicited high titer antibodies in mice. These two antibodies could only recognize the VP2 of HBoV 1–4 in Western blot assays, rather than those of the two other parvoviruses human parvovirus B19 and human parvovirus 4 (PARV4). Based on our findings, we evaluated epitope-based peptide-IgM ELISAs as potential diagnostic tools for HBoVs IgM antibodies. We found that the P1+P2-IgM ELISA showed a higher sensitivity and specificity in HBoVs IgM detection than the assays using a single peptide.Conclusions/SignificanceThe identification of the conserved B-cell epitopes among human bocavirus species contributes to our understanding of immunological cross-reactivities of HBoVs, and provides important insights for the development of HBoV diagnostic tools.

Characterization of the Protective HIV-1 CTL Epitopes and the Corresponding HLA Class I Alleles: A Step towards Designing CTL Based HIV-1 Vaccine.

Human immunodeficiency virus (HIV) possesses a major threat to the human life largely due to the unavailability of an efficacious vaccine and poor access to the antiretroviral drugs against this deadly virus. High mutation rate in the viral genome underlying the antigenic variability of the viral proteome is the major hindrance as far as the antibody based vaccine development is concerned. Although the exact mechanism by which CTL epitopes and the restricting HLA alleles mediate their action towards slow disease progression is still not clear, the important CTL restricted epitopes for controlling viral infections can be utilized in future vaccine design. This study was designed for the characterization the HIV-1 optimal CTL epitopes and their corresponding HLA alleles. CTL epitope cluster distribution analysis revealed only two HIV-1 proteins, namely, Nef and Gag, which have significant cluster forming capacity. We have found the role of specific HLA supertypes such as HLA B∗07, HLA B∗58, and HLA A∗03 in selecting the hydrophobic and conserved amino acid positions within Nef and Gag proteins, to be presented as epitopes. The analyses revealed that the clusters of optimal epitopes for Nef and p24 proteins of HIV-1 could potentially serve as a source of vaccine.

Cryo-EM structure of a fully glycosylated soluble cleaved HIV-1 envelope trimer.

The HIV-1 envelope glycoprotein (Env) trimer contains the receptor binding sites and membrane fusion machinery that introduce the viral genome into the host cell. As the only target for broadly neutralizing antibodies (bnAbs), Env is a focus for rational vaccine design. We present a cryo-electron microscopy reconstruction and structural model of a cleaved, soluble Env trimer (termed BG505 SOSIP.664 gp140) in complex with a CD4 binding site (CD4bs) bnAb, PGV04, at 5.8 angstrom resolution. The structure reveals the spatial arrangement of Env components, including the V1/V2, V3, HR1, and HR2 domains, as well as shielding glycans. The structure also provides insights into trimer assembly, gp120-gp41 interactions, and the CD4bs epitope cluster for bnAbs, which covers a more extensive area and defines a more complex site of vulnerability than previously described.

In silico high throughput pre-clinical determination of monoclonal antibody immunogenicity (P3391)

Abstract One of the great surprises of the biologics revolution has been the discovery that recombinant human proteins, including humanized and fully-human monoclonal antibodies (MAb), can be immunogenic when administered to immune-competent subjects. Preclinical and clinical evaluations of the immunogenic potential for biologic drugs primarily focus on humoral immune responses; as a result, the critical contribution of T cells to the development of anti-drug antibodies (ADA) has been somewhat overlooked. Using the EpiMatrix T cell epitope mapping system, we have developed an interactive in silico screening and optimization platform that evaluates the overall immunogenic potential of a biologic as well as identifies individual T cell epitope clusters contributing to its immunogenicity. In contrast to other immunogenicity prediction tools, our platform considers the contribution of regulatory T cell epitopes (Tregitopes) to immunogenic potential. Tregitopes are highly conserved T cell epitopes derived from IgG that we and others have shown activate regulatory T cells and promote tolerance induction to associated antigens. Here we demonstrate the correlation of available clinical immunogenicity data with Tregitope-adjusted immunogenicity scoring for twenty approved MAbs. Further, we present a high-throughput platform from which these scores can be used to triage large pools of candidate MAbs during the discovery phase of antibody development.

HPV-16 E6 and E7 protein T cell epitopes prediction analysis based on distributions of HLA-A loci across populations: An in silico approach

Human papillomavirus type 16 (HPV-16) is the most prevalent virus in human cervical cancers, as it is present in more than half of all cases. Many studies have found continued expression of E6 and E7 proteins in the majority of cervical cancer cases, but not in normal tissues. These results indicated that the E6 and E7 proteins could be ideal candidate therapeutic vaccines against HPV-16 infection and cervical cancer. Using the Immune Epitope Database Analysis Resource, cytotoxic T lymphocyte (CTL) epitopes of the HPV-16 E6 and E7 proteins were predicted according to worldwide frequency distributions of HLA-A alleles (HLA-A*01:01, -A*02:01, -A*02:06, -A*03:01, -A*11:01, -A*24:02, -A*26:01, -A*31:01 and -A*33:03). Our results predicted a total of 81 epitopes of HPV-16 E6 (n=59) and E7 (n=22). Epitope cluster analysis showed that among the 20 clusters of HPV-16 E6, cluster 3 contained the most epitopes (10 epitopes), which was represented by HLA-A*31:01 and -A*33:03. Of the 10 clusters of HPV-16 E7, cluster 3 contained the most epitopes (5 epitopes), which was represented by HLA-A*01:01 and -A*26:01. Our results indicated that the combination of epitopes FAFRDLCIVYR52-62 of E6 (HLA-A*02:06, HLA-A*31:01, and HLA-A*33:03), PYAVCDKCLKF66-76 of E6 (HLA-A*11:01 and HLA-A*24:02), HGDTPTLHEY2-11 of E7 (HLA-A*01:01 and HLA-A*26:01), and YMLDLQPETT11-20 of E7 (HLA-A*02:01) could vaccinate >50% of all individuals worldwide. Our results propose CTL epitopes or combinations of them predicted in current study for candidate therapeutic vaccines to effectively control HPV-16 infection and development of cervical cancer.

HLA Supertypes Contribute in HIV Type 1 Cytotoxic T Lymphocyte Epitope Clustering in Nef and Gag Proteins

Induction of HIV-1-specific cytotoxic T lymphocyte (CTL) responses largely depends upon the presentation of CTL epitopes to the CD8(+) T cells aided by a large number of different HLA class I alleles. Although several studies showed the clustering pattern of HIV-1 CTL epitopes, the underlying reason for this tendency remains unresolved. Moreover, the hypothesis that the CTL epitope clusters tend to coincide with the conserved and hydrophobic regions of HIV-1 proteins has been challenged in recent times. The present study aims to characterize and compare the HIV-1 CTL epitope clusters in terms of restricting HLA alleles, hydrophobicity, and sequence conservation in a proteome-wide manner by including a large number of experimentally validated CTL epitopes from the HIV Molecular Immunology Database. CTL epitope cluster distribution analysis in a proteome-wide manner revealed that only two HIV-1 proteins, namely Nef and Gag, have significant cluster-forming capacity where their epitope localization coincides with the hydrophobic and conserved regions. Furthermore, analyses of proteasomal cleavage sites and HLA anchoring motif frequencies in the epitope-dense regions highlighted the role of specific HLA supertypes such as HLA B*07, HLA B*58, HLA A*02, and HLA A*03 in selecting the hydrophobic and conserved amino acid positions within Nef and Gag proteins to be presented as epitopes. Based on our results, we hypothesize that the cluster-forming tendency of HIV-1 CTL epitopes is not a proteome-wide feature confined to Nef and Gag proteins. Their cluster-forming tendency largely depends on the host HLA alleles that contribute significantly in selecting functionally constrained hydrophobic regions within the HIV-1 proteome.

Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding.

The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain; and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. Thus, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.

HIV-1 envelope resistance to proteasomal cleavage: implications for vaccine induced immune responses.

BackgroundAntigen processing involves many proteolytic enzymes such as proteasomes and cathepsins. The processed antigen is then presented on the cell surface bound to either MHC class I or class II molecules and induces/interacts with antigen-specific CD8+ and CD4+ T-cells, respectively. Preliminary immunological data from the RV144 phase III trial indicated that the immune responses were biased towards the Env antigen with a dominant CD4+ T-cell response.MethodsIn this study, we examined the susceptibility of HIV-1 Env-A244 gp120 protein, one of the protein boost subunits of the RV144 Phase III vaccine trial, to proteasomes and cathepsins and identified the generated peptide epitope repertoire by mass spectrometry. The peptide fragments were tested for cytokine production in CD4+ T-cell lines derived from RV144 volunteers.ResultsEnv-A244 was resistant to proteasomes, thus diminishing the possibility of the generation of class I epitopes by the classical MHC class I pathway. However, Env-A244 was efficiently cleaved by cathepsins generating peptide arrays identified by mass spectrometry that contained both MHC class I and class II epitopes as reported in the Los Alamos database. Each of the cathepsins generated distinct degradation patterns containing regions of light and dense epitope clusters. The sequence DKKQKVHALF that is part of the V2 loop of gp120 produced by cathepsins induced a polyfunctional cytokine response including the generation of IFN-γ from CD4+ T-cell lines-derived from RV144 vaccinees. This sequence is significant since antibodies to the V1/V2-loop region correlated inversely with HIV-1 infection in the RV144 trial.ConclusionsBased on our results, the susceptibility of Env-A244 to cathepsins and not to proteasomes suggests a possible mechanism for the generation of Env-specific CD4+T cell and antibody responses in the RV144 vaccinees.

Human Monoclonal Antibodies to a Novel Cluster of Conformational Epitopes on HCV E2 with Resistance to Neutralization Escape in a Genotype 2a Isolate

Human Monoclonal Antibodies to a Novel Cluster of Conformational Epitopes on HCV E2 with Resistance to Neutralization Escape in a Genotype 2a Isolate

The Membrane Protein of Severe Acute Respiratory Syndrome Coronavirus Acts as a Dominant Immunogen Revealed by a Clustering Region of Novel Functionally and Structurally Defined Cytotoxic T‐Lymphocyte Epitopes

Background. Severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged with highly contagious and life-threatening characteristics in 2002, remains a potential risk for future outbreaks. Membrane (M) and envelope (E) proteins are major structural proteins of the SARS-CoV. The M protein has been determined as a protective antigen in humoral responses. However, its potential roles in stimulating cellular immunity remain elusive. Methods. In this study, a panel of peptides derived from M and E proteins were tested by in vitro refolding, T2 cell-binding assays, and responses stimulated by cytotoxic T-lymphocyte (CTL) epitopes in HLA-A2.1/Kb transgenic mice and human peripheral blood mononuclear cells (PBMCs). Results. A nonameric epitope Mn2 and a decameric epitope Md3 derived from the M protein were identified and used for the evaluation of M protein-specific immunity. Responses stimulated by M protein-specific CTL epitopes have been found in the PBMCs of donors who had recovered from SARS infection. Additionally, the transmembrane domain of the M protein may contain a T cell epitope cluster revealed by the immunogenic and structural analysis of a panel of truncated peptides overlapping with Mn2 and Md3. Conclusions. The M protein of SARS-CoV holds dominant cellular immunogenicity. This, together with previous reports of a strong humoral response against the M protein, may help to further explain the immunogenicity of SARS and serves as potential targets for SARS-CoV vaccine design.

A Computational Approach for Identification of Epitopes in Dengue Virus Envelope Protein: A Step Towards Designing a Universal Dengue Vaccine Targeting Endemic Regions

A major problem in designing vaccine for the dengue virus has been the high antigenic variability in the envelope protein of different virus strains. In this study, a computational approach was adopted to identify a multi-epitope vaccine candidate against dengue virus that may be suitable for large populations in the dengue-endemic regions. Different bioinformatics tools were exploited that helped the identification of a conserved immunological hot-spot in the dengue envelope protein. The tools also rendered the prediction of immunogenicity and population coverage to the proposed 'in silico' vaccine candidate against dengue. A peptide region, spanning 19 amino acids, was identified in the envelope protein which found to be conserved in all four types of dengue viruses. Ten proteasomal cleavage sites were identified within the 19-mer conserved peptide sequence and a total of 8 overlapping putative cytotoxic T cell (CTL) epitopes were identified. The immunogenicity of these epitopes was evaluated in terms of their binding affinities to and dissociation half-time from respective human leukocyte antigen (HLA) molecules. The HLA allele frequencies were studied among populations in the dengue endemic regions and compared with respect to HLA restriction patterns of the overlapping epitopes. The cumulative population coverage for these epitopes as vaccine candidates was high ranging from approximately 80% to 92%. Structural analysis suggested that a 9-mer epitope fitted well into the peptide-binding groove of HLA-A*0201. In conclusion, the 19-mer epitope cluster was shown to have the potential for use as a vaccine candidate against dengue.

Antibody-mediated synergy and interference in the neutralization of SARS-CoV at an epitope cluster on the spike protein

Incomplete neutralization of virus, especially when it occurs in the presence of excess neutralizing antibody, represents a biological phenomenon that impacts greatly on antibody-mediated immune prophylaxis of viral infection and on successful vaccine design. To understand the mechanism by which a virus escapes from antibody-mediated neutralization, we have investigated the interactions of non-neutralizing and neutralizing antibodies at an epitope cluster on the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV). The epitope cluster was mapped at the C-terminus of the spike protein; it consists of structurally intertwined epitopes recognized by two neutralizing monoclonal antibodies (mAbs), 341C and 540C, and a non-neutralizing mAb, 240C. While mAb 341C binds to a mostly linear epitope composed of residues 507PAT 509 and V 349, mAb 240C binds to an epitope that partially overlaps the former by at least two residues (P 507 and A 508). The epitope corresponding to mAb 540C is a conformational one, involving residues L 504 and N 505. In neutralization assays, non-neutralizing 240C disrupted virus neutralization by mAb 341C and/or mAb 540C, whereas a combination of mAbs 341C and 540C blocked virus infectivity synergistically. These findings indicate that the epitope cluster on the spike protein may serve as an evolutionarily conserved platform at which a dynamic interplay between neutralizing and non-neutralizing antibodies occurs, thereby determining the outcome of SARS-CoV infection.